Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Insights into the mechanism of Huanglongbing tolerance in the Australian finger lime (Citrus australasica)

Insights into the mechanism of Huanglongbing tolerance in the Australian finger lime (Citrus... TYPE Original Research PUBLISHED 21 October 2022 DOI 10.3389/fpls.2022.1019295 Insights into the mechanism of Huanglongbing tolerance in the OPEN ACCESS EDITED BY Marcio C. Silva-Filho, Australian finger lime University of São Paulo, Brazil REVIEWED BY (Citrus australasica) Alessandra Alves De Souza, Secretariat of Agriculture and Food Supply of São Paulo State, Brazil 1 1,2 1 † † Kyle C. Weber , Lamiaa M. Mahmoud , Daniel Stanton , Marcos Antonio Machado, Instituto Agrono ˆ mico de Campinas 1 3 1 Stacy Welker , Wenming Qiu , Jude W. Grosser , (IAC), Brazil 1 1 Amit Levy and Manjul Dutt *CORRESPONDENCE Manjul Dutt Citrus Research and Education Center, University of Florida, Lake Alfred, FL, United States, manjul@ufl.edu Pomology Department, Faculty of Agriculture, Mansoura University, Mansoura, Egypt, These authors have contributed Institute of Fruit and Tea, Hubei Academy of Agricultural Sciences, Wuhan, China equally to this work SPECIALTY SECTION This article was submitted to Plant Breeding, The Australian finger lime (Citrus australasica) is tolerant to Huanglongbing a section of the journal (HLB; Citrus greening). This species can be utilized to develop HLB tolerant Frontiers in Plant Science citrus cultivars through conventional breeding and biotechnological RECEIVED 14 August 2022 approaches. In this report, we conducted a comprehensive analysis of ACCEPTED 22 September 2022 PUBLISHED 21 October 2022 transcriptomic data following a non-choice infection assay to understand the CITATION CaLas tolerance mechanisms in the finger lime. After filtering 3,768 Weber KC, Mahmoud LM, Stanton D, differentially expressed genes (DEGs), 2,396 were downregulated and 1,372 Welker S, Qiu W, Grosser JW, Levy A were upregulated in CaLas-infected finger lime compared to CaLas-infected and Dutt M (2022) Insights into the mechanism of Huanglongbing HLB-susceptible ‘Valencia’ sweet orange. Comparative analyses revealed tolerance in the Australian finger lime several DEGs belonging to cell wall, b-glucanase, proteolysis, R genes, (Citrus australasica). signaling, redox state, peroxidases, glutathione-S-transferase, secondary Front. Plant Sci. 13:1019295. doi: 10.3389/fpls.2022.1019295 metabolites, and pathogenesis-related (PR) proteins categories. Our results COPYRIGHT indicate that the finger lime has evolved specific redox control systems to ©2022Weber,Mahmoud,Stanton, mitigate the reactive oxygen species and modulate the plant defense response. Welker,Qiu,Grosser, Levyand Dutt. We also identified candidate genes responsible for the production of Cys-rich This is an open-access article distributed under the terms of the secretory proteins and Pathogenesis-related 1 (PR1-like) proteins that are Creative Commons Attribution License highly upregulated in infected finger lime relative to noninfected and (CC BY). The use, distribution or reproduction in other forums is infected ‘Valencia’ sweet orange. Additionally, the anatomical analysis of permitted, provided the original phloem and stem tissues in finger lime and ‘Valencia’ suggested better author(s) and the copyright owner(s) regeneration of phloem tissues in finger lime in response to HLB infection. are credited and that the original publication in this journal is cited, in Analysis of callose formation following infection revealed a significant accordance with accepted academic difference in the production of callose plugs between the stem phloem of practice. No use, distribution or reproduction is permitted which does CaLas+ ‘Valencia’ sweet orange and finger lime. Understanding the mechanism not comply with these terms. of resistance will help the scientific community design strategies to protect trees from CaLas infection and assist citrus breeders in developing durable HLB tolerant citrus varieties. KEYWORDS citrus, transcriptome, Huanglongbing, host response, pathogen-related proteins, callose deposition Frontiers in Plant Science 01 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 (Forster and Smith, 2010). Australian lime species such as Citrus Introduction australasica, and the hybrid of Citrus australis and Citrus virgata The genus Citrus originated in tropical and subtropical (Sydney hybrid) have been reported to be HLB resistant (Ramadugu et al., 2016; Alves et al., 2020; Huang et al., southeastern Asia (Wu et al., 2014). When consumed fresh, citrus fruit are good sources of dietary fiber (Marıń et al., 2007) 2021a). Thus, these species can provide pathogen resistance- related genes that can be used to confer HLB tolerance into and antioxidants (Wang et al., 2022), and they have anticancer and anti-inflammatory properties (Benavente-Garcı́aand conventional citrus cultivars to produce HLB-tolerant citrus hybrid scions and rootstocks. Although extensive research is Castillo, 2008). The United States is one of the citrus being conducted to use HLB tolerance traits for the development producers, with production concentrated in Florida, California, of new citrus cultivars, there is a lack of knowledge on the and Texas. Sweet orange constitutes most of the citrus mechanism underlying the perceived tolerance in these citrus production acreage, with the remainder being that of species. Resistance and tolerance observed in the different grapefruit, mandarin, lemons and limes (Kramer et al., 2022). cultivars can be defined by various factors, including the Citrus is susceptible to plethora of diseases and pests, with absence of CaLas multiplication and replication, delayed huanglongbing (HLB), a phloem-limiting bacterial disease infection, or recovery from infection by enhancing plant caused by the bacterium Candidatus Liberibacter asiaticus defensive systems. (CaLas), being the most destructive (Duan et al., 2009). In the CaLas is a gram-negative bacterium that employs secretion United States, this disease has been prevalent since 2005 (Bové, systems that deliver virulence proteins, known as effectors, to 2006), when it was first detected in south Florida’s Miami-Dade manipulate its hosts (Clark et al., 2018) through modulation of County (Halbert, 2005). Widespread monoculture of a few select host physiology and suppressing plant defense mechanisms. citrus varieties has reduced the genetic diversity of cultivated Effectors promote pathogen colonization and disease citrus, allowing HLB to spread quickly among the population. development and create environmental conditions favorable Since its initial discovery in 2005, HLB has spread rapidly for colonization and proliferation (Jones and Dangl, 2006; Dou throughout Florida to every citrus-growing county. and Zhou, 2012; Feng and Zhou, 2012). The plant defense Additionally, HLB is now present in Texas and California, response system involves pattern-triggered immunity (PTI), where it threatens the important central valley (Milne et al., which is triggered by microbe-associated molecular patterns 2018; Graham et al., 2020). Most of the commercial citrus (MAMPs) via cell surface-localized pattern-recognition cultivars grown in the United States, including several named receptors (PRRs), and effector-triggered immunity (ETI), cultivars and selections of sweet orange, mandarin, and which is induced by pathogen effector proteins via grapefruit, are highly susceptible to HLB. intracellular receptors that detect intercellular pathogen- The health of infected trees invariably declines, accompanied derived molecules and intracellular receptors that activate by reduced fruit yield and quality (Ferguson et al., 2021) and plant defense response upon detection of pathogen-secreted severely infected trees eventually die (Zhang et al., 2020b). Long- effector proteins that function inside the plant cell (Ngou term management of tree health through enhanced nutrition et al., 2021). Previous research showed that CaLas encodes (Zambon et al., 2019) and psyllid vector control using various several Sec-delivered effectors (SDEs), many of which are control strategies have been proposed and evaluated (Mattos-Jr conserved across CaLas isolates. Sec-delivered effector 1 et al., 2020). Tolerance to HLB has been reported in some citrus (SDE1), a secreted protein biomarker used for the detection of cultivars, such as citron and its hybrids (e.g., lemons), and in HLB, is highly expressed in infected citrus tissue at a relatively some trifoliate orange trees and their hybrids (Peng et al., 2020). early infection stage (Pagliaccia et al., 2017; Tran et al., 2020). HLB-tolerant scions and rootstocks, conventionally bred or Proteases secreted by pathogens have been shown to be transgenic, remain the best option for the control and important virulence factors that affect plant defense, and management of HLB (Qiu et al., 2020). Sugar Belle, a recently cysteine (Cys) proteases have been demonstrated to participate released mandarin hybrid, has also been observed to be HLB in different pathosystems (Zhang et al., 2020a). Citrus papain- tolerant (Killiny et al., 2018). Several wild sexually compatible like cysteine proteases (PLCPs) were found as a defense cultivars, such as Citrus ichangensis ‘2586’ (Wu et al., 2020), inducible in CaLas-infected trees, suggesting they are involved Citrus latipes (Folimonova et al., 2009), several accessions of sour in the citrus defense responses (Clark et al., 2018). Additionally, pummelo (Citrus grandis; Zou et al. (2019) and kaffir lime several lysosomal Cys proteases were shown to be involved in (Citrus hystrix; Hu et al. (2017), are also tolerant to HLB. various apoptosis models, although the mechanisms of their Additionally, several sexually incompatible citrus relatives are involvement are not yet clear (Rozman-Pungerčar et al., 2003). also tolerant to HLB (Miles et al., 2017). Plasma membrane-localized receptor-like kinases (RLKs) play a The Australian limes spread from Southeast Asia to role in plant recognition of microbes and in perceiving and Australasia during the early Pliocene epoch, approximately transducing these external stimuli to further activate the 4Ma (Wu et al., 2018). There are seven species of Australian associated downstream signaling pathways (Jose et al., 2020). limes that are all native to Australia and the New Guinea islands Frontiers in Plant Science 02 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 RLKs are categorized into several subfamilies, including leucine- integrity of the RNA were analyzed using electrophoresis on a rich repeat (LRR) RLKs (LRR-RLKs), Cys-rich repeat (CRKs), 1.0% agarose gel and then examined using an Agilent 2100 domains of unknown function 26 RLKs, S-domain RLKs, and Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). others (Quezada et al., 2019). High-quality RNA samples with an RNA integrity number In this study, we shed light on the potential mechanism of (RIN) > 6.5 were used for cDNA synthesis and RNA HLB tolerance in the finger lime.Tothisend,wegraft- sequencing (RNAseq). Single-stranded cDNA was synthesized inoculated one-year-mature finger lime and ‘Valencia’ (Citrus using a RevertAid First Strand cDNA Synthesis Kit (Thermo sinensis) sweet orange trees with CaLas and evaluated their Fisher Scientific, Massachusetts, USA). The cDNA transcriptome to provide insights into the mechanism of concentration was determined using a NanoDrop 1000 tolerance to HLB. The transcriptome data was also validated Spectrophotometer (Thermo Fisher Scientific). in five-year-mature trees growing in the field. The cDNA libraries were sequenced using an Illumina HiSeq platform configured for a 2x150 read length. The generated base callings of the cDNA reads were presented in a paired-ended Materials and methods format. The reads were cleaned, and their adapters were removed using AdapterRemoval v2.2.2 (Lindgreen, 2012), with Plant materials the default parameters. Short and poor-quality reads were filtered using Trimmomatic v0.39 (Bolger et al., 2014). The Certified HLB-free budwood of C. australasica clone DPI 50-36 following parameters were applied: a minimum length of 100 (Finger lime) and ‘Valencia’ sweet orange clone SPB-1-14-19 were bases, a trailing and leading length equal to 16 bases, a sliding obtained from Florida’s Division of Plant Industry budwood window of 16:25, and 5 threads. After processing, the final read repository and budded onto 6-month-old Swingle citrumelo count and average qualities were checked using FastQC v0.11.8 rootstock. One-year-old budded trees were subsequently side (Andrews, 2010). grafted (Figure S1)with CaLas-infected ‘Valencia’ sweet orange scions (Ct value of 23.2 ± 0.3). The trees were periodically evaluated for infection, and 1 to 2 year-old infected trees were utilized for Mapping of the reads, transcript counts, subsequent experiments (Table S1). and DEG analysis The cleaned reads were mapped to the C. sinensis genome Monitoring CaLas in finger lime and using STAR v2.6.0C (Dobin et al., 2013) with the default ‘valencia’ sweet orange plants parameters, except for the need to define a sorted BAM output. The C. sinensis genome and annotations used in STAR To diagnose the CaLas titer in the potentially infected were obtained from Phytozome (https://phytozome-next.jgi.doe. greenhouse-grown trees, genomic DNA was isolated periodically gov/) The BAM files were indexed using SAMtools v1.7 (Li et al., from the leaf petioles and midveins of fully expanded leaves using a 2009). The BAM files were assessed for transcript counts using GeneJET Plant Genomic DNA Purification Kit (Thermo Fisher featureCounts v1.6.0 (Liao et al., 2014) with default settings Scientific Waltham, MA, USA). Leaves were also collected in the except for the section of exon type and five threads. The list of late fall (November) and early spring (March) from 5-year-old counts was extracted from the featureCounts software output file finger lime DPI 50-36 and ‘Valencia’ SPB-1-14-19 trees growing in and organized to compare infected finger lime vs.infected the field (Swingle rootstock) to estimate the CaLas titer in the ‘Valencia’ sweet orange. A metadata file for the comparison sampled tissues for three years. The DNA concentration was was also generated for differentially expressed gene (DEG) normalized to 25 ng/mL before performing qPCR using a analysis via DESeq2 v3.10, an R Bioconductor package (Love StepOnePlus Real-Time PCR System (Thermo Fisher et al., 2014). The counts for each comparison were normalized, Scientific). Detection of CaLas genomic DNA was determined by and gene dispersion was estimated using DESeq2. The list of qPCR using TaqMan Gene Expression Master Mix and CQUL DEGs was filtered to remove any DEGs with a |log (fold- primers (Table S2)to amplify the CaLas rplJ/rplL ribosomal protein change)|< 2 and an adjusted P value (false discovery rate gene (Wang et al., 2006). (FDR)) ≥ 0.05. RNA extraction, cDNA synthesis and Gene ontology enrichment and sequencing pathway analysis Two years following infection, RNA was extracted using Statistically significant DEGs were analyzed using AgriGO TRIzol following the manufacturer’s protocol. The purity and v2 (Tian et al., 2017). To correctly assign GO terms, the Frontiers in Plant Science 03 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 following parameters were selected: Genome - Citrus sinensis, AMC), given that protease activity correlates with an increase in statistical test - Fisher’s exact test, adjusted according to the detectable relative light units (RFUs) over time (Barrett, 1980; Benjamini–Yekutieli method (Benjamini et al., 2006)for Tchoupe et al., 1991). The leaf samples were extracted in a buffer discovering FDRs in a multiple comparison with an consisting of 100 mM sodium acetate (pH 5.5), 2.5 mM DTT and 1 alpha=0.05, and a minimum mapping of 5. The statistically mM EDTA. The samples were centrifuged, and the supernatants significant GO terms from AgriGO v2 were then inputted into were incubated at a 1:2 ratio together with a mixture consisting of 100 REVIGO software to remove redundant GO terms (Supek et al., mM sodium acetate (pH 5.5), 2.5 mM DTT, 1 mM EDTA, 0.5% 2011). Functional analysis was conducted using the C. sinensis DMSO, and 37.5 mM Z-F-R-AMC. All the samples were incubated pathways file (m02) in MapMan (Thimm et al., 2004). The at 30°C for5minutes. Anotherset of samples were co-incubated for functional categories were viewed using PageMan and analyzed 3 hours with 10 µM synthetic epoxide peptide E-64 (L-3- for statistical significance using a nonparametric test (Usadel trans-carboxyoxiran-2-carbonyl]-L-Leu-agmatin]; [N- et al., 2006). Pathway analysis was performed using the pathways (transepoxysuccinyl)-L-leucine 4-guanidinobutylamide]) as function of MapMan (Mapman version 3.0.0). inhibitor of cysteine protease at 37°C. At the end of the incubation, ™ ™ fluorescence was measured in a Thermo Scientific GENESYS 30 Visible Spectrophotometer atlex = 380 and lem = 460 nm. Negative Quantitative PCR and DEG validation (no enzyme) and blank samples were also prepared along with the positive samples by the addition of E-64 inhibitor and solvent, The real-time PCR (qPCR) reaction mix consisted of 1 µL of respectively. The percent inhibition was calculated by using the ® ™ DNA (25 ng/µL), SYBR Green PowerUp PCR Master Mix following formula: (Applied Biosystems, Foster City, CA), and selected gene- Inhibition % = [Absorbance (blank)-Absorbance (test)]/ specific primers (Integrated DNA Technologies, Inc., Absorbance (blank) x 100 Coralville, IA, USA) in a final mixture of 20 mL, according to the manufacturers’ instructions. qPCR was performed in a StepOnePlus Real-Time PCR System (Thermo Fisher Evaluation of CaLas-infected ‘valencia’ Scientific, Massachusetts, USA). The citrus b-actin sweet orange and finger lime and housekeeping gene was used as a reference gene (Qiu et al., quantification of phloem callose deposits 2020); each sample was analyzed in triplicate. Relative gene -DDCt expression was calculated using the 2 method described Healthy and CaLas-infected finger lime and ‘Valencia’ sweet th th previously (Livak and Schmittgen, 2001). The relative mRNA orange leaves (position 5 -8 from the apical meristem) were levels were compared to those of the endogenous C. sinensis collected from the greenhouse. The petioles were cut and fixed in ACTIN gene (Qiu et al., 2020) and calculated using the 2 4% paraformaldehyde in 1x PBS. The samples were rinsed three DDCT method (Livak and Schmittgen, 2001). To confirm the times in 1x PBS and then dehydrated in an ethanol (EtOH) series validity of the DEGs, we selected eight upregulated and eight for 1 hour each. The samples were transitioned from 100% EtOH downregulated genes from the DEG data, then analyzed on the to 100% tert-butanol (3:1, 1:1, and 1:3) at room temperature same samples that were sequenced and the relative expression of (RT) for 8-16 hours each and then cleared in 100% tert-butanol those genes were compared with RNAseq results. As there is no for one hour prior to paraffininfiltration. The samples were publicly available genome assembly of the finger lime yet, we infiltrated using increasing concentrations (3:1, 1:1, 1:3) of selected those genes based on the sweet orange genome. Paraplast Plus paraffin (Fisher Scientific, Waltham, MA, USA) Additionally, some of the DEGs that show significant for 24 hours each and then incubated for 48-36 hours in 100% difference were validated in twelve samples collected from paraffin, which was changed three times. The samples were finger lime and ‘Valencia’ sweet orange trees growing in the embedded in paraffin and allowed to harden for 24 hours at field. The gene expression of the trees was compared with that of 4°C. Afterward, ten micrometer sections were cut using a Leica uninfected control trees growing in a protected greenhouse. The 2155 microtome (Leica Biosystems, Deer Park IL, USA), and the control trees were confirmed to be negative for CaLas before sections were floated on a drop of water on a slide. The slides subsequent comparison. A list of the primers used in this study is were subsequently incubated overnight on a slide warmer at presented in Tables S3, S4. 37°C to allow the sections to adhere to the slide. The slides were dewaxed in 100% Histoclear II (National Diagnostics, Atlanta, GA, USA) for an hour each, and the solution was changed twice. Proteolytic enzyme assays The sections were stained with 0.05% toluidine blue O for 30 seconds and then rinsed in dH O. The slides were dehydrated in Assessment of Cys protease activitywasperformedbyrecording an EtOH series for 5-10 minutes each. Coverslips were mounted the liberation of fluorogenic peptide substrate VIII (Z-Phe-Arg- using Fisher Scientific’s mounting media with toluene (Fisher AMC. Z: N-carbobenzyloxy; 7-amino-4-methylcoumarin; Z-F-R- Scientific, Waltham, MA, USA). The slides were observed under Frontiers in Plant Science 04 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 an Olympus BX61 epifluorescence microscope (Olympus, were collected from 8-year-mature trees, and the total DNA Center Valley, PA, USA), and images were captured using a 14 obtained from leaf petioles and midribs was analyzed using MP OMAX digital camera (OMAX, Irvine, CA). The phloem qPCR. Our results indicate that the field-grown finger lime trees and xylem ring distances were measured using FIJI (Schindelin were always HLB negative (undetermined cycle threshold (Ct) or et al., 2012) Figure S2, and the phloem ratio (Pa/Xa) and xylem had high Ct values (37.88 ± 0.28)), whereas ‘Valencia’ sweet ratio (Xa/Pa) for petioles and stems were calculated. Three orange trees had low Ct values of 25.14 ± 0.82 (Figure 1C), samples were used for evaluation and the data were recorded indicating active HLB infection. as average for three images of each sample. Subsequently, field-collected HLB-infected ‘Valencia’ sweet Phloem sieve plate callose was measured according to a orange scions were side grafted onto healthy one-year-mature previous protocol (Ferrara et al., 2015). Stem phloem tissue grafted finger lime and ‘Valencia’ sweet orange trees to observe samples were collected from CaLas+ ‘Valencia’ sweet orange and disease progression under controlled conditions. To assess finger lime trees. The tissue samples were obtained from the bacterial population levels in the leaves, qPCR was periodically stems of the mature trees with a scalpel, approximately 8 cm performed to screen the CaLas titer in the samples collected from the leaves. Three stem phloem samples were collected from from greenhouse trees Table S1. Screening for the presence of five trees of each plant type. Each tissue sample was placed into CaLas revealed that when trees were forcibly inoculated with an 85% EtOH solution for fixation immediately after collection CaLas, both finger lime and ‘Valencia’ sweet orange trees were and incubated overnight for de-staining. The samples were then infected (Figure 1D). However, the rate of infection differed transferred to a 0.01% Tween-20 solution to rehydrate for 1 between the two accessions. At 24 months following infection, hour. Finally, the samples were transferred to a 0.01% aniline the Ct value of the finger lime trees was, on average, 33.2 ± 1.3, blue staining solution. After staining for 1 hour, images of the while the ‘Valencia’ sweet orange Ct value was, on average, 23.4 tissue samples were collected. A Leica SP8 laser-scanning ± 2.8. confocal microscope was used to collect the images, with A transcriptome analysis was subsequently conducted to settings that have been described previously (Welker and Levy, understand the possible biological reasons for the HLB tolerance 2022). Three images were taken from the central region of each of finger lime compared with susceptible citrus such as ‘Valencia’ tissue sample. Using a FIJI macro, counts of callose deposits were sweet orange. The RNA from CaLas-infected samples of finger obtained as described previously (Welker et al., 2022). lime and ‘Valencia’ sweet orange (three technical replicates each) was sequenced using the Illumina HiSeq next-generation sequencing platform. The average total number of raw reads Statistical analysis produced was 37,550,067 and 31,506,847 for the CaLas-infected finger lime replicates and the CaLas-infected ‘Valencia’ sweet The data were analyzed using JMP Pro v16 software, with a orange replicates, respectively (Table 1); the cleaning process of post hoc Tukey–Kramer honestly significant difference (HSD) the reads resulted in average numbers of reads of 29,854,551 test or t tests to compare the means of the different treatments. (79.51%) and 26,230,524 (83.25%), respectively. The cleaned Statistical significance was established at P < 0.05. Pearson reads were mapped onto the C. sinensis genome. Genomic Correlation Coefficient (r) was calculated to validate the mapping of the RNA reads revealed that, on average, modulationingeneexpressionfor RNAseq data and 24,807,563 (84.84%) and 22,595,692 (86.14%) clean reads were quantitative PCR using JMP Pro v16 software. As for mapped. The mapped reads were analyzed by differential statistical Testing of Phloem Callose Deposits, ANOVA was expression analysis. After filtering the differentially expressed performed using R statistical software to assess the significance genes (DEGs) according to |log (fold-change)| < 2 and an of the model and interactions, any non-zero counts of callose adjusted P value (FDR) ≥ 0.05, 3,768 remained. Of the 3,768 plugs were analyzed with a negative binomial regression in R (R DEGs, 2,396 were downregulated in HLB-infected finger lime Core Team, 2013). After log-transforming the counts to meet the compared to HLB-infected ‘Valencia’ sweet orange, while 1,372 assumption of normality, ANOVA was performed on the mean were upregulated (Figure 2A). counts of each tissue type group. Domain differences between finger lime Results and ‘valencia’ sweet orange infected with HLB Finger lime trees have enhanced tolerance to CaLas There were20GOcategoriesthatweresignificantly upregulated, and 19 GO categories downregulated between the To understand HLB levels in mature finger lime and HLB-infected finger lime samples and the HLB-infected ‘Valencia’ sweet orange trees growing in the field, leaf samples ‘Valencia’ sweet orange samples (Table 2). Among the Frontiers in Plant Science 05 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 FIGURE 1 (A) HLB infected ‘Valencia’ sweet orange in the field exhibiting the characteristic blotchy mottle pattern in the leaves. (B) Finger lime leaves from trees growing in the field with no visible disease symptom. (C) Detection of CaLas in leaf tissues of Finger lime and ‘Valencia’ trees by qPCR. Leaf samples were collected from 8-year-old field trees at the beginning of the study. (D) CaLas detection from leaf samples collected periodically from trees, side grafted with HLB infected budwood and growing in the green house. * represents the sampling time for RNAseq analysis. Different letters above the error bar indicate statistically significant differences, while the same letters signify no significant differences using the Tukey–Kramer honestly significant difference test (Tukey HSD; p <0.05). domains of the upregulated DEGs, eleven were representatives of (GO:0008037). Other domains with lower enrichment values the molecular function category. Of these 11 domains, the most are listed in Table 2. There were no domains of upregulated significant were heme binding (GO:0020037), tetrapyrrole DEGs within the cell component category. binding (GO:0046906), and iron ion binding (GO:0005506). The top downregulated GO terms of the molecular function The other domains of the upregulated DEGs represented types category included “catalytic activity” (GO: 0003824) and of biological processes: signaling (GO:0023052), amine “ADP binding” (GO: 0043531). “Amine metabolic process” metabolic process (GO:0009308), and cell recognition (GO:0009308), “polysaccharide catabolic process” (GO:0000272), TABLE 1 Summary of sequencing, cleaning, and mapping of reads following sequencing the HLB infected finger lime and HLB infected ‘Valencia’ samples. Run Name Raw read count Read count after cleaning Surviving Read Percent Mapped reads Mapped reads percent FL1 42,957,054 31,809,635 74.05% 26,440,485 83.12% FL2 34,840,983 28,780,882 82.61% 23,935,454 83.16% FL3 34,852,164 28,973,136 83.13% 24,046,751 83.00% FL Avg 37,550,067 29,854,551 79.51% 24,807,563 83.09% Val1 32,285,138 26,821,818 83.08% 22,756,095 84.84% Val2 32,676,339 27,320,775 83.61% 23,775,386 87.02% Val3 29,559,065 24,548,978 83.05% 21,255,596 86.58% Val Avg 31,506,847 26,230,524 83.25% 22,595,692 86.14% Frontiers in Plant Science 06 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 FIGURE 2 (A) Volcano plot of the upregulated and downregulated DEGs. Genes with an adjusted p value of less than 0.05 found with DESeq were assigned as differentially expressed. (B) Graphical view of the most statistically significant upregulated and downregulated enriched GO terms in Finger line trees as compared to ‘Valencia’ trees. Statistically significant DEGs were analyzed using AgriGO v2 and REVIGO. (C) Differentially expressed genes as identified following MAPMAN analysis. Regulation of stress-related gene pathways by CaLas infection in the infected Finger Lime (Left). Overview of the differentially expressed genes related to the metabolic pathways in Finger lime and ‘Valencia’ sweet orange (Right). Genes that were significantly upregulated following CaLas infection are displayed in blue, and downregulated genes are displayed in red. Frontiers in Plant Science 07 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 TABLE 2 Significant GO terms represented in the HLB infected finger lime vs HLB infected ‘Valencia’ comparison. GoTerm Type Description PosCount PosEV NegCount NegEV GO:0003824 F catalytic activity 0 0 655 -5.6576 GO:0005506 F iron ion binding 57 -7.1549 63 -3.585 GO:0043531 F ADP binding 56 -4.0269 72 -2.8539 GO:0009308 P amine metabolic process 13 -3.5229 13 -2.0915 GO:0000272 P polysaccharide catabolic process 6 -2.0655 8 -2.0915 GO:0042221 P response to chemical 0 0 33 -2.0915 GO:0071554 P cell wall organization or biogenesis 0 0 24 -2.0915 GO:0004857 F enzyme inhibitor activity 0 0 25 -1.7696 GO:0030554 F adenyl nucleotide binding 0 0 205 -1.7696 GO:0071555 P cell wall organization 0 0 18 -1.7212 GO:0020037 F heme binding 54 -4.4815 60 -1.6576 GO:0046906 F tetrapyrrole binding 54 -4.4815 60 -1.6576 GO:0005618 C cell wall 0 0 215 -1.6198 GO:0030312 C external encapsulating structure 0 0 19 -1.6198 GO:0016705 F oxidoreductase activity paired donors 48 -4.4685 52 -1.5376 GO:0016491 F oxidoreductase activity 48 -3.1739 153 -1.5376 GO:0010333 F terpene synthase activity 0 0 17 -1.5376 GO:0016838 F carbon-oxygen lyase activity 0 0 17 -1.4559 GO:0030599 F pectinesterase activity 0 0 15 -1.4437 GO:0023052 P Signaling 44 -3.9586 0 0 GO:0008037 P cell recognition 16 -2.3979 0 0 GO:0048544 P recognition of pollen 16 -2.3979 0 0 GO:0051704 P multi-organism process 17 -2.3979 0 0 GO:0000003 P Reproduction 16 -1.9586 0 0 GO:0016758 F hexosyltransferase activity 41 -1.7959 0 0 GO:0005515 F protein binding 202 -1.4318 0 0 GO:0016757 F glycosyltransferase activity 44 -1.4318 0 0 GO:0030247 F polysaccharide binding 12 -1.4318 0 0 GO:0004568 F chitinase activity 6 -1.3768 0 0 GO:0006026 P aminoglycan catabolic process 6 -1.3665 0 0 GO:0032501 P multicellular organismal process 17 -1.3665 0 0 and “response to chemical” (GO:0042221) were the top categories. Out of the 3,768 DEGs, 1,162 were assigned to disease downregulated GO terms in the biological process category. response categories. Nearly all the DEGs belonged to one of the Unlike the upregulated GO terms, the downregulated GO terms following categories: cell wall, b-glucanase, proteolysis, R genes, were assigned to two cellular component categories: “cell wall” signaling, respiratory burst, abiotic stress, redox state, (GO: 0005618) and “external encapsulating structure” (GO: peroxidases, glutathione-S-transferase, secondary metabolites, 0030312). A graphical view of these data can be found and pathogenesis-related (PR) proteins Figure 2C.When in Figure 2B. comparing the DEGs in functional categories between HLB- infected finger lime and HLB-infected ‘Valencia’ sweet orange, we found that that genes involved in flavonoids, isoflavones, Functional differences between HLB- cytokinin synthesis and degradation, ethylene synthesis and infected finger lime and ‘valencia’ degradation, sugar and nutrient signaling, and the transport of sweet orange sugars as well as genes encoding isoflavone reductase, UDP glucosyl and glucoronyl transferases, cytochrome p450, GRAS DEG functional analysis is important to understand the transcription factors, MAD box transcription factors, WRKY biochemical responses elicited by these genes and the roles transcription factors, DNA methyltransferase, ubiquitin E3, they play in the overall function of the plant. The significant receptor kinases, LRR XI, and DUF were upregulated in finger DEGs were analyzed via PageMan to investigate the functional lime. However, genes assigned to categories related to the cell Frontiers in Plant Science 08 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 wall, pectin esterase, pectin methylesterification (PME), from the field. The response of the infected trees in the field was phenylpropanoids, lignin biosynthesis, short chain largely consistent with data obtained from the greenhouse trees, dehydrogenases/reductases, and posttranslational modification indicating that the RNA-seq data reported here are consistent of kinases and receptors (such as cytoplasmic kinase VII), as well for both sets of samples (greenhouse and field samples). as a few unassigned ones, were found to be underrepresented in Several DEGs encoding many other R proteins that finger lime. Given the functional differences between the predominantly contain a nucleotide-binding site (NBS) and/or upregulated genes among the groups, adding context to better LRR domain were differentially expressed in CaLas-infected understand relationships within a pathway can help determine finger lime. Indeed, 114 pathogenesis-associated DEGs the differences between HLB-infected finger lime and HLB- constituted 3% of all the DEGs mapped, 48 of which were infected ‘Valencia’ sweet orange. upregulated. Twenty-nine of these upregulated genes were PR protein-encoding DEGs of the Tir-NBS-LRR class, 4 were of the NBS-LRR class, and 7 were of the CC-NBS-LRR class. In Pathogen interaction factors were addition, there were 33 Tir-NBS-LRR class genes, 7 CC-NBS- upregulated in finger limes LRR gene class genes, and 14 NBS-LRR class genes that were downregulated. Transcript levels of orange1.1g035344m, similar It has been widely reported that microbe infection induces to the stable antimicrobial peptide (SAMP) (Huang et al., 2021b) plant defense through two mechanisms, namely, PTI and ETI, was not detected in our RNAseq DEG data although play a substantial role in plant disease resistance (Deng et al., orange1.1g033887m, an alternate isoform was upregulated in 2018). Seven cell wall LRR family protein-related DEGs were HLB+ ‘Valencia’. identified as being enriched. Of the DEGs mapped to the Among these proteins, PR proteins and Cys-rich secretory genome, 8.0% were kinase-related DEGs. We identified proteins are thought to be involved in the plant defense response to multiple cysteine (Cys)-rich receptor-like protein kinases pathogen infection and plant tolerance (Van Loon, 1997; Baindara (CRKs) upregulated in infected finger lime (Table 3). No et al., 2017). Interestingly, we identified candidate genes encoding changes in expression (log (fold-change)) were recorded for several Cys-rich secretory proteins or Pathogenesis-related 1 the mitogen-activated protein kinase-encoding DEG (MAPK) protein (PR1-like) that were highly upregulated in the infected in finger lime and ‘Valencia’ sweet orange when the uninfected finger lime relative to CaLas-uninfected and infected ‘Valencia’ tissues were compared with the infected tissues. sweet orange (Table 4). Of these Cys-rich secretory proteins, To validate the reliability of the RNA-seq data in terms of the orange1.1g043403m was highly upregulated in the non-infected overexpression of CRKs, we randomly selected 9 DEGs encoding finger lime trees, and the expression was recorded as two-fold Cys-rich RLKs for confirmation by qPCR (Figure 3) on samples increase after CaLas infection. The results of the field experiment of infected finger lime and ‘Valencia’ sweet orange obtained confirmed this gene was highly upregulated in finger lime trees TABLE 3 DEGs involved in cysteine-rich receptor-like protein kinase of CaLas infected finger lime and ‘Valencia’ sweet orange. Gene symbol Log2 fold change Citrus sinensis ID Arabidopsis homolog FL (HLB+) Val(HLB+) Cysteine-rich RLK- 8* 1.55 0.23 orange1.1g007239m AT4G23160.1 Cysteine-rich RLK- 10 4.17 0.26 orange1.1g041433m AT4G23180.1 Cysteine-rich RLK- 10 31.5 2.13 orange1.1g041917m AT4G23180.1 Cysteine-rich RLK- 10 1.45 0 orange1.1g039168m AT4G23180.1 Cysteine-rich RLK- 10 0.9 9.5 orange1.1g010329m AT4G23180.1 Cysteine-rich RLK- 10 9.25 0.88 orange1.1g037707m AT4G23180.1 Cysteine-rich RLK- 10 11.66 0.06 orange1.1g042892m AT4G23180.1 Cysteine-rich RLK- 10 0.66 0 orange1.1g005893m AT4G23180.1 Cysteine-rich RLK- 10 14.58 3.17 orange1.1g009186m AT4G23180.1 Cysteine-rich RLK- 16 5.5 0.69 orange1.1g021682m AT4G23130.2 Cysteine-rich RLK- 18 2.25 0.09 orange1.1g040682m AT4G23260.1 Cysteine-rich RLK- 25 2.03 0.02 orange1.1g017211m AT4G05200.1 Cysteine-rich RLK- 25 5.06 0.6 orange1.1g017150m AT4G05200.1 Cysteine-rich RLK- 25 2.03 0.02 orange1.1g017211m AT4G05200.1 Cysteine-rich RLK- 34 7.49 1.47 orange1.1g006125m AT4G11530.1 *Cysteine-rich RLK: cysteine-rich receptor-like protein kinase. Frontiers in Plant Science 09 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 AB C DE F GH I FIGURE 3 Relative transcript levels of cysteine-rich RLK (RECEPTOR-like protein kinase) as calculated by real-time PCR compared with the CaLas free ‘Valencia’. The CaLas infected samples were collected from five year old trees growing in the field and the CaLas free (control) samples were collected from trees growing in a protected greenhouse. (A–I) Relative RLK expression as detected in this study compared with CaLas free 'Valencia'. The control trees were confirmed negative for CaLas before further comparison. Data are means ± SE of twelve samples. Different letters above the error bar indicate statistically significant differences, while the same letters signify no significant differences using the Tukey–Kramer honestly significant difference test (Tukey HSD; p <0.05). when compared with the infected ‘Valencia’ sweet orange limonia)), pummelo (Hirado Buntan pummelo and Siamese Sweet (Figure 4). Additionally, we investigated the presence of this gene pummelo (Citrus maxima)), grapefruit (Ruby Red and Duncan (orange1.1g043403m) in several field grown citrus species and (Citrus × paradisi)), sweet orange (‘Valencia’ and Parson Brown cultivars, and we detected lower relative expression in all the (Citrus sinensis)), Volkamer lemon (Citrus volkameriana), Sydney evaluated trees compared to finger lime. The only species that hybrid (Citrus x virgata), Citrus papuana,and Citrus inodora, presented high expression was the Australian desert lime (Citrus showed lower expression of this gene (orange1.1g043403m) than glauca), and this expression was negatively associated with CaLas did finger lime (Figure S3). Although Poncirus trifoliata is highly presence (Figures S3, S4). In contrast, multiple citrus relatives, such tolerant to HLB (Figure S4), therelativeexpression of the Cys-rich as kumquat (Nagami (Fortunella margarita)and Meiwa secretory protein transcripts identified in this study was lower than (Fortunella crassifolia)), Poncirus trifoliata (50-7 and Flying that recorded in finger lime. Dragon), Mandarin (Ponkan and Cleopatra (Citrus reticulata), Additionally, many of the DEGs characterized encoded lime (key lime (Citrus aurantifolia) and Rangpur lime (Citrus transcription factors, kinase activity-related proteins, or were Frontiers in Plant Science 10 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 TABLE 4 DEGs involved in Cysteine-rich secretory proteins or Pathogenesis-related protein of CaLas in finger lime and ‘Valencia’ sweet orange. Gene symbol Log2 fold change Gene function Citrus sinensis ID Arabidopsis homolog FL (HLB+) Val (HLB+) CAP1 1.83 0.25 Pathogenesis-related proteins orange1.1g031237m AT4G33720.1 CAP2 5330 15.35 orange1.1g043403m AT4G33720.1 CAP3 0.22 15.55 orange1.1g037670m AT5G66590.1 LCR69 41.64 6.89 orange1.1g034999m AT2G02100.1 CAP, Cysteine-rich secretory proteins; Antigen 5; and Pathogenesis-related 1 protein) superfamily protein, LCR69: low-molecular-weight cysteine-rich 69. involved in proteolysis. Sixty-seven DEGs were categorized as family, which are involved in secondary metabolism, hormone encoding transcription factors involved in the biotic stress signal transduction, plant development, abiotic stress tolerance, response, constituting 1.8% of all the mapped DEGs, 27 of which and disease resistance, six of which were upregulated (MYB12, were found to be upregulated in finger lime. The most notable were MYB17, MYB62, MYB38, MYB102,and MYB112). Eleven of the MYB, WRKY, zinc-finger, and bZIP transcription factors. There DEGs identified belonged to the WRKY transcription factor family, were 32 DEGs related to the Myb domain transcription factor the members of which play important roles in plant development AB C D FIGURE 4 Relative transcript levels of CAP (Cysteine-rich secretory proteins; Antigen 5; and Pathogenesis-related 1 protein) superfamily protein is calculated by real-time PCR compared with the CaLas free ‘Valencia’. (A) CAP1, (B) CAP2, (C) CAP3 and (D) LCR69. The CaLas infected samples were collected from five years old samples growing in the field and the CaLas free (control) samples were collected from a protected greenhouse. The control samples were confirmed negative for CaLas before further comparison. Data are means ± SE of twelve samples. Different letters above the error bar indicate statistically significant differences, while the same letters signify no significant differences using the Tukey-Kramer honestly significant difference test (Tukey HSD; p <0.05). Frontiers in Plant Science 11 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 and stress responses. Nine of the WRKY transcription factors were (orange1.1g018968m) was upregulated in finger lime. There were 11 upregulated (WRKY14, WRKY23, WRKY28, WRKY31, WRKY47, serine protease (SP)-encoding, 2 Kunitz Family Trypsin- and protease WRKY48, WRKY50, WRKY72 and WRKY75). Additionally, a inhibitor protein-encoding, and 15 Subtilase-related DEGS Dof-Type zinc finger DNA-binding family protein (DAG1) and downregulated in finger lime. In contrast, 20 Ubiquitin E3 Scf F-box two BZIP transcription factor family proteins (TGA9 and BZIP42) and one Ubiquitin E3 Scf Skp (orange1.1g030652m) genes were were found to be upregulated in finger lime, while bZIP58, BZIP61, overexpressed in finger lime, while 16 Ubiquitin E3 Ring, 1 and PAN were downregulated. Ubiquitin E2, 2 Ubiquitin Proteasome, 1 Ubiquitin 4, and 1 Polyubiquitin 10 genes were downregulated. We detected none to minimal expression of three xylem Cys Genes involved in hormone signaling protease 1 (XCP1) and one of xylem Cys protease 2 encoding pathways were differentially expressed genes in the finger lime. Comparatively, these genes were upregulated in response to CaLas infection in ‘Valencia’ sweet Hormone signaling is important in plant physiology and regulates orange. In contrast, one Xylem Serine Peptidase 1 gene many aspects from the transduction of messages through plants to (orange1.1g004503m) was highly upregulated in the infected adaptation to the environment to the timing offruit development and finger lime trees (Table 5). ripening. Out of the mapped genes, 2.6% (99 DEGs) were associated Additionally, the infected ‘Valencia’ sweet orange trees with hormone synthesis. Nine abscisic acid-induced genes were showed higher inhibition capacity of E64 compared with that identified. Three of these were Hva22-like protein-encoding DEGs of the finger lime trees and CaLas-free trees (Figure 5A). We (orange1.1g042117m, orange1.1g038094m, and orange1.1g030361m) selected some of Cys protease transcription factors for validation and were upregulated. Two Gram Domain-Containing Protein 2- via real-time PCR. We found that the relative expression of encodedDEGsweredownregulated.Abscisicaciddegradation-related genes encoding three transcription factors of Cys protease was DEGs were downregulated only in finger lime; these genes included downregulated following CaLas infection in finger lime epoxycarotenoid dioxygenase, aldehyde oxidase, and zeaxanthin compared with ‘Valencia’ sweet orange (Figuress 5B–D). oxidase. Auxin induced-regulated related DEGs were expressed to a higher degree in ‘Valencia’ sweet orange than in finger lime, with 25 out of the 34 DEGs upregulated in ‘Valencia’ sweet orange. The Genes involved in cellular development upregulated genes in finger lime included those encoding ILR1, TIR1, were differentially regulated DFL1, and GH3.1 proteins. Of the six brassinosteroid-related DEGs, cytochrome P450 (orange1.1g037705m) was upregulated, while the One hundred and one DEGs related to processes involved in cell STE1-, HYD1-, and SMT1-encoding genes were downregulated. wall synthesis and support were identified from our transcriptome There were 33 ethylene-related DEGs mapped, 17 of which data. Only 25 of these genes were upregulated in finger lime. Ten were upregulated, including Gibberellin 2-Oxidase, 2-Oxoglutarate, cellulose synthase-encoding genes were mapped, of which those Kar-Up Oxidoreductase 1, Integrase-Type DNA-Binding, GASA1 encoding D1, G2, and G3 cellulose synthases were found to be and SRG1. Seven jasmonate-related genes were also identified, upregulatedcomparedtothoseof ‘Valencia’ sweet orange. Five genes which were upregulated and included three of LOX2 and JAZ1 related to cellulase and 1,4-b-glucanase degradation, namely, gene. Finally, we identified ten salicylic acid (SA)-related genes, and orange1.1g042201m, orange1.1g036635m, orange1.1g041590m, three genes encoding methyltransferase were found to be orange1.1g010632m, and orange1.1g048736m, were upregulated in upregulated, namely, orange1.1g017363m, orange1.1g044676m, finger lime. Expansin A1 (orange1.1g025919m) and A20 and orange1.1g043411m in the infected finger lime. (orange1.1g025617m) were also upregulated in finger lime. Of the 17 PME-related genes identified by mapping, the PME inhibitor (orange1.1g010441m) was the only DEG upregulated in finger lime. Protease related genes were generally Additionally, six arabinogalactan protein (AGP)-related DEGs were downregulated in the finger lime identified,five of which were downregulated, while the gene encoding FLA3 protein (orange1.1g042255m) was upregulated. Among the DEGs mapped, 139 were associated with biotic proteolysis, accounting for 3.6% of all the mapped genes. Fifty-one of these genes, including orange1.1g044297m, which encodes a P-Loop qPCR validated the RNA-seq data containing Nucleoside Triphosphate Hydrolases Superfamily Protein, were upregulated in finger lime. However, the Aaa-type gene Aaa- To evaluate the accuracy of the RNA-seq data and the presence of Atpase 1, several Cytochrome Bc1 synthesis-related genes, aspartate technical artifacts or errors introduced during the RNA-seq library protease,autophagy-related DEGs,and ametalloprotease-related DEG preparations, the expression of several highly conserved genes and were downregulated. Of the 10 Cys protease-related DEGs that were genes encoding transcription factors were assayed through qPCR. mapped, only one of the Cys protease inhibitor-encoding genes Eight of the selected genes (orange1.1g025919m, orange1. Frontiers in Plant Science 12 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 TABLE 5 DEGs involved in cysteine protease production in finger lime and ‘Valencia’ sweet orange. Gene symbol* Log2 fold change Gene function Citrus sinensis ID Arabidopsis homolog FL (HLB+) Val (HLB+) XCP1 0 5.02 cysteine proteases orange1.1g048025m AT4G35350.1 XCP1 0.07 1.83 orange1.1g018781m AT4G35350.1 XCP1 0.08 17.44 orange1.1g019063m AT3G49340.1 XCP2 0.47 18.78 orange1.1g018649m AT1G20850.1 CP 0.03 12.78 orange1.1g032006m AT4G15880.1 CP 2.12 34.03 orange1.1g028661m AT1G50670.1 CP 0.05 16.56 orange1.1g019112m AT3G49340.1 XSP 1 98.82 10.01 xylem serine peptidase 1 orange1.1g004503m AT4G00230.1 *XCP1- xylem cysteine Protease 1, XCP2- xylem cysteine Protease 2 and CP- Cys protease. 1g004503m, orange1.1g025919m, orange1.1g000943m, several genes were downregulated (orange1.1g019200m, orange1.1g044721m, orange1.1g041335m, orange1.1g040540m, orange1.1g020892m, orange1.1g041918m, orange1.1g027498m, orange1.1g020713m, orange1.1g008415m) were upregulated in orange1.1g004923m, orange1.1g005031m, orange1.1g047288m, finger lime (Figure 6). However, the RNA-seq data revealed that orange1.1g008038m) (Figure 6). The qPCR results were consistent AB C D FIGURE 5 (A) Inhibition capacity of Finger lime and ‘Valencia’ T1- FL (HLB+) + substrate, T2- FL (HLB+) + substrate, T3- VAL (HLB+) + substrate + E64 and T4- Val (HLB+) + substrate + E64. The inhibition capacity was compared with the HLB negative leaves. Relative transcript levels of Cysteine proteinases superfamily protein transcription factors were calculated by real-time PCR and compared with the CaLas free ‘Valencia’. The CaLas infected trees were collected from five year old trees growing in the field and the CaLas free (control) samples here instead of trees were collected from trees kept in a protected greenhouse. The control trees were confirmed negative for CaLas before further comparison. Data are means ±SE of twelve samples. (B–D) Relative gene expression of selected cysteine proteases genes in the infected finger lime compared with infected 'Valencia'. Different letters above the error bar indicate statistically significant differences, while the same letters signify no significant differences using the Tukey-Kramer honestly significant difference test (Tukey HSD; p <0.05). Frontiers in Plant Science 13 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 with the RNA-seq data, and the mRNA expression of these genes was The relative transcript levels of CsPP2-B15 were highly either significantly up- or downregulated in the infected finger lime upregulated in the infected ‘Valencia’ sweet orange leaves compared with the infected ‘Valencia’ sweet orange. compared with the finger lime leaves (Figures 8D–F). Phloem and xylem morphological Discussion differences sheds light on finger lime HLB tolerance HLB is a devastating disease affecting citrus worldwide. Currently, many strategies have been explored for HLB We observed phloem and xylem morphological differences mitigation, including application of antimicrobials, macro, in the CaLas– and CaLas+ petioles of finger lime and ‘Valencia’ micronutrients and plant defense inducers, control of insect sweet orange (Figures 7A, B). Because phloem and xylem vectors, thermotherapy, biocontrol, and eradication of HLB thickness are dependent on tissue type and age, we calculated symptomatic citrus trees (Yang and Ancona, 2022). However, a relative phloem thickness (RPT) by dividing the average these strategies have shown limited success in field applications, phloem thickness by the average xylem thickness within the and effective long term HLB management remains a challenge. same sample. There were significant differences between healthy Thus, breeding for HLB tolerance may provide the most effective and HLB-infected finger lime petioles (p value = 0.0001) and and sustainable solution to combat HLB (Bové, 2006). In the between healthy and HLB-infected stems (p value = 0.0044) field, CaLas-infected ‘Valencia’ sweet orange trees display (Figure 7D). CaLas-infected finger lime had a significantly symptoms of stunted growth, yellow shoots, and blotchy higher RPT compared to healthy finger lime. This was also mottled leaves while adjacent finger lime grow normally and true for ‘Valencia’ sweet orange petioles (p value =0.0385) produce fruits without any apparent visible HLB symptoms (Figure 7F). The xylem/phloem ratio (Xa/Pa) showed (Figures 1A, B). Diaphorina citri,the vector transmitting significant difference in finger lime in both petioles (p value = CaLas has a feeding preference for certain citrus cultivars. 0.0006) and stems (p value = 0.0038) (Figure 7C), however, there ‘Valencia’ sweet orange is considered a preferred host while was slight differences in Xa/Pa in ‘Valencia’ petioles (p value = finger limes are considered not suitable (Felisberto et al., 2019). 0.0427) and no significant difference in the stems (Figure 7E). Poor Diaphorina citri colonization levels observed on finger lime Together, our results suggest that phloem may regenerate in trees in a recent long term field study confirms these finger lime in response to HLB infection. observations (Ramadugu et al., 2016). Under controlled conditions, finger limes trees do get infected following budding with CaLas-infected budwood (Alves et al., 2020). Quantification of phloem callose However, the mechanism for this perceived tolerance to CaLas deposits indicated differential in the host is unknown and our study provides insights into the callose deposition tolerance mechanism. Finger limes are monoembryonic and open pollinated seedlings can vary in their tolerance to HLB We found increased accumulation of callose in the infected (Ramadugu et al., 2016). Finger lime trees (clone DPI 50-36) ‘Valencia’ sweet orange compared with the infected finger lime. have remained HLB negative under field conditions for more Analysis of the callose formation counts revealed a significant than a decade. This study was designed to understand this difference in the percentage of images with few callose plugs in the perceived tolerance by graft inoculating budded trees of the stem phloem of CaLas+ ‘Valencia’ sweet orange (0%) and finger same clone under controlled greenhouse conditions. lime (26%); Table 6. Among the images of phloem that did contain The possible genetic mechanisms of the symptoms of CaLas callose formations, ‘Valencia’ sweet orange had significantly higher in citrus trees have been previously discussed by several groups mean counts of formations per image (Figures 8A–C). (Albrecht and Bowman, 2008; Kim et al., 2009; Rawat et al., 2015; Martinelli and Dandekar, 2017; Curtolo et al., 2020; Ma et al., 2022). Nehela and Killiny (2020) summarized three main Phloem protein genes are potential mechanisms during CaLas infection: (I) disorder of downregulated in finger limes carbohydrate metabolism affecting the flow of nutrients and source–sink disruption due to starch accumulation in leaves; (II) Some of the transcripts encoding for phloem proteins in the phytohormones alteration in response to stress; and (III) samples were analyzed via qPCR. CsPP2-B1 transcripts were activation of detoxification proteins, particularly glutathione-S- detected in both finger lime and ‘Valencia’ but were not transferases (GSTs) and modulation of antioxidant pathways. Understanding the virulence mechanisms employed by statistically significant when the uninfected and infected trees were compared. CsPP2-B13 transcripts were almost undetectable CaLas against the host is an important step to identify an in finger lime samples, while they were upregulated in ‘Valencia’. approach to increase plant defense. Insect-transmitted bacteria Frontiers in Plant Science 14 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 FIGURE 6 Verification of expression levels of selected upregulated (A) or downregulated (B) DEGs in the infected Finger lime (FL-HLB) compared to infected ‘Valencia’ (Val-HLB) as determined by qPCR (2-DDCt). Different letters (a, b) represent a significant difference at p ≤ 0.05 using Tukey– Kramer honestly significant difference (HSD) and error bars represent SE (n = 3). Pearson Correlation Coefficient (r)> greater than 0.5 is considered positive and strong. such as CaLas utilize the general Sec secretion system to release developmental processes within the host to benefitthe effectors (Sugio et al., 2011). However, the mechanism of the pathogen (Jones and Dangl, 2006). Previous research has CaLas effectors secretion remains poorly understood. Protein shown that CaLas encodes several SDEs, many of which are effectors often suppress plant defense or manipulate conserved across CaLas isolates and of which were found to be Frontiers in Plant Science 15 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 AB C D EF FIGURE 7 Morphological differences between healthy and HLB-infected Finger lime and ‘Valencia’. Brightfield images of petiole and stems for each healthy and HLB-infected cultivar, Finger lime (A), ‘Valencia’ (B). Phloem Ratio (C) and Xylem Ratio (D) in Finger lime and Phloem Ratio (E) and Xylem Ratio (F) in ‘Valencia’. Bars represent standard error. NS, not significant. highly present in infected citrus tissue at a relatively early TABLE 6 Percent of images with zero callose plugs per 10x field image of the stem phloem of CaLas+ ‘Valencia’ sweet orange and infection stage (Pagliaccia et al., 2017; Tran et al., 2020). finger lime. Several studies have demonstrated that the activity of leucine-rich repeat proteins (LRR-RLKs) serves as an early Plant type Images with zero callose plugs warning system for detecting the presence of potential Finger lime 26%* pathogens and activating protective immune-related signaling Valencia 0% in plants (Matsushima and Miyashita, 2012). Interestingly, receptor kinases, LRRs and cysteine (Cys)-rich receptor-like *p> value <0.001. Frontiers in Plant Science 16 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 FIGURE 8 Non-zero counts of callose plugs per 10x field image of the stem phloem of CaLas+ Finger lime (A) and ‘Valencia’ sweet orange (B) sampled after 2 years following infection. The mean callose formation count per 10x field image was significant. (C) Relative transcript levels of phloem proteins are calculated by real-time PCR and compared with the CaLas free ‘Valencia’. The CaLas infected samples were collected from five year old trees growing in the field and the CaLas free (control) samples were collected from trees kept in a protected greenhouse. The control trees were confirmed negative for CaLas before further comparison. Data are means ± SE of twelve samples (D–F). Different letters above the error bar indicate statistically significant differences, while the same letters signify no significant differences using the Tukey-Kramer honestly significant difference test (Tukey HSD; p <0.05). protein kinases (CRKs) were found to be upregulated in HLB would not interact with the external receptors of the plant cell. infected finger lime. Our findings are similar to those reported Thus, it has little or no interaction with PTI mechanisms. In by Peng et al. (2020), where the involvement of multiple Arabidopsis, most CRKs are differentially regulated by SA, ROS, nucleotide-binding site-containing and LRR-encoding genes in and pathogen infection (Czernic et al., 1999; Du and Chen, 2000; the HLB tolerance/resistance process in Poncirus trifoliata was Ohtake et al., 2000; Chen et al., 2003; Zhang et al., 2013). Curtolo reported. CRKs are characterized by the presence of one to four et al. (2020) reported that the CaLas defense mechanisms were copies of Domain of Unknown Function 26 (DUF26) and a C– controlled by a class of receptor-related genes and the induction X8–C–X2–C motif in the extracellular receptor region at the N- of WRKY transcription factors. These observations suggest that terminus (Mou et al., 2021). These conserved Cys residues might CRKs could play a vital role in the regulatory network regulating be required to form the three-dimensional structure of the the finger lime response to CaLas infection, suggesting that protein through disulfide bonds (Chen, 2001) and can mediate members of the CRK gene family can be effective targets for protein–protein interactions (Rayapuram et al., 2012). Several the improvement of citrus tolerance to HLB disease. CRKs have been functionally characterized in response to Recently, Ma et al. (2022) reported that Huanglongbing pathogen infection. We detected overexpression of CsCRK10, (HLB) could by controlled by ROS detoxification via induction CsCRK16, CsCRK25 and CsCRK34 in the infected finger lime. of antioxidant pathways and plant growth hormones Functional analysis of the RLKs in CaLas infected plants would (particularly GA). Additionally, the involvement of GA be difficult because CaLas is an intracellular bacterium and signaling in HLB resistance has also been reported by Rawat inoculated directly by the psyllids into the phloem tissues and et al. (2015) and Curtolo et al. (2020). Curtolo et al. (2020) Frontiers in Plant Science 17 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 suggested that the genetic mechanism of HLB tolerance was HLB infection induced the differential expression of multiple associated with the downregulation of gibberellin (GA) synthesis genes encoding enzymes and proteins involved in the synthesis, and cell wall strengthening. In the current study, we reported the assembly, and modification of the cell wall. Overall, many genes upregulation of several candidate genes responsible for involved in cellulose synthesis and 1,4-b-glucan degradation as controlling GA synthesis and production of bioactive GA in well as those encoding cellulase glycosyl hydrolases, the infected finger lime such as gibberellin 2-oxidase 8 polygalacturonases and fasciclin-like AGPs were upregulated in (GA2ox8), gibberellin 3-oxidase 1 (GA3ox1) and cysteine-rich the infected finger lime. Several other genes involved in cellulose protein or GA-stimulated transcript (GAST1 protein homolog synthesis, such as cellulose synthase D1, G2 and G3, were also 4). Additionally, our data showed that CaLas induced ROS upregulated in CaLas-infected finger lime. CaLas infection in detoxification pathways and activated glutathione-S- ‘Valencia’ sweet orange resulted in the repression of genes transferases (GSTs), catalase and thioredoxin in finger lime. encoding cellulose synthase-like D4 (CSLD4) and CSLC7 that Glutathione-S-transferases was suggested to be as an important mediate synthesis of b-1,4 linkages in the hemicellulose backbones modulator of citrus tolerance to HLB disease (Martinelli and (Aritua et al., 2013). In thephloem, thesieve platepores Dandekar, 2017). We also recorded the overexpression of several accumulate callose, a b-1-3 glucan, and starch, which restrict plant growth regulator-related-genes that may have direct or symplastic transport, in response to CaLas infection (Koh et al., indirect function in ROS mitigation (Figure 9). 2012; Welker et al., 2022). Callose deposition and phloem protein Plants accumulate inducible defense-related proteins in (PP) plugging of the sieve tubes are defensive measures to form response to biotic and abiotic stress. Antimicrobial peptides physical barriers that prevent the spread of pathogens (Musetti (AMPs) and other Cys-rich peptides are major components of et al., 2010). Excessive callose deposition is considered the main innate immunity in various groups of organisms, including reason for phloem blockage in HLB disease. This blockage limits insects, mammals and plants (Pushpanathan et al., 2013; thetransferoforganic compoundsfrom the sitesof Slavokhotova et al., 2017). Some eukaryotic AMPs are largely photosynthesis (source) to where photosynthate is stored (sink), Cys-rich peptides, which are defined as defensins (Baindara which affects plant growth and mimics the symptoms of a nutrient et al., 2017). A potent AMP has recently been identified from disorder. We observed decreased callose accumulation in finger finger lime (Huang et al., 2021b). A Cys-rich secretory protein/ lime phloem tissue. Previous research revealed that some phloem PR1-like protein was also found to be abundant in finger lime proteins play an important role in callose deposition (Wen et al., and in HLB-tolerant Australian desert lime (C. glauca; 2018). When the gene expression of HLB infected and healthy Ramadugu et al. (2016)). We also identified a low-molecular- controls was compared, PP2 genes were found to be upregulated weight defensin (Cys-rich 69: LCR69) in this study that is highly in the infected trees (Musetti et al., 2010; Ghosh et al., 2018). In the upregulated in finger lime trees. Defensins usually contain six to comparison between finger lime and ‘Valencia’ sweet orange, we eight conserved Cys residues and thus are referred to as Cys-rich found that phloem proteins were significantly induced by CaLas peptides (CRPs) (Silverstein et al., 2007). infection in ‘Valencia’ sweet orange, while they were repressed in Cell wall integrity sensing is a mechanism by which plants finger lime. Specifically, CsPP2-B15 was found to be highly can be induced to mitigate biotic stress. Various strategies upregulated in the infected ‘Valencia’ sweet orange compared to involving resistance of the cell wall against plant pathogens the finger lime. These findings agree with those of Wen et al. have evolved, such as the remodeling of the cell wall and (2018), who found that CsPP2-B15 expression was upregulated in alterations to cell wall-associated protein distribution and infected leaves of Jincheng orange (C. sinensis Osbeck) and accumulation (Leszczuk et al., 2019). Both cell wall synthesis downregulated in the HLB tolerant sour pummelo (C. grandis and proteins associated with the cell wall have roles in structural Osbeck). Similarly, CsPP2-15 was also found to be downregulated support, and the presence or absence of these proteins may in the tolerant C. ichangensis (Wu et al., 2020). This is in addition indicate disease susceptibility or infection. The susceptibility of to our observations that the ‘Valencia’ sweet orange phloem ‘Valencia’ trees to CaLas infection resulted in a decreased ability produces more callose. It should be noted that the enhanced to synthesize new cellular components, whereas the finger lime production of callose and phloem proteins in the susceptible plants was not affected in the same manner and thus could continue is purely a mechanical response: it chokes off the transport of with cell metabolism and growth. These findings are agreed with nutrients and other vital molecules and at the same time, does not Fan et al. (2012) who compared resistant rough lemon and prevent the spread of CaLas. Killiny et al. (2022) suggested that the susceptible sweet orange following CaLas infection. They increased ROS could be one of the factors affecting callose reported that cell wall-related pathways were upregulated in deposition as reported for CaLas-infected citrus trees. rough lemon during the late stage of infection, while there was The vascular cambium increases stem diameter via periclinal downregulation in the susceptible sweet orange. Thus, the rough divisions and the circumference by anticlinal divisions, resulting in lemon trees generated healthy new growth following infection the development of secondary phloem and xylem (Chaffey, 1999). despite older leaves having some blotchy mottle, while the Proteolytic enzymes, including serine, Cysteine and threonine growth was inhibited in sweet orange trees. proteases, have been implicated in the regulation of vascular Frontiers in Plant Science 18 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 FIGURE 9 Schematic diagram elucidating the potential mechanism of CaLas tolerance in the Finger lime, and susceptibility in ‘Valencia’ sweet orange. The figure was created in BioRender.com. Frontiers in Plant Science 19 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 tissue differentiation. Beers and Zhao (2001) screened seven Conclusions protease genes, including members of the serine, Cysteine, and aspartic acid protease families, and reported that the expression of To date, there are no effective practical strategies to mitigate three genes (XCP1, XCP2, and XSP1) was xylem specific. XCP1 and citrus greening disease (HLB). Understanding the mechanisms XCP2 are predicted to encode papain-like Cys proteases, and XSP1 against CaLas can contribute to the development of effective is predicted to encode a subtilisin-like SP. Clark et al. (2018) approaches for combatting HLB. This report provides insights evaluated six candidates of Cys protease and reported that the C. into different mechanisms of HLB tolerance in finger lime, an sinensis protein annotated as “xylem Cys protease 1”, a member of HLB tolerant citrus species. Finger lime trees protect themselves the PLCP family, was confirmed by yeast two-hybrid (Y2H) assays from CaLas infection and disease symptoms through the control of as interacting with CaLas effectors (SDE1). Papain-like Cys ROS-overproduction by enhancing redox control factors such as proteases are important regulators involved in numerous plant glutathione-S-transferases (GSTs), catalase, thioredoxin and growth biological processes, including programmed cell death (PCD) and hormones to mitigate HLB symptoms. Primary recognition of ROS leaf senescence. It has been demonstrated that activation of cysteine leads to stronger activation of defense responses against CaLas proteases induced programmed cell death (PCD) pathway of plant infection. The over-accumulation of Cys proteases and their cells (Minami and Fukuda, 1995; Ye and Varner, 1996). inhibitors in ‘Valencia’ linked with the degradation of citrus Particularly, two xylem-specificPLCPs (AtXCP1 and AtXCP2) defense proteins such as the cysteine rich proteins. Finger lime were found to be expressed at a high level in xylem during the did not exhibit any imbalance in the expression of the Cys proteases PCD process (Liu et al., 2018). We did not find any significant and their inhibitors. Several defense-related-factors were expression of many of the xylem Cys protease 1 genes in our finger upregulated in the finger lime such as R-proteins, Cys-rich lime transcriptome data; however, these genes were upregulated in secretory proteins or PR proteins and hormone signaling-related the infected ‘Valencia’ sweet orange. Cys proteases and their genes. Additionally, we observed that ‘Valencia’ sweet orange inhibitors were highly expressed in ‘Valencia’ sweet orange field phloem constitutively produced more callose and expressed more samples. Under stress conditions, the activity of PLCPs and their phloem proteins than finger lime following infection. Taken regulatory factors can be disrupted, resulting in unknown together, this study provides evidence that HLB can be managed consequences at the cellular level (Liu et al., 2018). We suggest by mitigating ROS and control of the over-accumulation of cysteine that the overexpression of proteases and inhibitors in ‘Valencia’ protease related genes that induce cell death (Figure 9). sweet orange reflects an imbalance in metabolism. CaLas infection can alter the function of the transcription factors responsible for the Cys proteases, which led to an abundance of Cys proteases in Data availability statement ‘Valencia’ cells. To maintain a relatively balanced intercellular level of proteases, the activities of the inhibitors that can directly control The datasets presented in this study can be found in online Cys protease activity are also induced in the cells. The inhibition repositories. The names of the repository/repositories and ratio of Cys proteases revealed the overproduction of Cys proteases accession number(s) can be found below: https://www.ncbi. in ‘Valencia’ sweet orange leaves. Yu and Killiny, (2018) reported nlm.nih.gov/, PRJNA755969. the presence of proteases and endoglucanase in the saliva of D. citri, potentially linked with the ability of the insects to degrade/inhibit citrus defense proteins. We hypothesized that the overaccumulation Author Contributions of cysteine proteases causes degradation of defense proteins and/or induces PCD and are linked to the enhanced susceptibility of KW, LM, DS and MD - wrote the manuscript. LM - gene expression analysis. KW - transcriptome data analysis. DS – ‘Valencia’ trees to CaLas. SPs appear to be widespread in the xylem sap of different species (Buhtz et al., 2004). SPs have Microscopy. SW – callose density estimation. LM and WQ – RNA extraction. LM - statistical analysis. AL and JG – resources. various functions in plant cells, including functions in the pathogen response (Tornero et al., 1997; Jordá et al., 1999). MD – designed the study, obtained funding, and supervised the project. All authors contributed to the article and approved the Specific citrus SPs were detected, suggesting that protease enzymatic activity is finely tuned during CaLas infection (Franco submitted version. et al., 2020). The overexpression of XSP1 in finger lime suggests that there is specific mechanism underlying the defense against CaLas infection in finger lime tissues. Since the role of several genes Funding reported in this study have not been elucidated before, further comprehensive studies are needed to understand in detail their role This work was supported by the Florida State legislative in the finger lime HLB defense pathways. funding for the UF/IFAS Citrus Initiative. Frontiers in Plant Science 20 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 Acknowledgments Publisher’s note We thank E. Nielsen, C. Hardy, H Rubio, J. Thomson and K. All claims expressed in this article are solely those of the Plant for aiding in various aspects of the work. Microscopy work authors and do not necessarily represent those of their affiliated was carried out at the Microscopy Core Facility of the Citrus organizations, or those of the publisher, the editors and the Research and Education Center. We thank Novogene reviewers. Any product that may be evaluated in this article, or Corporation Inc. for library construction and RNA sequencing. claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Supplementary material Conflict of interest The Supplementary Material for this article can be found The authors declare that the research was conducted in the online at: https://www.frontiersin.org/articles/10.3389/ absence of any commercial or financial relationships that could fpls.2022.1019295/full#supplementary-material be construed as a potential conflict of interest. References Albrecht, U., and Bowman, K. D. (2008). Gene expression in Citrus sinensis (L.) kinase in Arabidopsis. Plant Mol. Biol. 53, 61–74. doi: 10.1023/b: osbeck following infection with the bacterial pathogen candidatus liberibacter plan.0000009265.72567.58 asiaticus causing huanglongbing in Florida. Plant Sci. 175, 291–306. doi: 10.1016/ Clark, K., Franco, J. Y., Schwizer, S., Pang, Z., Hawara, E., Liebrand, T. W. H., j.plantsci.2008.05.001 et al. (2018). An effector from the huanglongbing-associated pathogen targets citrus Alves, M. N., Lopes, S. A., Raiol-Junior, L. L., Wulff, N. A., Girardi, E. A., proteases. Nat. Commun. 9, 1718. doi: 10.1038/s41467-018-04140-9 Ollitrault, P., et al. (2020). Resistance to 'Candidatus liberibacter asiaticus,' the Curtolo, M., de Souza Pacheco, I., Boava, L. P., Takita, M. A., Granato, L. M., huanglongbing associated bacterium, in sexually and/or graft-compatible citrus Galdeano, D. M., et al. (2020). Wide-ranging transcriptomic analysis of poncirus relatives. Front. Plant Sci. 11. doi: 10.3389/fpls.2020.617664 trifoliata, citrus sunki, citrus sinensis and contrasting hybrids reveals HLB tolerance Andrews, S. (2010) FASTQC. a quality control tool for high throughput sequence mechanisms. Sci. Rep. 10, 1–14. doi: 10.1038/s41598-020-77840-2 data. Available at: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ Czernic, P., Visser, B., Sun, W., Savouré, A., Deslandes, L., Marco, Y., et al. (Accessed 6 December 2021). (1999). Characterization of an arabidopsis thaliana receptor-like protein kinase Aritua, V., Achor, D., Gmitter, F. G., Albrigo, G., and Wang, N. (2013). gene activated by oxidative stress and pathogen attack. Plant J. 18, 321–327. doi: Transcriptional and microscopic analyses of citrus stem and root responses to 10.1046/j.1365-313X.1999.00447.x Candidatus liberibacter asiaticus infection. PloS One 8, e73742. doi: 10.1371/ Deng, B., Wang, W., Deng, L., Yao, S., Ming, J., and Zeng, K. (2018). journal.pone.0073742 Comparative RNA-seq analysis of citrus fruit in response to infection with three Baindara, P., Kapoor, A., Korpole, S., and Grover, V. (2017). Cysteine-rich low major postharvest fungi. Postharvest Biol. Technol. 146, 134–146. doi: 10.1016/ molecular weight antimicrobial peptides from brevibacillus and related genera for j.postharvbio.2018.08.012 biotechnological applications. World J. Microbiol. Biotechnol. 33, 124. doi: 10.1007/ Dobin, A., Davis, C. A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., et al. s11274-017-2291-9 (2013). STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21. Barrett, A. J. (1980). Fluorimetric assays for cathepsin b and cathepsin h with doi: 10.1093/bioinformatics/bts635 methylcoumarylamide substrates. Biochem. J. 187, 909–912. doi: 10.1042/ Dou, D., and Zhou, J. M. (2012). Phytopathogen effectors subverting host bj1870909 immunity: different foes, similar battleground. Cell Host Microbe 12, 484–495. Beers, E. P., and Zhao, C. (2001). “Arabidopsis as a model for investigating gene doi: 10.1016/j.chom.2012.09.003 activity and function in vascular tissues,” in Progress in biotechnology. Eds. N. Duan, Y., Zhou, L., Hall, D. G., Li, W., Doddapaneni, H., Lin, H., et al. (2009). Morohoshi and A. Komamine (Amsterdam: Elsevier), 43–52. Complete genome sequence of citrus huanglongbing bacterium, ‘Candidatus Benavente-Garcıa,́ O., and Castillo, J. (2008). Update on uses and properties of liberibacter asiaticus’ obtained through metagenomics. Mol. Plant-Microbe citrus flavonoids: new findings in anticancer, cardiovascular, and anti- Interact. 22, 1011–1020. doi: 10.1094/mpmi-22-8-1011 inflammatory activity. J. Agric. Food Chem. 56, 6185–6205. doi: 10.1021/jf8006568 Du, L., and Chen, Z. (2000). Identification of genes encoding receptor-like Benjamini, Y., Krieger, A. M., and Yekutieli, D. (2006). Adaptive linear step-up protein kinases as possible targets of pathogen-and salicylic acid-induced WRKY procedures that control the false discovery rate. Biometrika 93, 491–507. DNA-binding proteins in arabidopsis. Plant J. 24, 837–847. doi: 10.1046/j.1365- doi: 10.1093/biomet/93.3.491 313x.2000.00923.x Bolger, A. M., Lohse, M., and Usadel, B. (2014). Trimmomatic: a flexible Fan, J., Chen, C., Yu, Q., Khalaf, A., Achor, D. S., Brlansky, R. H., et al. (2012). trimmer for illumina sequence data. Bioinformatics 30, 2114–2120. doi: 10.1093/ Comparative transcriptional and anatomical analyses of tolerant rough lemon and bioinformatics/btu170 susceptible sweet orange in response to ‘Candidatus liberibacter asiaticus’ infection. Mol. Plant-Microbe Interact. 25, 1396–1407. doi: 10.1094/MPMI-06-12-0150-R Bové, J. M. (2006). Huanglongbing: a destructive, newly-emerging, century-old disease of citrus. J. Plant Pathol. 88, 7–37. Felisberto, P. A., Girardi, E. A., Peña, L., Felisberto, G., Beattie, G. A. C., and Lopes, S. A. (2019). Unsuitability of indigenous south American rutaceae as Buhtz, A., Kolasa, A., Arlt, K., Walz, C., and Kehr, J. (2004). Xylem sap protein potential hosts of Diaphorina citri. Pest Manage. Sci. 75, 1911–1920. composition is conserved among different plant species. Planta 219, 610–618. doi: 10.1002/ps.5304 doi: 10.1007/s00425-004-1259-9 Feng, F., and Zhou, J. M. (2012). Plant–bacterial pathogen interactions mediated Chaffey, N. (1999). Wood formation in forest trees: from Arabidopsis to Zinnia. by type III effectors. Curr. Opin. Plant Biol. 15, 469–476. doi: 10.1016/ Trends Plant Sci. 4, 203–204. doi: 10.1016/s1360-1385(99)01417-x j.pbi.2012.03.004 Chen, Z. (2001). A superfamily of proteins with novel cysteine-rich repeats. Ferguson, K., da Cruz, M. A., Ferrarezi, R., Dorado, C., Bai, J., and Cameron, R. Plant Physiol. 126, 473–476. doi: 10.1104/pp.126.2.473 G. (2021). Impact of huanglongbing (HLB) on grapefruit pectin yield and quality Chen, K., Du, L., and Chen, Z. (2003). Sensitization of defense responses and during grapefruit maturation. Food Hydrocoll. 113, 106553. doi: 10.1016/ activation of programmed cell death by a pathogen-induced receptor-like protein j.foodhyd.2020.106553 Frontiers in Plant Science 21 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 Ferrara, T., Schneider, V. K., Kishi, L. T., Carmona, A. K., Alves, M. F. M., Lindgreen, S. (2012). AdapterRemoval: easy cleaning of next-generation Belasque-Júnior, J., et al. (2015). Characterization of a recombinant cathepsin b- sequencing reads. BMC Res. Notes 5, 337. doi: 10.1186/1756-0500-5-337 like cysteine peptidase from Diaphorina citri kuwayama (Hemiptera: Liviidae): a Liu, H., Hu, M., Wang, Q., Cheng, L., and Zhang, Z. (2018). Role of papain-like putative target for control of citrus huanglongbing. PloS One 10, e0145132. cysteine proteases in plant development. Front. Plant Sci. 9. doi: 10.3389/ doi: 10.1371/journal.pone.0145132 fpls.2018.01717 Folimonova, S. Y., Robertson, C. J., Garnsey, S. M., Gowda, S., and Dawson, W. Livak, K. J., and Schmittgen, T. D. (2001). Analysis of relative gene expression O. (2009). Examination of the responses of different genotypes of citrus to data using real-time quantitative PCR and the 2–DDCT method. Methods 25, 402– huanglongbing (citrus greening) under different conditions. Phytopathology 99, 408. doi: 10.1006/meth.2001.1262 1346–1354. doi: 10.1094/phyto-99-12-1346 Love, M. I., Huber, W., and Anders, S. (2014). Moderated estimation of fold Forster, P. I., and Smith, M. W. (2010). Citrus wakonai PI forst. & MW change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550. Sm.(Rutaceae), a new species from goodenough island, Papua new Guinea. doi: 10.1186/s13059-014-0550-8 Austrobaileya 8, 133–138. Ma, W., Pang, Z., Huang, X., Xu, J., Pandey, S. S., Li, J., et al. (2022). Citrus Franco, J. Y., Thapa, S. P., Pang, Z., Gurung, F. B., Liebrand, T. W. H., Stevens, huanglongbing is a pathogen-triggered immune disease that can be mitigated with D. M., et al. (2020). Citrus vascular proteomics highlights the role of peroxidases antioxidants and gibberellin. Nat. Commun. 13, 1–13. doi: 10.1038/s41467-022- and serine proteases during huanglongbing disease progression. Mol. Cell. Proteom. 28189-9 19, 1936–1952. doi: 10.1074/mcp.RA120.002075 Marın,́ F. R., Soler-Rivas, C., Benavente-Garcıa,́ O., Castillo, J., and Pérez- Ghosh, D., Motghare, M., and Gowda, S. (2018). Citrus greening: overview of the Alvarez, J. A. (2007). By-products from different citrus processes as a source of most severe disease of citrus. Adv. Agric. Res. Technol. J. 2, 83–100. customized functional fibres. Food Chem. 100, 736–741. doi: 10.1016/ Graham, J., Gottwald, T., and Setamou, M. (2020). Status of huanglongbing j.foodchem.2005.04.040 (HLB) outbreaks in Florida, California and Texas. Trop. Plant Pathol. 45, 265–278. Martinelli, F., and Dandekar, A. M. (2017). Genetic mechanisms of the devious doi: 10.1007/s40858-020-00335-y intruder candidatus liberibacter in citrus. Front. Plant Sci. 8, 904. doi: 10.3389/ Halbert, S. (2005). Citrus greening/Huanglongbingx (Gainesville, Florida: Pest fpls.2017.00904 Alert, Florida Department of Agriculture and Consumer Services, Division of Plant Matsushima, N., and Miyashita, H. (2012). Leucine-rich repeat (LRR) domains Industry). containing intervening motifs in plants. Biomolecules 2, 288–311. doi: 10.3390/ Huang, C. Y., Araujo, K., Sánchez, J. N., Kund, G., Trumble, J., Roper, C., et al. biom2020288 (2021b). A stable antimicrobial peptide with dual functions of treating and Mattos-Jr, D., Kadyampakeni, D. M., da Silva, J. R., Vashisth, T., and Boaretto, R. preventing citrus huanglongbing. Proc. Natl. Acad. Sci. U.S.A. 118, e2019628118. M. (2020). Reciprocal effects of huanglongbing infection and nutritional status of doi: 10.1073/pnas.2019628118 citrus trees: a review. Trop. Plant Pathol. 45, 586–596. doi: 10.1007/s40858-020- Huang, C. Y., Niu, D., Kund, G., Jones, M., Albrecht, U., Nguyen, L., et al. 00389-y (2021a). Identification of citrus immune regulators involved in defence against Miles, G. P., Stover, E., Ramadugu, C., Keremane, M. L., and Lee, R. F. (2017). huanglongbing using a new functional screening system. Plant Biotechnol. J. 19, Apparent tolerance to huanglongbing in citrus and citrus-related germplasm. 757–766. doi: 10.1111/pbi.13502 HortScience 52, 31–39. doi: 10.21273/hortsci11374-16 Hu, Y., Zhong, X., Liu, X., Lou, B., Zhou, C., and Wang, X. (2017). Comparative Milne, A. E., Teiken, C., Deledalle, F., van den Bosch, F., Gottwald, T., and transcriptome analysis unveils the tolerance mechanisms of Citrus hystrix in McRoberts, N. (2018). Growers' risk perception and trust in control options for response to 'Candidatus liberibacter asiaticus' infection. PloS One 12, e0189229. huanglongbing citrus-disease in Florida and California. Crop Prot. 114, 177–186. doi: 10.1371/journal.pone.0189229 doi: 10.1016/j.cropro.2018.08.028 Jones, J. D. G., and Dangl, J. L. (2006). The plant immune system. Nature 444, Minami, A., and Fukuda, H. (1995). Transient and specific expression of a 323–329. doi: 10.1038/nature05286 cysteine endopeptidase associated with autolysis during differentiation of zinnia Jordá, L., Coego, A., Conejero, V., and Vera, P. (1999). A genomic cluster mesophyll cells into tracheary elements. Plant Cell Physiol. 36, 1599–1606. containing four differentially regulated subtilisin-like processing protease genes is doi: 10.1093/oxfordjournals.pcp.a078926 in tomato plants. J. Biol. Chem. 274, 2360–2365. doi: 10.1074/jbc.274.4.2360 Mou, S., Meng, Q., Gao, F., Zhang, T., He, W., Guan, D., et al. (2021). A cysteine- Jose,J., Ghantasala,S., andRoy Choudhury, S. (2020).Arabidopsis rich receptor-like protein kinase CaCKR5 modulates immune response against transmembrane receptor-like kinases (RLKs): a bridge between extracellular Ralstonia solanacearum infection in pepper. BMC Plant Biol. 21, 382. doi: 10.1186/ signal and intracellular regulatory machinery. Int. J. Mol. Sci. 21, 4000. s12870-021-03150-y doi: 10.3390/ijms21114000 Musetti, R., Paolacci, A., Ciaffi, M., Tanzarella, O. A., Polizzotto, R., Tubaro, F., Killiny, N., Jones, S. E., and Gonzalez-Blanco, P. (2022). Silencing of d- et al. (2010). Phloem cytochemical modification and gene expression following the aminolevulinic acid dehydratase via virus induced gene silencing promotes recovery of apple plants from apple proliferation disease. Phytopathology 100, 390– callose deposition in plant phloem. Plant Signaling Behav., 1:2024733. doi: 399. doi: 10.1094/phyto-100-4-0390 10.1080/15592324.2021.2024733 Nehela, Y., and Killiny, N. (2020). Revisiting the complex pathosystem of Killiny, N., Jones, S. E., Nehela, Y., Hijaz, F., Dutt, M., Gmitter, F. G., et al. huanglongbing: Deciphering the role of citrus metabolites in symptom (2018). All roads lead to Rome: Towards understanding different avenues of development. Metabolites 10, 409. doi: 10.3390/metabo10100409 tolerance to huanglongbing in citrus cultivars. Plant Physiol. Biochem. 129, 1–10. Ngou, B. P. M., Ahn, H. K., Ding, P., and Jones, J. D. G. (2021). Mutual doi: 10.1016/j.plaphy.2018.05.005 potentiation of plant immunity by cell-surface and intracellular receptors. Nature Kim, J.-S., Sagaram, U. S., Burns, J. K., Li, J. L., and Wang, N. (2009). Response of 592, 110–115. doi: 10.1038/s41586-021-03315-7 sweet orange (Citrus sinensis)to ‘Candidatus liberibacter asiaticus’ infection: Ohtake, Y., Takahashi, T., and Komeda, Y. (2000). Salicylic acid induces the microscopy and microarray analyses. Phytopathology 99, 50–57. doi: 10.1094/ expression of a number of receptor-like kinase genes in arabidopsis thaliana. Plant PHYTO-99-1-0050 Cell Physiol. 41, 1038–1044. doi: 10.1093/pcp/pcd028 Koh, E. J., Zhou, L., Williams, D. S., Park, J., Ding, N., Duan, Y. P., et al. (2012). Pagliaccia, D., Shi, J., Pang, Z., Hawara, E., Clark, K., Thapa, S. P., et al. (2017). A Callose deposition in the phloem plasmodesmata and inhibition of phloem pathogen secreted protein as a detection marker for citrus huanglongbing. Front. transportincitrusleavesinfected with “Candidatus liberibacter asiaticus”. Microbiol. 8. doi: 10.3389/fmicb.2017.02041 Protoplasma 249, 687–697. doi: 10.1007/s00709-011-0312-3 Peng, Z., Bredeson, J. V., Wu, G. A., Shu, S., Rawat, N., Du, D., et al. (2020). A Kramer, J., Simnitt, S., and Weber, C. (2022) Fruit and tree nuts outlook chromosome-scale reference genome of trifoliate orange (Poncirus trifoliata) (Washington, DC: USDA ERS). Available at: https://www.ers.usda.gov/webdocs/ provides insights into disease resistance, cold tolerance and genome evolution in outlooks/103650/fts-374.pdf (Accessed 30 June 2022). Citrus. Plant J. 104, 1215–1232. doi: 10.1111/tpj.14993 Leszczuk, A., Koziol, A., Szczuka, E., and Zdunek, A. (2019). Analysis of AGP Pushpanathan, M., Gunasekaran, P., and Rajendhran, J. (2013). Antimicrobial contribution to the dynamic assembly and mechanical properties of cell wall during peptides: versatile biological properties, Antimicrobial peptides: versatile Biol. pollen tube growth. Plant Sci 281, 9–18. properties. Int. J. Pept. 2013, 675391. doi: 10.1155/2013/675391 Liao, Y., Smyth, G. K., and Shi, W. (2014). featureCounts: an efficient general Qiu, W., Soares, J., Pang, Z., Huang, Y., Sun, Z., Wang, N., et al. (2020). Potential purpose program for assigning sequence reads to genomic features. Bioinformatics mechanisms of AtNPR1 mediated resistance against huanglongbing (HLB) in 30, 923–930. doi: 10.1093/bioinformatics/btt656 Citrus. int. J. Mol. Sci. 21, 2009. doi: 10.3390/ijms21062009 Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., et al. (2009). Quezada, E.-H., Garcıa,́ G.-X., Arthikala, M.-K., Melappa, G., Lara, M., and The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079. Nanjareddy, K. (2019). Cysteine-rich receptor-like kinase gene family identification doi: 10.1093/bioinformatics/btp352 in the phaseolus genome and comparative analysis of their expression profiles Frontiers in Plant Science 22 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 specific to mycorrhizal and rhizobial symbiosis. Genes 10, 59. doi: 10.3390/ Van Loon, L. C. (1997). Induced resistance in plants and the role of genes10010059 pathogenesis-related proteins. Eur. J. Plant Pathol. 103, 753–765. doi: 10.1023/ a:1008638109140 Ramadugu, C., Keremane, M. L., Halbert, S. E., Duan, Y. P., Roose, M. L., Stover, E., et al. (2016). Long-term field evaluation reveals huanglongbing resistance in Wang, Y., Liu, X. J., Chen, J. B., Cao, J. P., Li, X., and Sun, C. D. (2022). Citrus Citrus relatives. Plant Dis. 100, 1858–1869. doi: 10.1094/pdis-03-16-0271-re flavonoids and their antioxidant evaluation. Crit. Rev. Food Sci. Nutr. 62, 3833– 3854. doi: 10.1080/10408398.2020.1870035 Rawat, N., Kiran, S.P., Du,D., Gmitter, F. G.,and Deng,Z.(2015). Comprehensive meta-analysis, co-expression, and miRNA nested network Wang, Z., Yin, Y., Hu, H., Yuan, Q., Peng, G., and Xia, Y. (2006). Development analysis identifies gene candidates in citrus against huanglongbing disease. BMC and application of molecular-based diagnosis for 'Candidatus liberibacter asiaticus', Plant Biol. 15, 1–21. doi: 10.1186/s12870-015-0568-4 the causal pathogen of citrus huanglongbing. Plant Pathol. 55, 630–638. doi: 10.1111/j.1365-3059.2006.01438.x Rayapuram, C., Jensen, M. K., Maiser, F., Shanir, J. V., Hornshøj, H., Rung, J. H., et al. (2012). Regulation of basal resistance by a powdery mildew-induced cysteine- Welker, S., and Levy, A. (2022). Comparing machine learning and binary rich receptor-like protein kinase in barley. Mol. Plant Pathol. 13, 135–147. thresholding methods for quantification of callose deposits in the citrus phloem. doi: 10.1111/j.1364-3703.2011.00736.x Plants 11, 624. doi: 10.3390/plants11050624 R Core Team (2013). R: A language and environment for statistical computing Welker, S., Pierre, M., Santiago, J. P., Dutt, M., Vincent, C., and Levy, A. (2022). (Vienna: R Foundation for Statistical Computing). Phloem transport limitation in huanglongbing -affected sweet orange is dependent on phloem-limited bacteria and callose. Tree Physiol. 42, 379–390. doi: 10.1093/ Rozman-Pungerčar, J., Kopitar-Jerala, N., Bogyo, M., Turk, D., Vasiljeva, O., treephys/tpab134 Stefe, I., et al. (2003). Inhibition of papain-like cysteine proteases and legumain by caspase-specific inhibitors: when reaction mechanism is more important than Wen, Q., Xie, Z., Wu, L., He, Y., Chen, S., and Zou, X. (2018). Clone and specificity. Cell Death Differ. 10, 881–888. doi: 10.1038/sj.cdd.4401247 expression analysis of the citrus phloem protein 2 gene CsPP2B15 responding to huanglongbing infection in citrus. Acta Hortic. Sin. 45, 2347–2357. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., et al. (2012). Fiji: an open-source platform for biological-image analysis. Nat. Wu, H., Hu, Y., Fu, S., Zhou, C., and Wang, X. (2020). Coordination of multiple Methods 9, 676–682. doi: 10.1038/nmeth.2019 regulation pathways contributes to the tolerance of a wild citrus species (Citrus ichangensis ‘2586’) against huanglongbing. Physiol. Mol. Plant Pathol. 109, 101457. Silverstein, K. A. T., Moskal, W. A., Wu, H. C., Underwood, B. A., Graham, M. doi: 10.1016/j.pmpp.2019.101457 A., Town, C. D., et al. (2007). Small cysteine-rich peptides resembling antimicrobial peptides have been under-predicted in plants. Plant J. 51, 262–280. doi: 10.1111/ Wu, G. A., Prochnik, S., Jenkins, J., Salse, J., Hellsten, U., Murat, F., et al. (2014). j.1365-313x.2007.03136.x Sequencing of diverse mandarin, pummelo and orange genomes reveals complex history of admixture during citrus domestication. Nat. Biotechnol. 32, 656–662. Slavokhotova, A. A., Shelenkov, A. A., Korostyleva, T. V., Rogozhin, E. A., doi: 10.1038/nbt.2906 Melnikova, N. V., Kudryavtseva, A. V., et al. (2017). Defense peptide repertoire of Stellaria media predicted by high throughput next generation sequencing. Wu, G. A., Terol, J., Ibanez, V., López-Garcıa,́ A., Pérez-Román, E., Borredá, C., Biochimie 135, 15–27. doi: 10.1016/j.biochi.2016.12.017 et al. (2018). Genomics of the origin and evolution of Citrus. Nature 554, 311–316. doi: 10.1038/nature25447 Sugio, A., MacLean, A. M., Kingdom, H. N., Grieve, V. M., Manimekalai, R., and Hogenhout, S. A. (2011). Diverse targets of phytoplasma effectors: from plant Yang, C., and Ancona, V. (2022). An overview of the mechanisms against development to defense against insects. Annu. Rev. Phytopathol. 49, 175–195. "Candidatus liberibacter asiaticus": virulence targets, citrus defenses, and doi: 10.1146/annurev-phyto-072910-095323 microbiome. Front. Microbiol. 13. doi: 10.3389/fmicb.2022.850588 ̌ ̌ Supek, F., Bosnjak, ̌ M., Skunca, N., and Smuc, T. (2011). REVIGO summarizes Ye, Z. H., and Varner, J. E. (1996). Induction of cysteine and serine proteases and visualizes long lists of gene ontology terms. PloS One 6, e21800. doi: 10.1371/ during xylogenesis in zinnia elegans. Plant Mol. Biol. 30, 1233–1246. doi: 10.1007/ journal.pone.0021800 BF00019555 Tchoupé, J. R., Moreau, T., Gauthier, F., and Bieth, J. G. (1991). Photometric or Yu, X., and Killiny, N. (2018). The secreted salivary proteome of Asian citrus psyllid fluorometric assay of cathepsin b, l and h and papain using substrates with an diaphorina citri. Physiol. Entomology 43 (4), 324–333. doi: 10.1111/phen.12263 aminotrifluoromethylcoumarin leaving group. Biochim. Biophys. Acta Protein Zambon, F. T., Kadyampakeni, D. M., and Grosser, J. W. (2019). Ground Struct. Mol. Enzymol. 1076, 149–151. doi: 10.1016/0167-4838(91)90232-o application of overdoses of manganese have a therapeutic effect on sweet orange Thimm, O., Bläsing, O., Gibon, Y., Nagel, A., Meyer, S., Krüger, P., et al. (2004). trees infected with candidatus liberibacter asiaticus. HortScience 54, 1077–1086. Mapman: a user-driven tool to display genomics data sets onto diagrams of doi: 10.21273/HORTSCI13635-18 metabolic pathways and other biological processes. Plant J. 37, 914–939. Zhang, X., Han, X., Shi, R., Yang, G., Qi, L., Wang, R., et al. (2013). Arabidopsis doi: 10.1111/j.1365-313x.2004.02016.x cysteine-rich receptor-like kinase 45 positively regulates disease resistance to Tian, T., Liu, Y., Yan, H., You, Q., Yi, X., Du, Z., et al. (2017). agriGO v2.0: a GO Pseudomonas syringae. plant physiol. Biochem. 73, 383–391. doi: 10.1016/ analysis toolkit for the agricultural community 2017 update. Nucleic Acids Res. 45, j.plaphy.2013.10.024 W122–W129. doi: 10.1093/nar/gkx382 Zhang, Q., Li, W., Yang, J., Xu, J., Meng, Y., and Shan, W. (2020a). Two Tornero, P., Conejero, V., and Vera, P. (1997). Identification of a new pathogen- Phytophthora parasitica cysteine protease genes, PpCys44 and PpCys45, trigger cell induced member of the subtilisin-like processing protease family from plants. J. death in various Nicotiana spp. and act as virulence factors. Mol. Plant Pathol. 21, Biol. Chem. 272, 14412–14419. doi: 10.1074/jbc.272.22.14412 541–554. doi: 10.1111/mpp.12915 Tran, T.-T., Clark, K., Ma, W., and Mulchandani, A. (2020). Detection of a Zhang, X. H., Pizzo, N., Abutineh, M., Jin, X.-L., Naylon, S., Meredith, T. L., et al. secreted protein biomarker for citrus huanglongbing using a single-walled carbon (2020b). Molecular and cellular analysis of orange plants infected with nanotubes-based chemiresistive biosensor. Biosens. Bioelectron. 147, 111766. huanglongbing (citrus greening disease). Plant Growth Regul. 92, 333–343. doi: 10.1016/j.bios.2019.111766 doi: 10.1007/s10725-020-00642-z Usadel, B., Nagel, A., Steinhauser, D., Gibon, Y., Bläsing, O. E., Redestig, H., et al. Zou, X., Bai, X., Wen, Q., Xie, Z., Wu, L., Peng, A., et al. (2019). Comparative (2006). PageMan: an interactive ontology tool to generate, display, and annotate analysis of tolerant and susceptible citrus reveals the role of methyl salicylate overview graphs for profiling experiments. BMC Bioinform. 7, 535. doi: 10.1186/ signaling in the response to huanglongbing. J. Plant Growth Regul. 38, 1516–1528. 1471-2105-7-535 doi: 10.1007/s00344-019-09953-6 Frontiers in Plant Science 23 frontiersin.org http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Frontiers in Plant Science Pubmed Central

Insights into the mechanism of Huanglongbing tolerance in the Australian finger lime (Citrus australasica)

Loading next page...
 
/lp/pubmed-central/insights-into-the-mechanism-of-huanglongbing-tolerance-in-the-lCwojTo2K1

References (203)

Publisher
Pubmed Central
Copyright
Copyright © 2022 Weber, Mahmoud, Stanton, Welker, Qiu, Grosser, Levy and Dutt
eISSN
1664-462X
DOI
10.3389/fpls.2022.1019295
Publisher site
See Article on Publisher Site

Abstract

TYPE Original Research PUBLISHED 21 October 2022 DOI 10.3389/fpls.2022.1019295 Insights into the mechanism of Huanglongbing tolerance in the OPEN ACCESS EDITED BY Marcio C. Silva-Filho, Australian finger lime University of São Paulo, Brazil REVIEWED BY (Citrus australasica) Alessandra Alves De Souza, Secretariat of Agriculture and Food Supply of São Paulo State, Brazil 1 1,2 1 † † Kyle C. Weber , Lamiaa M. Mahmoud , Daniel Stanton , Marcos Antonio Machado, Instituto Agrono ˆ mico de Campinas 1 3 1 Stacy Welker , Wenming Qiu , Jude W. Grosser , (IAC), Brazil 1 1 Amit Levy and Manjul Dutt *CORRESPONDENCE Manjul Dutt Citrus Research and Education Center, University of Florida, Lake Alfred, FL, United States, manjul@ufl.edu Pomology Department, Faculty of Agriculture, Mansoura University, Mansoura, Egypt, These authors have contributed Institute of Fruit and Tea, Hubei Academy of Agricultural Sciences, Wuhan, China equally to this work SPECIALTY SECTION This article was submitted to Plant Breeding, The Australian finger lime (Citrus australasica) is tolerant to Huanglongbing a section of the journal (HLB; Citrus greening). This species can be utilized to develop HLB tolerant Frontiers in Plant Science citrus cultivars through conventional breeding and biotechnological RECEIVED 14 August 2022 approaches. In this report, we conducted a comprehensive analysis of ACCEPTED 22 September 2022 PUBLISHED 21 October 2022 transcriptomic data following a non-choice infection assay to understand the CITATION CaLas tolerance mechanisms in the finger lime. After filtering 3,768 Weber KC, Mahmoud LM, Stanton D, differentially expressed genes (DEGs), 2,396 were downregulated and 1,372 Welker S, Qiu W, Grosser JW, Levy A were upregulated in CaLas-infected finger lime compared to CaLas-infected and Dutt M (2022) Insights into the mechanism of Huanglongbing HLB-susceptible ‘Valencia’ sweet orange. Comparative analyses revealed tolerance in the Australian finger lime several DEGs belonging to cell wall, b-glucanase, proteolysis, R genes, (Citrus australasica). signaling, redox state, peroxidases, glutathione-S-transferase, secondary Front. Plant Sci. 13:1019295. doi: 10.3389/fpls.2022.1019295 metabolites, and pathogenesis-related (PR) proteins categories. Our results COPYRIGHT indicate that the finger lime has evolved specific redox control systems to ©2022Weber,Mahmoud,Stanton, mitigate the reactive oxygen species and modulate the plant defense response. Welker,Qiu,Grosser, Levyand Dutt. We also identified candidate genes responsible for the production of Cys-rich This is an open-access article distributed under the terms of the secretory proteins and Pathogenesis-related 1 (PR1-like) proteins that are Creative Commons Attribution License highly upregulated in infected finger lime relative to noninfected and (CC BY). The use, distribution or reproduction in other forums is infected ‘Valencia’ sweet orange. Additionally, the anatomical analysis of permitted, provided the original phloem and stem tissues in finger lime and ‘Valencia’ suggested better author(s) and the copyright owner(s) regeneration of phloem tissues in finger lime in response to HLB infection. are credited and that the original publication in this journal is cited, in Analysis of callose formation following infection revealed a significant accordance with accepted academic difference in the production of callose plugs between the stem phloem of practice. No use, distribution or reproduction is permitted which does CaLas+ ‘Valencia’ sweet orange and finger lime. Understanding the mechanism not comply with these terms. of resistance will help the scientific community design strategies to protect trees from CaLas infection and assist citrus breeders in developing durable HLB tolerant citrus varieties. KEYWORDS citrus, transcriptome, Huanglongbing, host response, pathogen-related proteins, callose deposition Frontiers in Plant Science 01 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 (Forster and Smith, 2010). Australian lime species such as Citrus Introduction australasica, and the hybrid of Citrus australis and Citrus virgata The genus Citrus originated in tropical and subtropical (Sydney hybrid) have been reported to be HLB resistant (Ramadugu et al., 2016; Alves et al., 2020; Huang et al., southeastern Asia (Wu et al., 2014). When consumed fresh, citrus fruit are good sources of dietary fiber (Marıń et al., 2007) 2021a). Thus, these species can provide pathogen resistance- related genes that can be used to confer HLB tolerance into and antioxidants (Wang et al., 2022), and they have anticancer and anti-inflammatory properties (Benavente-Garcı́aand conventional citrus cultivars to produce HLB-tolerant citrus hybrid scions and rootstocks. Although extensive research is Castillo, 2008). The United States is one of the citrus being conducted to use HLB tolerance traits for the development producers, with production concentrated in Florida, California, of new citrus cultivars, there is a lack of knowledge on the and Texas. Sweet orange constitutes most of the citrus mechanism underlying the perceived tolerance in these citrus production acreage, with the remainder being that of species. Resistance and tolerance observed in the different grapefruit, mandarin, lemons and limes (Kramer et al., 2022). cultivars can be defined by various factors, including the Citrus is susceptible to plethora of diseases and pests, with absence of CaLas multiplication and replication, delayed huanglongbing (HLB), a phloem-limiting bacterial disease infection, or recovery from infection by enhancing plant caused by the bacterium Candidatus Liberibacter asiaticus defensive systems. (CaLas), being the most destructive (Duan et al., 2009). In the CaLas is a gram-negative bacterium that employs secretion United States, this disease has been prevalent since 2005 (Bové, systems that deliver virulence proteins, known as effectors, to 2006), when it was first detected in south Florida’s Miami-Dade manipulate its hosts (Clark et al., 2018) through modulation of County (Halbert, 2005). Widespread monoculture of a few select host physiology and suppressing plant defense mechanisms. citrus varieties has reduced the genetic diversity of cultivated Effectors promote pathogen colonization and disease citrus, allowing HLB to spread quickly among the population. development and create environmental conditions favorable Since its initial discovery in 2005, HLB has spread rapidly for colonization and proliferation (Jones and Dangl, 2006; Dou throughout Florida to every citrus-growing county. and Zhou, 2012; Feng and Zhou, 2012). The plant defense Additionally, HLB is now present in Texas and California, response system involves pattern-triggered immunity (PTI), where it threatens the important central valley (Milne et al., which is triggered by microbe-associated molecular patterns 2018; Graham et al., 2020). Most of the commercial citrus (MAMPs) via cell surface-localized pattern-recognition cultivars grown in the United States, including several named receptors (PRRs), and effector-triggered immunity (ETI), cultivars and selections of sweet orange, mandarin, and which is induced by pathogen effector proteins via grapefruit, are highly susceptible to HLB. intracellular receptors that detect intercellular pathogen- The health of infected trees invariably declines, accompanied derived molecules and intracellular receptors that activate by reduced fruit yield and quality (Ferguson et al., 2021) and plant defense response upon detection of pathogen-secreted severely infected trees eventually die (Zhang et al., 2020b). Long- effector proteins that function inside the plant cell (Ngou term management of tree health through enhanced nutrition et al., 2021). Previous research showed that CaLas encodes (Zambon et al., 2019) and psyllid vector control using various several Sec-delivered effectors (SDEs), many of which are control strategies have been proposed and evaluated (Mattos-Jr conserved across CaLas isolates. Sec-delivered effector 1 et al., 2020). Tolerance to HLB has been reported in some citrus (SDE1), a secreted protein biomarker used for the detection of cultivars, such as citron and its hybrids (e.g., lemons), and in HLB, is highly expressed in infected citrus tissue at a relatively some trifoliate orange trees and their hybrids (Peng et al., 2020). early infection stage (Pagliaccia et al., 2017; Tran et al., 2020). HLB-tolerant scions and rootstocks, conventionally bred or Proteases secreted by pathogens have been shown to be transgenic, remain the best option for the control and important virulence factors that affect plant defense, and management of HLB (Qiu et al., 2020). Sugar Belle, a recently cysteine (Cys) proteases have been demonstrated to participate released mandarin hybrid, has also been observed to be HLB in different pathosystems (Zhang et al., 2020a). Citrus papain- tolerant (Killiny et al., 2018). Several wild sexually compatible like cysteine proteases (PLCPs) were found as a defense cultivars, such as Citrus ichangensis ‘2586’ (Wu et al., 2020), inducible in CaLas-infected trees, suggesting they are involved Citrus latipes (Folimonova et al., 2009), several accessions of sour in the citrus defense responses (Clark et al., 2018). Additionally, pummelo (Citrus grandis; Zou et al. (2019) and kaffir lime several lysosomal Cys proteases were shown to be involved in (Citrus hystrix; Hu et al. (2017), are also tolerant to HLB. various apoptosis models, although the mechanisms of their Additionally, several sexually incompatible citrus relatives are involvement are not yet clear (Rozman-Pungerčar et al., 2003). also tolerant to HLB (Miles et al., 2017). Plasma membrane-localized receptor-like kinases (RLKs) play a The Australian limes spread from Southeast Asia to role in plant recognition of microbes and in perceiving and Australasia during the early Pliocene epoch, approximately transducing these external stimuli to further activate the 4Ma (Wu et al., 2018). There are seven species of Australian associated downstream signaling pathways (Jose et al., 2020). limes that are all native to Australia and the New Guinea islands Frontiers in Plant Science 02 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 RLKs are categorized into several subfamilies, including leucine- integrity of the RNA were analyzed using electrophoresis on a rich repeat (LRR) RLKs (LRR-RLKs), Cys-rich repeat (CRKs), 1.0% agarose gel and then examined using an Agilent 2100 domains of unknown function 26 RLKs, S-domain RLKs, and Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). others (Quezada et al., 2019). High-quality RNA samples with an RNA integrity number In this study, we shed light on the potential mechanism of (RIN) > 6.5 were used for cDNA synthesis and RNA HLB tolerance in the finger lime.Tothisend,wegraft- sequencing (RNAseq). Single-stranded cDNA was synthesized inoculated one-year-mature finger lime and ‘Valencia’ (Citrus using a RevertAid First Strand cDNA Synthesis Kit (Thermo sinensis) sweet orange trees with CaLas and evaluated their Fisher Scientific, Massachusetts, USA). The cDNA transcriptome to provide insights into the mechanism of concentration was determined using a NanoDrop 1000 tolerance to HLB. The transcriptome data was also validated Spectrophotometer (Thermo Fisher Scientific). in five-year-mature trees growing in the field. The cDNA libraries were sequenced using an Illumina HiSeq platform configured for a 2x150 read length. The generated base callings of the cDNA reads were presented in a paired-ended Materials and methods format. The reads were cleaned, and their adapters were removed using AdapterRemoval v2.2.2 (Lindgreen, 2012), with Plant materials the default parameters. Short and poor-quality reads were filtered using Trimmomatic v0.39 (Bolger et al., 2014). The Certified HLB-free budwood of C. australasica clone DPI 50-36 following parameters were applied: a minimum length of 100 (Finger lime) and ‘Valencia’ sweet orange clone SPB-1-14-19 were bases, a trailing and leading length equal to 16 bases, a sliding obtained from Florida’s Division of Plant Industry budwood window of 16:25, and 5 threads. After processing, the final read repository and budded onto 6-month-old Swingle citrumelo count and average qualities were checked using FastQC v0.11.8 rootstock. One-year-old budded trees were subsequently side (Andrews, 2010). grafted (Figure S1)with CaLas-infected ‘Valencia’ sweet orange scions (Ct value of 23.2 ± 0.3). The trees were periodically evaluated for infection, and 1 to 2 year-old infected trees were utilized for Mapping of the reads, transcript counts, subsequent experiments (Table S1). and DEG analysis The cleaned reads were mapped to the C. sinensis genome Monitoring CaLas in finger lime and using STAR v2.6.0C (Dobin et al., 2013) with the default ‘valencia’ sweet orange plants parameters, except for the need to define a sorted BAM output. The C. sinensis genome and annotations used in STAR To diagnose the CaLas titer in the potentially infected were obtained from Phytozome (https://phytozome-next.jgi.doe. greenhouse-grown trees, genomic DNA was isolated periodically gov/) The BAM files were indexed using SAMtools v1.7 (Li et al., from the leaf petioles and midveins of fully expanded leaves using a 2009). The BAM files were assessed for transcript counts using GeneJET Plant Genomic DNA Purification Kit (Thermo Fisher featureCounts v1.6.0 (Liao et al., 2014) with default settings Scientific Waltham, MA, USA). Leaves were also collected in the except for the section of exon type and five threads. The list of late fall (November) and early spring (March) from 5-year-old counts was extracted from the featureCounts software output file finger lime DPI 50-36 and ‘Valencia’ SPB-1-14-19 trees growing in and organized to compare infected finger lime vs.infected the field (Swingle rootstock) to estimate the CaLas titer in the ‘Valencia’ sweet orange. A metadata file for the comparison sampled tissues for three years. The DNA concentration was was also generated for differentially expressed gene (DEG) normalized to 25 ng/mL before performing qPCR using a analysis via DESeq2 v3.10, an R Bioconductor package (Love StepOnePlus Real-Time PCR System (Thermo Fisher et al., 2014). The counts for each comparison were normalized, Scientific). Detection of CaLas genomic DNA was determined by and gene dispersion was estimated using DESeq2. The list of qPCR using TaqMan Gene Expression Master Mix and CQUL DEGs was filtered to remove any DEGs with a |log (fold- primers (Table S2)to amplify the CaLas rplJ/rplL ribosomal protein change)|< 2 and an adjusted P value (false discovery rate gene (Wang et al., 2006). (FDR)) ≥ 0.05. RNA extraction, cDNA synthesis and Gene ontology enrichment and sequencing pathway analysis Two years following infection, RNA was extracted using Statistically significant DEGs were analyzed using AgriGO TRIzol following the manufacturer’s protocol. The purity and v2 (Tian et al., 2017). To correctly assign GO terms, the Frontiers in Plant Science 03 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 following parameters were selected: Genome - Citrus sinensis, AMC), given that protease activity correlates with an increase in statistical test - Fisher’s exact test, adjusted according to the detectable relative light units (RFUs) over time (Barrett, 1980; Benjamini–Yekutieli method (Benjamini et al., 2006)for Tchoupe et al., 1991). The leaf samples were extracted in a buffer discovering FDRs in a multiple comparison with an consisting of 100 mM sodium acetate (pH 5.5), 2.5 mM DTT and 1 alpha=0.05, and a minimum mapping of 5. The statistically mM EDTA. The samples were centrifuged, and the supernatants significant GO terms from AgriGO v2 were then inputted into were incubated at a 1:2 ratio together with a mixture consisting of 100 REVIGO software to remove redundant GO terms (Supek et al., mM sodium acetate (pH 5.5), 2.5 mM DTT, 1 mM EDTA, 0.5% 2011). Functional analysis was conducted using the C. sinensis DMSO, and 37.5 mM Z-F-R-AMC. All the samples were incubated pathways file (m02) in MapMan (Thimm et al., 2004). The at 30°C for5minutes. Anotherset of samples were co-incubated for functional categories were viewed using PageMan and analyzed 3 hours with 10 µM synthetic epoxide peptide E-64 (L-3- for statistical significance using a nonparametric test (Usadel trans-carboxyoxiran-2-carbonyl]-L-Leu-agmatin]; [N- et al., 2006). Pathway analysis was performed using the pathways (transepoxysuccinyl)-L-leucine 4-guanidinobutylamide]) as function of MapMan (Mapman version 3.0.0). inhibitor of cysteine protease at 37°C. At the end of the incubation, ™ ™ fluorescence was measured in a Thermo Scientific GENESYS 30 Visible Spectrophotometer atlex = 380 and lem = 460 nm. Negative Quantitative PCR and DEG validation (no enzyme) and blank samples were also prepared along with the positive samples by the addition of E-64 inhibitor and solvent, The real-time PCR (qPCR) reaction mix consisted of 1 µL of respectively. The percent inhibition was calculated by using the ® ™ DNA (25 ng/µL), SYBR Green PowerUp PCR Master Mix following formula: (Applied Biosystems, Foster City, CA), and selected gene- Inhibition % = [Absorbance (blank)-Absorbance (test)]/ specific primers (Integrated DNA Technologies, Inc., Absorbance (blank) x 100 Coralville, IA, USA) in a final mixture of 20 mL, according to the manufacturers’ instructions. qPCR was performed in a StepOnePlus Real-Time PCR System (Thermo Fisher Evaluation of CaLas-infected ‘valencia’ Scientific, Massachusetts, USA). The citrus b-actin sweet orange and finger lime and housekeeping gene was used as a reference gene (Qiu et al., quantification of phloem callose deposits 2020); each sample was analyzed in triplicate. Relative gene -DDCt expression was calculated using the 2 method described Healthy and CaLas-infected finger lime and ‘Valencia’ sweet th th previously (Livak and Schmittgen, 2001). The relative mRNA orange leaves (position 5 -8 from the apical meristem) were levels were compared to those of the endogenous C. sinensis collected from the greenhouse. The petioles were cut and fixed in ACTIN gene (Qiu et al., 2020) and calculated using the 2 4% paraformaldehyde in 1x PBS. The samples were rinsed three DDCT method (Livak and Schmittgen, 2001). To confirm the times in 1x PBS and then dehydrated in an ethanol (EtOH) series validity of the DEGs, we selected eight upregulated and eight for 1 hour each. The samples were transitioned from 100% EtOH downregulated genes from the DEG data, then analyzed on the to 100% tert-butanol (3:1, 1:1, and 1:3) at room temperature same samples that were sequenced and the relative expression of (RT) for 8-16 hours each and then cleared in 100% tert-butanol those genes were compared with RNAseq results. As there is no for one hour prior to paraffininfiltration. The samples were publicly available genome assembly of the finger lime yet, we infiltrated using increasing concentrations (3:1, 1:1, 1:3) of selected those genes based on the sweet orange genome. Paraplast Plus paraffin (Fisher Scientific, Waltham, MA, USA) Additionally, some of the DEGs that show significant for 24 hours each and then incubated for 48-36 hours in 100% difference were validated in twelve samples collected from paraffin, which was changed three times. The samples were finger lime and ‘Valencia’ sweet orange trees growing in the embedded in paraffin and allowed to harden for 24 hours at field. The gene expression of the trees was compared with that of 4°C. Afterward, ten micrometer sections were cut using a Leica uninfected control trees growing in a protected greenhouse. The 2155 microtome (Leica Biosystems, Deer Park IL, USA), and the control trees were confirmed to be negative for CaLas before sections were floated on a drop of water on a slide. The slides subsequent comparison. A list of the primers used in this study is were subsequently incubated overnight on a slide warmer at presented in Tables S3, S4. 37°C to allow the sections to adhere to the slide. The slides were dewaxed in 100% Histoclear II (National Diagnostics, Atlanta, GA, USA) for an hour each, and the solution was changed twice. Proteolytic enzyme assays The sections were stained with 0.05% toluidine blue O for 30 seconds and then rinsed in dH O. The slides were dehydrated in Assessment of Cys protease activitywasperformedbyrecording an EtOH series for 5-10 minutes each. Coverslips were mounted the liberation of fluorogenic peptide substrate VIII (Z-Phe-Arg- using Fisher Scientific’s mounting media with toluene (Fisher AMC. Z: N-carbobenzyloxy; 7-amino-4-methylcoumarin; Z-F-R- Scientific, Waltham, MA, USA). The slides were observed under Frontiers in Plant Science 04 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 an Olympus BX61 epifluorescence microscope (Olympus, were collected from 8-year-mature trees, and the total DNA Center Valley, PA, USA), and images were captured using a 14 obtained from leaf petioles and midribs was analyzed using MP OMAX digital camera (OMAX, Irvine, CA). The phloem qPCR. Our results indicate that the field-grown finger lime trees and xylem ring distances were measured using FIJI (Schindelin were always HLB negative (undetermined cycle threshold (Ct) or et al., 2012) Figure S2, and the phloem ratio (Pa/Xa) and xylem had high Ct values (37.88 ± 0.28)), whereas ‘Valencia’ sweet ratio (Xa/Pa) for petioles and stems were calculated. Three orange trees had low Ct values of 25.14 ± 0.82 (Figure 1C), samples were used for evaluation and the data were recorded indicating active HLB infection. as average for three images of each sample. Subsequently, field-collected HLB-infected ‘Valencia’ sweet Phloem sieve plate callose was measured according to a orange scions were side grafted onto healthy one-year-mature previous protocol (Ferrara et al., 2015). Stem phloem tissue grafted finger lime and ‘Valencia’ sweet orange trees to observe samples were collected from CaLas+ ‘Valencia’ sweet orange and disease progression under controlled conditions. To assess finger lime trees. The tissue samples were obtained from the bacterial population levels in the leaves, qPCR was periodically stems of the mature trees with a scalpel, approximately 8 cm performed to screen the CaLas titer in the samples collected from the leaves. Three stem phloem samples were collected from from greenhouse trees Table S1. Screening for the presence of five trees of each plant type. Each tissue sample was placed into CaLas revealed that when trees were forcibly inoculated with an 85% EtOH solution for fixation immediately after collection CaLas, both finger lime and ‘Valencia’ sweet orange trees were and incubated overnight for de-staining. The samples were then infected (Figure 1D). However, the rate of infection differed transferred to a 0.01% Tween-20 solution to rehydrate for 1 between the two accessions. At 24 months following infection, hour. Finally, the samples were transferred to a 0.01% aniline the Ct value of the finger lime trees was, on average, 33.2 ± 1.3, blue staining solution. After staining for 1 hour, images of the while the ‘Valencia’ sweet orange Ct value was, on average, 23.4 tissue samples were collected. A Leica SP8 laser-scanning ± 2.8. confocal microscope was used to collect the images, with A transcriptome analysis was subsequently conducted to settings that have been described previously (Welker and Levy, understand the possible biological reasons for the HLB tolerance 2022). Three images were taken from the central region of each of finger lime compared with susceptible citrus such as ‘Valencia’ tissue sample. Using a FIJI macro, counts of callose deposits were sweet orange. The RNA from CaLas-infected samples of finger obtained as described previously (Welker et al., 2022). lime and ‘Valencia’ sweet orange (three technical replicates each) was sequenced using the Illumina HiSeq next-generation sequencing platform. The average total number of raw reads Statistical analysis produced was 37,550,067 and 31,506,847 for the CaLas-infected finger lime replicates and the CaLas-infected ‘Valencia’ sweet The data were analyzed using JMP Pro v16 software, with a orange replicates, respectively (Table 1); the cleaning process of post hoc Tukey–Kramer honestly significant difference (HSD) the reads resulted in average numbers of reads of 29,854,551 test or t tests to compare the means of the different treatments. (79.51%) and 26,230,524 (83.25%), respectively. The cleaned Statistical significance was established at P < 0.05. Pearson reads were mapped onto the C. sinensis genome. Genomic Correlation Coefficient (r) was calculated to validate the mapping of the RNA reads revealed that, on average, modulationingeneexpressionfor RNAseq data and 24,807,563 (84.84%) and 22,595,692 (86.14%) clean reads were quantitative PCR using JMP Pro v16 software. As for mapped. The mapped reads were analyzed by differential statistical Testing of Phloem Callose Deposits, ANOVA was expression analysis. After filtering the differentially expressed performed using R statistical software to assess the significance genes (DEGs) according to |log (fold-change)| < 2 and an of the model and interactions, any non-zero counts of callose adjusted P value (FDR) ≥ 0.05, 3,768 remained. Of the 3,768 plugs were analyzed with a negative binomial regression in R (R DEGs, 2,396 were downregulated in HLB-infected finger lime Core Team, 2013). After log-transforming the counts to meet the compared to HLB-infected ‘Valencia’ sweet orange, while 1,372 assumption of normality, ANOVA was performed on the mean were upregulated (Figure 2A). counts of each tissue type group. Domain differences between finger lime Results and ‘valencia’ sweet orange infected with HLB Finger lime trees have enhanced tolerance to CaLas There were20GOcategoriesthatweresignificantly upregulated, and 19 GO categories downregulated between the To understand HLB levels in mature finger lime and HLB-infected finger lime samples and the HLB-infected ‘Valencia’ sweet orange trees growing in the field, leaf samples ‘Valencia’ sweet orange samples (Table 2). Among the Frontiers in Plant Science 05 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 FIGURE 1 (A) HLB infected ‘Valencia’ sweet orange in the field exhibiting the characteristic blotchy mottle pattern in the leaves. (B) Finger lime leaves from trees growing in the field with no visible disease symptom. (C) Detection of CaLas in leaf tissues of Finger lime and ‘Valencia’ trees by qPCR. Leaf samples were collected from 8-year-old field trees at the beginning of the study. (D) CaLas detection from leaf samples collected periodically from trees, side grafted with HLB infected budwood and growing in the green house. * represents the sampling time for RNAseq analysis. Different letters above the error bar indicate statistically significant differences, while the same letters signify no significant differences using the Tukey–Kramer honestly significant difference test (Tukey HSD; p <0.05). domains of the upregulated DEGs, eleven were representatives of (GO:0008037). Other domains with lower enrichment values the molecular function category. Of these 11 domains, the most are listed in Table 2. There were no domains of upregulated significant were heme binding (GO:0020037), tetrapyrrole DEGs within the cell component category. binding (GO:0046906), and iron ion binding (GO:0005506). The top downregulated GO terms of the molecular function The other domains of the upregulated DEGs represented types category included “catalytic activity” (GO: 0003824) and of biological processes: signaling (GO:0023052), amine “ADP binding” (GO: 0043531). “Amine metabolic process” metabolic process (GO:0009308), and cell recognition (GO:0009308), “polysaccharide catabolic process” (GO:0000272), TABLE 1 Summary of sequencing, cleaning, and mapping of reads following sequencing the HLB infected finger lime and HLB infected ‘Valencia’ samples. Run Name Raw read count Read count after cleaning Surviving Read Percent Mapped reads Mapped reads percent FL1 42,957,054 31,809,635 74.05% 26,440,485 83.12% FL2 34,840,983 28,780,882 82.61% 23,935,454 83.16% FL3 34,852,164 28,973,136 83.13% 24,046,751 83.00% FL Avg 37,550,067 29,854,551 79.51% 24,807,563 83.09% Val1 32,285,138 26,821,818 83.08% 22,756,095 84.84% Val2 32,676,339 27,320,775 83.61% 23,775,386 87.02% Val3 29,559,065 24,548,978 83.05% 21,255,596 86.58% Val Avg 31,506,847 26,230,524 83.25% 22,595,692 86.14% Frontiers in Plant Science 06 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 FIGURE 2 (A) Volcano plot of the upregulated and downregulated DEGs. Genes with an adjusted p value of less than 0.05 found with DESeq were assigned as differentially expressed. (B) Graphical view of the most statistically significant upregulated and downregulated enriched GO terms in Finger line trees as compared to ‘Valencia’ trees. Statistically significant DEGs were analyzed using AgriGO v2 and REVIGO. (C) Differentially expressed genes as identified following MAPMAN analysis. Regulation of stress-related gene pathways by CaLas infection in the infected Finger Lime (Left). Overview of the differentially expressed genes related to the metabolic pathways in Finger lime and ‘Valencia’ sweet orange (Right). Genes that were significantly upregulated following CaLas infection are displayed in blue, and downregulated genes are displayed in red. Frontiers in Plant Science 07 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 TABLE 2 Significant GO terms represented in the HLB infected finger lime vs HLB infected ‘Valencia’ comparison. GoTerm Type Description PosCount PosEV NegCount NegEV GO:0003824 F catalytic activity 0 0 655 -5.6576 GO:0005506 F iron ion binding 57 -7.1549 63 -3.585 GO:0043531 F ADP binding 56 -4.0269 72 -2.8539 GO:0009308 P amine metabolic process 13 -3.5229 13 -2.0915 GO:0000272 P polysaccharide catabolic process 6 -2.0655 8 -2.0915 GO:0042221 P response to chemical 0 0 33 -2.0915 GO:0071554 P cell wall organization or biogenesis 0 0 24 -2.0915 GO:0004857 F enzyme inhibitor activity 0 0 25 -1.7696 GO:0030554 F adenyl nucleotide binding 0 0 205 -1.7696 GO:0071555 P cell wall organization 0 0 18 -1.7212 GO:0020037 F heme binding 54 -4.4815 60 -1.6576 GO:0046906 F tetrapyrrole binding 54 -4.4815 60 -1.6576 GO:0005618 C cell wall 0 0 215 -1.6198 GO:0030312 C external encapsulating structure 0 0 19 -1.6198 GO:0016705 F oxidoreductase activity paired donors 48 -4.4685 52 -1.5376 GO:0016491 F oxidoreductase activity 48 -3.1739 153 -1.5376 GO:0010333 F terpene synthase activity 0 0 17 -1.5376 GO:0016838 F carbon-oxygen lyase activity 0 0 17 -1.4559 GO:0030599 F pectinesterase activity 0 0 15 -1.4437 GO:0023052 P Signaling 44 -3.9586 0 0 GO:0008037 P cell recognition 16 -2.3979 0 0 GO:0048544 P recognition of pollen 16 -2.3979 0 0 GO:0051704 P multi-organism process 17 -2.3979 0 0 GO:0000003 P Reproduction 16 -1.9586 0 0 GO:0016758 F hexosyltransferase activity 41 -1.7959 0 0 GO:0005515 F protein binding 202 -1.4318 0 0 GO:0016757 F glycosyltransferase activity 44 -1.4318 0 0 GO:0030247 F polysaccharide binding 12 -1.4318 0 0 GO:0004568 F chitinase activity 6 -1.3768 0 0 GO:0006026 P aminoglycan catabolic process 6 -1.3665 0 0 GO:0032501 P multicellular organismal process 17 -1.3665 0 0 and “response to chemical” (GO:0042221) were the top categories. Out of the 3,768 DEGs, 1,162 were assigned to disease downregulated GO terms in the biological process category. response categories. Nearly all the DEGs belonged to one of the Unlike the upregulated GO terms, the downregulated GO terms following categories: cell wall, b-glucanase, proteolysis, R genes, were assigned to two cellular component categories: “cell wall” signaling, respiratory burst, abiotic stress, redox state, (GO: 0005618) and “external encapsulating structure” (GO: peroxidases, glutathione-S-transferase, secondary metabolites, 0030312). A graphical view of these data can be found and pathogenesis-related (PR) proteins Figure 2C.When in Figure 2B. comparing the DEGs in functional categories between HLB- infected finger lime and HLB-infected ‘Valencia’ sweet orange, we found that that genes involved in flavonoids, isoflavones, Functional differences between HLB- cytokinin synthesis and degradation, ethylene synthesis and infected finger lime and ‘valencia’ degradation, sugar and nutrient signaling, and the transport of sweet orange sugars as well as genes encoding isoflavone reductase, UDP glucosyl and glucoronyl transferases, cytochrome p450, GRAS DEG functional analysis is important to understand the transcription factors, MAD box transcription factors, WRKY biochemical responses elicited by these genes and the roles transcription factors, DNA methyltransferase, ubiquitin E3, they play in the overall function of the plant. The significant receptor kinases, LRR XI, and DUF were upregulated in finger DEGs were analyzed via PageMan to investigate the functional lime. However, genes assigned to categories related to the cell Frontiers in Plant Science 08 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 wall, pectin esterase, pectin methylesterification (PME), from the field. The response of the infected trees in the field was phenylpropanoids, lignin biosynthesis, short chain largely consistent with data obtained from the greenhouse trees, dehydrogenases/reductases, and posttranslational modification indicating that the RNA-seq data reported here are consistent of kinases and receptors (such as cytoplasmic kinase VII), as well for both sets of samples (greenhouse and field samples). as a few unassigned ones, were found to be underrepresented in Several DEGs encoding many other R proteins that finger lime. Given the functional differences between the predominantly contain a nucleotide-binding site (NBS) and/or upregulated genes among the groups, adding context to better LRR domain were differentially expressed in CaLas-infected understand relationships within a pathway can help determine finger lime. Indeed, 114 pathogenesis-associated DEGs the differences between HLB-infected finger lime and HLB- constituted 3% of all the DEGs mapped, 48 of which were infected ‘Valencia’ sweet orange. upregulated. Twenty-nine of these upregulated genes were PR protein-encoding DEGs of the Tir-NBS-LRR class, 4 were of the NBS-LRR class, and 7 were of the CC-NBS-LRR class. In Pathogen interaction factors were addition, there were 33 Tir-NBS-LRR class genes, 7 CC-NBS- upregulated in finger limes LRR gene class genes, and 14 NBS-LRR class genes that were downregulated. Transcript levels of orange1.1g035344m, similar It has been widely reported that microbe infection induces to the stable antimicrobial peptide (SAMP) (Huang et al., 2021b) plant defense through two mechanisms, namely, PTI and ETI, was not detected in our RNAseq DEG data although play a substantial role in plant disease resistance (Deng et al., orange1.1g033887m, an alternate isoform was upregulated in 2018). Seven cell wall LRR family protein-related DEGs were HLB+ ‘Valencia’. identified as being enriched. Of the DEGs mapped to the Among these proteins, PR proteins and Cys-rich secretory genome, 8.0% were kinase-related DEGs. We identified proteins are thought to be involved in the plant defense response to multiple cysteine (Cys)-rich receptor-like protein kinases pathogen infection and plant tolerance (Van Loon, 1997; Baindara (CRKs) upregulated in infected finger lime (Table 3). No et al., 2017). Interestingly, we identified candidate genes encoding changes in expression (log (fold-change)) were recorded for several Cys-rich secretory proteins or Pathogenesis-related 1 the mitogen-activated protein kinase-encoding DEG (MAPK) protein (PR1-like) that were highly upregulated in the infected in finger lime and ‘Valencia’ sweet orange when the uninfected finger lime relative to CaLas-uninfected and infected ‘Valencia’ tissues were compared with the infected tissues. sweet orange (Table 4). Of these Cys-rich secretory proteins, To validate the reliability of the RNA-seq data in terms of the orange1.1g043403m was highly upregulated in the non-infected overexpression of CRKs, we randomly selected 9 DEGs encoding finger lime trees, and the expression was recorded as two-fold Cys-rich RLKs for confirmation by qPCR (Figure 3) on samples increase after CaLas infection. The results of the field experiment of infected finger lime and ‘Valencia’ sweet orange obtained confirmed this gene was highly upregulated in finger lime trees TABLE 3 DEGs involved in cysteine-rich receptor-like protein kinase of CaLas infected finger lime and ‘Valencia’ sweet orange. Gene symbol Log2 fold change Citrus sinensis ID Arabidopsis homolog FL (HLB+) Val(HLB+) Cysteine-rich RLK- 8* 1.55 0.23 orange1.1g007239m AT4G23160.1 Cysteine-rich RLK- 10 4.17 0.26 orange1.1g041433m AT4G23180.1 Cysteine-rich RLK- 10 31.5 2.13 orange1.1g041917m AT4G23180.1 Cysteine-rich RLK- 10 1.45 0 orange1.1g039168m AT4G23180.1 Cysteine-rich RLK- 10 0.9 9.5 orange1.1g010329m AT4G23180.1 Cysteine-rich RLK- 10 9.25 0.88 orange1.1g037707m AT4G23180.1 Cysteine-rich RLK- 10 11.66 0.06 orange1.1g042892m AT4G23180.1 Cysteine-rich RLK- 10 0.66 0 orange1.1g005893m AT4G23180.1 Cysteine-rich RLK- 10 14.58 3.17 orange1.1g009186m AT4G23180.1 Cysteine-rich RLK- 16 5.5 0.69 orange1.1g021682m AT4G23130.2 Cysteine-rich RLK- 18 2.25 0.09 orange1.1g040682m AT4G23260.1 Cysteine-rich RLK- 25 2.03 0.02 orange1.1g017211m AT4G05200.1 Cysteine-rich RLK- 25 5.06 0.6 orange1.1g017150m AT4G05200.1 Cysteine-rich RLK- 25 2.03 0.02 orange1.1g017211m AT4G05200.1 Cysteine-rich RLK- 34 7.49 1.47 orange1.1g006125m AT4G11530.1 *Cysteine-rich RLK: cysteine-rich receptor-like protein kinase. Frontiers in Plant Science 09 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 AB C DE F GH I FIGURE 3 Relative transcript levels of cysteine-rich RLK (RECEPTOR-like protein kinase) as calculated by real-time PCR compared with the CaLas free ‘Valencia’. The CaLas infected samples were collected from five year old trees growing in the field and the CaLas free (control) samples were collected from trees growing in a protected greenhouse. (A–I) Relative RLK expression as detected in this study compared with CaLas free 'Valencia'. The control trees were confirmed negative for CaLas before further comparison. Data are means ± SE of twelve samples. Different letters above the error bar indicate statistically significant differences, while the same letters signify no significant differences using the Tukey–Kramer honestly significant difference test (Tukey HSD; p <0.05). when compared with the infected ‘Valencia’ sweet orange limonia)), pummelo (Hirado Buntan pummelo and Siamese Sweet (Figure 4). Additionally, we investigated the presence of this gene pummelo (Citrus maxima)), grapefruit (Ruby Red and Duncan (orange1.1g043403m) in several field grown citrus species and (Citrus × paradisi)), sweet orange (‘Valencia’ and Parson Brown cultivars, and we detected lower relative expression in all the (Citrus sinensis)), Volkamer lemon (Citrus volkameriana), Sydney evaluated trees compared to finger lime. The only species that hybrid (Citrus x virgata), Citrus papuana,and Citrus inodora, presented high expression was the Australian desert lime (Citrus showed lower expression of this gene (orange1.1g043403m) than glauca), and this expression was negatively associated with CaLas did finger lime (Figure S3). Although Poncirus trifoliata is highly presence (Figures S3, S4). In contrast, multiple citrus relatives, such tolerant to HLB (Figure S4), therelativeexpression of the Cys-rich as kumquat (Nagami (Fortunella margarita)and Meiwa secretory protein transcripts identified in this study was lower than (Fortunella crassifolia)), Poncirus trifoliata (50-7 and Flying that recorded in finger lime. Dragon), Mandarin (Ponkan and Cleopatra (Citrus reticulata), Additionally, many of the DEGs characterized encoded lime (key lime (Citrus aurantifolia) and Rangpur lime (Citrus transcription factors, kinase activity-related proteins, or were Frontiers in Plant Science 10 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 TABLE 4 DEGs involved in Cysteine-rich secretory proteins or Pathogenesis-related protein of CaLas in finger lime and ‘Valencia’ sweet orange. Gene symbol Log2 fold change Gene function Citrus sinensis ID Arabidopsis homolog FL (HLB+) Val (HLB+) CAP1 1.83 0.25 Pathogenesis-related proteins orange1.1g031237m AT4G33720.1 CAP2 5330 15.35 orange1.1g043403m AT4G33720.1 CAP3 0.22 15.55 orange1.1g037670m AT5G66590.1 LCR69 41.64 6.89 orange1.1g034999m AT2G02100.1 CAP, Cysteine-rich secretory proteins; Antigen 5; and Pathogenesis-related 1 protein) superfamily protein, LCR69: low-molecular-weight cysteine-rich 69. involved in proteolysis. Sixty-seven DEGs were categorized as family, which are involved in secondary metabolism, hormone encoding transcription factors involved in the biotic stress signal transduction, plant development, abiotic stress tolerance, response, constituting 1.8% of all the mapped DEGs, 27 of which and disease resistance, six of which were upregulated (MYB12, were found to be upregulated in finger lime. The most notable were MYB17, MYB62, MYB38, MYB102,and MYB112). Eleven of the MYB, WRKY, zinc-finger, and bZIP transcription factors. There DEGs identified belonged to the WRKY transcription factor family, were 32 DEGs related to the Myb domain transcription factor the members of which play important roles in plant development AB C D FIGURE 4 Relative transcript levels of CAP (Cysteine-rich secretory proteins; Antigen 5; and Pathogenesis-related 1 protein) superfamily protein is calculated by real-time PCR compared with the CaLas free ‘Valencia’. (A) CAP1, (B) CAP2, (C) CAP3 and (D) LCR69. The CaLas infected samples were collected from five years old samples growing in the field and the CaLas free (control) samples were collected from a protected greenhouse. The control samples were confirmed negative for CaLas before further comparison. Data are means ± SE of twelve samples. Different letters above the error bar indicate statistically significant differences, while the same letters signify no significant differences using the Tukey-Kramer honestly significant difference test (Tukey HSD; p <0.05). Frontiers in Plant Science 11 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 and stress responses. Nine of the WRKY transcription factors were (orange1.1g018968m) was upregulated in finger lime. There were 11 upregulated (WRKY14, WRKY23, WRKY28, WRKY31, WRKY47, serine protease (SP)-encoding, 2 Kunitz Family Trypsin- and protease WRKY48, WRKY50, WRKY72 and WRKY75). Additionally, a inhibitor protein-encoding, and 15 Subtilase-related DEGS Dof-Type zinc finger DNA-binding family protein (DAG1) and downregulated in finger lime. In contrast, 20 Ubiquitin E3 Scf F-box two BZIP transcription factor family proteins (TGA9 and BZIP42) and one Ubiquitin E3 Scf Skp (orange1.1g030652m) genes were were found to be upregulated in finger lime, while bZIP58, BZIP61, overexpressed in finger lime, while 16 Ubiquitin E3 Ring, 1 and PAN were downregulated. Ubiquitin E2, 2 Ubiquitin Proteasome, 1 Ubiquitin 4, and 1 Polyubiquitin 10 genes were downregulated. We detected none to minimal expression of three xylem Cys Genes involved in hormone signaling protease 1 (XCP1) and one of xylem Cys protease 2 encoding pathways were differentially expressed genes in the finger lime. Comparatively, these genes were upregulated in response to CaLas infection in ‘Valencia’ sweet Hormone signaling is important in plant physiology and regulates orange. In contrast, one Xylem Serine Peptidase 1 gene many aspects from the transduction of messages through plants to (orange1.1g004503m) was highly upregulated in the infected adaptation to the environment to the timing offruit development and finger lime trees (Table 5). ripening. Out of the mapped genes, 2.6% (99 DEGs) were associated Additionally, the infected ‘Valencia’ sweet orange trees with hormone synthesis. Nine abscisic acid-induced genes were showed higher inhibition capacity of E64 compared with that identified. Three of these were Hva22-like protein-encoding DEGs of the finger lime trees and CaLas-free trees (Figure 5A). We (orange1.1g042117m, orange1.1g038094m, and orange1.1g030361m) selected some of Cys protease transcription factors for validation and were upregulated. Two Gram Domain-Containing Protein 2- via real-time PCR. We found that the relative expression of encodedDEGsweredownregulated.Abscisicaciddegradation-related genes encoding three transcription factors of Cys protease was DEGs were downregulated only in finger lime; these genes included downregulated following CaLas infection in finger lime epoxycarotenoid dioxygenase, aldehyde oxidase, and zeaxanthin compared with ‘Valencia’ sweet orange (Figuress 5B–D). oxidase. Auxin induced-regulated related DEGs were expressed to a higher degree in ‘Valencia’ sweet orange than in finger lime, with 25 out of the 34 DEGs upregulated in ‘Valencia’ sweet orange. The Genes involved in cellular development upregulated genes in finger lime included those encoding ILR1, TIR1, were differentially regulated DFL1, and GH3.1 proteins. Of the six brassinosteroid-related DEGs, cytochrome P450 (orange1.1g037705m) was upregulated, while the One hundred and one DEGs related to processes involved in cell STE1-, HYD1-, and SMT1-encoding genes were downregulated. wall synthesis and support were identified from our transcriptome There were 33 ethylene-related DEGs mapped, 17 of which data. Only 25 of these genes were upregulated in finger lime. Ten were upregulated, including Gibberellin 2-Oxidase, 2-Oxoglutarate, cellulose synthase-encoding genes were mapped, of which those Kar-Up Oxidoreductase 1, Integrase-Type DNA-Binding, GASA1 encoding D1, G2, and G3 cellulose synthases were found to be and SRG1. Seven jasmonate-related genes were also identified, upregulatedcomparedtothoseof ‘Valencia’ sweet orange. Five genes which were upregulated and included three of LOX2 and JAZ1 related to cellulase and 1,4-b-glucanase degradation, namely, gene. Finally, we identified ten salicylic acid (SA)-related genes, and orange1.1g042201m, orange1.1g036635m, orange1.1g041590m, three genes encoding methyltransferase were found to be orange1.1g010632m, and orange1.1g048736m, were upregulated in upregulated, namely, orange1.1g017363m, orange1.1g044676m, finger lime. Expansin A1 (orange1.1g025919m) and A20 and orange1.1g043411m in the infected finger lime. (orange1.1g025617m) were also upregulated in finger lime. Of the 17 PME-related genes identified by mapping, the PME inhibitor (orange1.1g010441m) was the only DEG upregulated in finger lime. Protease related genes were generally Additionally, six arabinogalactan protein (AGP)-related DEGs were downregulated in the finger lime identified,five of which were downregulated, while the gene encoding FLA3 protein (orange1.1g042255m) was upregulated. Among the DEGs mapped, 139 were associated with biotic proteolysis, accounting for 3.6% of all the mapped genes. Fifty-one of these genes, including orange1.1g044297m, which encodes a P-Loop qPCR validated the RNA-seq data containing Nucleoside Triphosphate Hydrolases Superfamily Protein, were upregulated in finger lime. However, the Aaa-type gene Aaa- To evaluate the accuracy of the RNA-seq data and the presence of Atpase 1, several Cytochrome Bc1 synthesis-related genes, aspartate technical artifacts or errors introduced during the RNA-seq library protease,autophagy-related DEGs,and ametalloprotease-related DEG preparations, the expression of several highly conserved genes and were downregulated. Of the 10 Cys protease-related DEGs that were genes encoding transcription factors were assayed through qPCR. mapped, only one of the Cys protease inhibitor-encoding genes Eight of the selected genes (orange1.1g025919m, orange1. Frontiers in Plant Science 12 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 TABLE 5 DEGs involved in cysteine protease production in finger lime and ‘Valencia’ sweet orange. Gene symbol* Log2 fold change Gene function Citrus sinensis ID Arabidopsis homolog FL (HLB+) Val (HLB+) XCP1 0 5.02 cysteine proteases orange1.1g048025m AT4G35350.1 XCP1 0.07 1.83 orange1.1g018781m AT4G35350.1 XCP1 0.08 17.44 orange1.1g019063m AT3G49340.1 XCP2 0.47 18.78 orange1.1g018649m AT1G20850.1 CP 0.03 12.78 orange1.1g032006m AT4G15880.1 CP 2.12 34.03 orange1.1g028661m AT1G50670.1 CP 0.05 16.56 orange1.1g019112m AT3G49340.1 XSP 1 98.82 10.01 xylem serine peptidase 1 orange1.1g004503m AT4G00230.1 *XCP1- xylem cysteine Protease 1, XCP2- xylem cysteine Protease 2 and CP- Cys protease. 1g004503m, orange1.1g025919m, orange1.1g000943m, several genes were downregulated (orange1.1g019200m, orange1.1g044721m, orange1.1g041335m, orange1.1g040540m, orange1.1g020892m, orange1.1g041918m, orange1.1g027498m, orange1.1g020713m, orange1.1g008415m) were upregulated in orange1.1g004923m, orange1.1g005031m, orange1.1g047288m, finger lime (Figure 6). However, the RNA-seq data revealed that orange1.1g008038m) (Figure 6). The qPCR results were consistent AB C D FIGURE 5 (A) Inhibition capacity of Finger lime and ‘Valencia’ T1- FL (HLB+) + substrate, T2- FL (HLB+) + substrate, T3- VAL (HLB+) + substrate + E64 and T4- Val (HLB+) + substrate + E64. The inhibition capacity was compared with the HLB negative leaves. Relative transcript levels of Cysteine proteinases superfamily protein transcription factors were calculated by real-time PCR and compared with the CaLas free ‘Valencia’. The CaLas infected trees were collected from five year old trees growing in the field and the CaLas free (control) samples here instead of trees were collected from trees kept in a protected greenhouse. The control trees were confirmed negative for CaLas before further comparison. Data are means ±SE of twelve samples. (B–D) Relative gene expression of selected cysteine proteases genes in the infected finger lime compared with infected 'Valencia'. Different letters above the error bar indicate statistically significant differences, while the same letters signify no significant differences using the Tukey-Kramer honestly significant difference test (Tukey HSD; p <0.05). Frontiers in Plant Science 13 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 with the RNA-seq data, and the mRNA expression of these genes was The relative transcript levels of CsPP2-B15 were highly either significantly up- or downregulated in the infected finger lime upregulated in the infected ‘Valencia’ sweet orange leaves compared with the infected ‘Valencia’ sweet orange. compared with the finger lime leaves (Figures 8D–F). Phloem and xylem morphological Discussion differences sheds light on finger lime HLB tolerance HLB is a devastating disease affecting citrus worldwide. Currently, many strategies have been explored for HLB We observed phloem and xylem morphological differences mitigation, including application of antimicrobials, macro, in the CaLas– and CaLas+ petioles of finger lime and ‘Valencia’ micronutrients and plant defense inducers, control of insect sweet orange (Figures 7A, B). Because phloem and xylem vectors, thermotherapy, biocontrol, and eradication of HLB thickness are dependent on tissue type and age, we calculated symptomatic citrus trees (Yang and Ancona, 2022). However, a relative phloem thickness (RPT) by dividing the average these strategies have shown limited success in field applications, phloem thickness by the average xylem thickness within the and effective long term HLB management remains a challenge. same sample. There were significant differences between healthy Thus, breeding for HLB tolerance may provide the most effective and HLB-infected finger lime petioles (p value = 0.0001) and and sustainable solution to combat HLB (Bové, 2006). In the between healthy and HLB-infected stems (p value = 0.0044) field, CaLas-infected ‘Valencia’ sweet orange trees display (Figure 7D). CaLas-infected finger lime had a significantly symptoms of stunted growth, yellow shoots, and blotchy higher RPT compared to healthy finger lime. This was also mottled leaves while adjacent finger lime grow normally and true for ‘Valencia’ sweet orange petioles (p value =0.0385) produce fruits without any apparent visible HLB symptoms (Figure 7F). The xylem/phloem ratio (Xa/Pa) showed (Figures 1A, B). Diaphorina citri,the vector transmitting significant difference in finger lime in both petioles (p value = CaLas has a feeding preference for certain citrus cultivars. 0.0006) and stems (p value = 0.0038) (Figure 7C), however, there ‘Valencia’ sweet orange is considered a preferred host while was slight differences in Xa/Pa in ‘Valencia’ petioles (p value = finger limes are considered not suitable (Felisberto et al., 2019). 0.0427) and no significant difference in the stems (Figure 7E). Poor Diaphorina citri colonization levels observed on finger lime Together, our results suggest that phloem may regenerate in trees in a recent long term field study confirms these finger lime in response to HLB infection. observations (Ramadugu et al., 2016). Under controlled conditions, finger limes trees do get infected following budding with CaLas-infected budwood (Alves et al., 2020). Quantification of phloem callose However, the mechanism for this perceived tolerance to CaLas deposits indicated differential in the host is unknown and our study provides insights into the callose deposition tolerance mechanism. Finger limes are monoembryonic and open pollinated seedlings can vary in their tolerance to HLB We found increased accumulation of callose in the infected (Ramadugu et al., 2016). Finger lime trees (clone DPI 50-36) ‘Valencia’ sweet orange compared with the infected finger lime. have remained HLB negative under field conditions for more Analysis of the callose formation counts revealed a significant than a decade. This study was designed to understand this difference in the percentage of images with few callose plugs in the perceived tolerance by graft inoculating budded trees of the stem phloem of CaLas+ ‘Valencia’ sweet orange (0%) and finger same clone under controlled greenhouse conditions. lime (26%); Table 6. Among the images of phloem that did contain The possible genetic mechanisms of the symptoms of CaLas callose formations, ‘Valencia’ sweet orange had significantly higher in citrus trees have been previously discussed by several groups mean counts of formations per image (Figures 8A–C). (Albrecht and Bowman, 2008; Kim et al., 2009; Rawat et al., 2015; Martinelli and Dandekar, 2017; Curtolo et al., 2020; Ma et al., 2022). Nehela and Killiny (2020) summarized three main Phloem protein genes are potential mechanisms during CaLas infection: (I) disorder of downregulated in finger limes carbohydrate metabolism affecting the flow of nutrients and source–sink disruption due to starch accumulation in leaves; (II) Some of the transcripts encoding for phloem proteins in the phytohormones alteration in response to stress; and (III) samples were analyzed via qPCR. CsPP2-B1 transcripts were activation of detoxification proteins, particularly glutathione-S- detected in both finger lime and ‘Valencia’ but were not transferases (GSTs) and modulation of antioxidant pathways. Understanding the virulence mechanisms employed by statistically significant when the uninfected and infected trees were compared. CsPP2-B13 transcripts were almost undetectable CaLas against the host is an important step to identify an in finger lime samples, while they were upregulated in ‘Valencia’. approach to increase plant defense. Insect-transmitted bacteria Frontiers in Plant Science 14 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 FIGURE 6 Verification of expression levels of selected upregulated (A) or downregulated (B) DEGs in the infected Finger lime (FL-HLB) compared to infected ‘Valencia’ (Val-HLB) as determined by qPCR (2-DDCt). Different letters (a, b) represent a significant difference at p ≤ 0.05 using Tukey– Kramer honestly significant difference (HSD) and error bars represent SE (n = 3). Pearson Correlation Coefficient (r)> greater than 0.5 is considered positive and strong. such as CaLas utilize the general Sec secretion system to release developmental processes within the host to benefitthe effectors (Sugio et al., 2011). However, the mechanism of the pathogen (Jones and Dangl, 2006). Previous research has CaLas effectors secretion remains poorly understood. Protein shown that CaLas encodes several SDEs, many of which are effectors often suppress plant defense or manipulate conserved across CaLas isolates and of which were found to be Frontiers in Plant Science 15 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 AB C D EF FIGURE 7 Morphological differences between healthy and HLB-infected Finger lime and ‘Valencia’. Brightfield images of petiole and stems for each healthy and HLB-infected cultivar, Finger lime (A), ‘Valencia’ (B). Phloem Ratio (C) and Xylem Ratio (D) in Finger lime and Phloem Ratio (E) and Xylem Ratio (F) in ‘Valencia’. Bars represent standard error. NS, not significant. highly present in infected citrus tissue at a relatively early TABLE 6 Percent of images with zero callose plugs per 10x field image of the stem phloem of CaLas+ ‘Valencia’ sweet orange and infection stage (Pagliaccia et al., 2017; Tran et al., 2020). finger lime. Several studies have demonstrated that the activity of leucine-rich repeat proteins (LRR-RLKs) serves as an early Plant type Images with zero callose plugs warning system for detecting the presence of potential Finger lime 26%* pathogens and activating protective immune-related signaling Valencia 0% in plants (Matsushima and Miyashita, 2012). Interestingly, receptor kinases, LRRs and cysteine (Cys)-rich receptor-like *p> value <0.001. Frontiers in Plant Science 16 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 FIGURE 8 Non-zero counts of callose plugs per 10x field image of the stem phloem of CaLas+ Finger lime (A) and ‘Valencia’ sweet orange (B) sampled after 2 years following infection. The mean callose formation count per 10x field image was significant. (C) Relative transcript levels of phloem proteins are calculated by real-time PCR and compared with the CaLas free ‘Valencia’. The CaLas infected samples were collected from five year old trees growing in the field and the CaLas free (control) samples were collected from trees kept in a protected greenhouse. The control trees were confirmed negative for CaLas before further comparison. Data are means ± SE of twelve samples (D–F). Different letters above the error bar indicate statistically significant differences, while the same letters signify no significant differences using the Tukey-Kramer honestly significant difference test (Tukey HSD; p <0.05). protein kinases (CRKs) were found to be upregulated in HLB would not interact with the external receptors of the plant cell. infected finger lime. Our findings are similar to those reported Thus, it has little or no interaction with PTI mechanisms. In by Peng et al. (2020), where the involvement of multiple Arabidopsis, most CRKs are differentially regulated by SA, ROS, nucleotide-binding site-containing and LRR-encoding genes in and pathogen infection (Czernic et al., 1999; Du and Chen, 2000; the HLB tolerance/resistance process in Poncirus trifoliata was Ohtake et al., 2000; Chen et al., 2003; Zhang et al., 2013). Curtolo reported. CRKs are characterized by the presence of one to four et al. (2020) reported that the CaLas defense mechanisms were copies of Domain of Unknown Function 26 (DUF26) and a C– controlled by a class of receptor-related genes and the induction X8–C–X2–C motif in the extracellular receptor region at the N- of WRKY transcription factors. These observations suggest that terminus (Mou et al., 2021). These conserved Cys residues might CRKs could play a vital role in the regulatory network regulating be required to form the three-dimensional structure of the the finger lime response to CaLas infection, suggesting that protein through disulfide bonds (Chen, 2001) and can mediate members of the CRK gene family can be effective targets for protein–protein interactions (Rayapuram et al., 2012). Several the improvement of citrus tolerance to HLB disease. CRKs have been functionally characterized in response to Recently, Ma et al. (2022) reported that Huanglongbing pathogen infection. We detected overexpression of CsCRK10, (HLB) could by controlled by ROS detoxification via induction CsCRK16, CsCRK25 and CsCRK34 in the infected finger lime. of antioxidant pathways and plant growth hormones Functional analysis of the RLKs in CaLas infected plants would (particularly GA). Additionally, the involvement of GA be difficult because CaLas is an intracellular bacterium and signaling in HLB resistance has also been reported by Rawat inoculated directly by the psyllids into the phloem tissues and et al. (2015) and Curtolo et al. (2020). Curtolo et al. (2020) Frontiers in Plant Science 17 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 suggested that the genetic mechanism of HLB tolerance was HLB infection induced the differential expression of multiple associated with the downregulation of gibberellin (GA) synthesis genes encoding enzymes and proteins involved in the synthesis, and cell wall strengthening. In the current study, we reported the assembly, and modification of the cell wall. Overall, many genes upregulation of several candidate genes responsible for involved in cellulose synthesis and 1,4-b-glucan degradation as controlling GA synthesis and production of bioactive GA in well as those encoding cellulase glycosyl hydrolases, the infected finger lime such as gibberellin 2-oxidase 8 polygalacturonases and fasciclin-like AGPs were upregulated in (GA2ox8), gibberellin 3-oxidase 1 (GA3ox1) and cysteine-rich the infected finger lime. Several other genes involved in cellulose protein or GA-stimulated transcript (GAST1 protein homolog synthesis, such as cellulose synthase D1, G2 and G3, were also 4). Additionally, our data showed that CaLas induced ROS upregulated in CaLas-infected finger lime. CaLas infection in detoxification pathways and activated glutathione-S- ‘Valencia’ sweet orange resulted in the repression of genes transferases (GSTs), catalase and thioredoxin in finger lime. encoding cellulose synthase-like D4 (CSLD4) and CSLC7 that Glutathione-S-transferases was suggested to be as an important mediate synthesis of b-1,4 linkages in the hemicellulose backbones modulator of citrus tolerance to HLB disease (Martinelli and (Aritua et al., 2013). In thephloem, thesieve platepores Dandekar, 2017). We also recorded the overexpression of several accumulate callose, a b-1-3 glucan, and starch, which restrict plant growth regulator-related-genes that may have direct or symplastic transport, in response to CaLas infection (Koh et al., indirect function in ROS mitigation (Figure 9). 2012; Welker et al., 2022). Callose deposition and phloem protein Plants accumulate inducible defense-related proteins in (PP) plugging of the sieve tubes are defensive measures to form response to biotic and abiotic stress. Antimicrobial peptides physical barriers that prevent the spread of pathogens (Musetti (AMPs) and other Cys-rich peptides are major components of et al., 2010). Excessive callose deposition is considered the main innate immunity in various groups of organisms, including reason for phloem blockage in HLB disease. This blockage limits insects, mammals and plants (Pushpanathan et al., 2013; thetransferoforganic compoundsfrom the sitesof Slavokhotova et al., 2017). Some eukaryotic AMPs are largely photosynthesis (source) to where photosynthate is stored (sink), Cys-rich peptides, which are defined as defensins (Baindara which affects plant growth and mimics the symptoms of a nutrient et al., 2017). A potent AMP has recently been identified from disorder. We observed decreased callose accumulation in finger finger lime (Huang et al., 2021b). A Cys-rich secretory protein/ lime phloem tissue. Previous research revealed that some phloem PR1-like protein was also found to be abundant in finger lime proteins play an important role in callose deposition (Wen et al., and in HLB-tolerant Australian desert lime (C. glauca; 2018). When the gene expression of HLB infected and healthy Ramadugu et al. (2016)). We also identified a low-molecular- controls was compared, PP2 genes were found to be upregulated weight defensin (Cys-rich 69: LCR69) in this study that is highly in the infected trees (Musetti et al., 2010; Ghosh et al., 2018). In the upregulated in finger lime trees. Defensins usually contain six to comparison between finger lime and ‘Valencia’ sweet orange, we eight conserved Cys residues and thus are referred to as Cys-rich found that phloem proteins were significantly induced by CaLas peptides (CRPs) (Silverstein et al., 2007). infection in ‘Valencia’ sweet orange, while they were repressed in Cell wall integrity sensing is a mechanism by which plants finger lime. Specifically, CsPP2-B15 was found to be highly can be induced to mitigate biotic stress. Various strategies upregulated in the infected ‘Valencia’ sweet orange compared to involving resistance of the cell wall against plant pathogens the finger lime. These findings agree with those of Wen et al. have evolved, such as the remodeling of the cell wall and (2018), who found that CsPP2-B15 expression was upregulated in alterations to cell wall-associated protein distribution and infected leaves of Jincheng orange (C. sinensis Osbeck) and accumulation (Leszczuk et al., 2019). Both cell wall synthesis downregulated in the HLB tolerant sour pummelo (C. grandis and proteins associated with the cell wall have roles in structural Osbeck). Similarly, CsPP2-15 was also found to be downregulated support, and the presence or absence of these proteins may in the tolerant C. ichangensis (Wu et al., 2020). This is in addition indicate disease susceptibility or infection. The susceptibility of to our observations that the ‘Valencia’ sweet orange phloem ‘Valencia’ trees to CaLas infection resulted in a decreased ability produces more callose. It should be noted that the enhanced to synthesize new cellular components, whereas the finger lime production of callose and phloem proteins in the susceptible plants was not affected in the same manner and thus could continue is purely a mechanical response: it chokes off the transport of with cell metabolism and growth. These findings are agreed with nutrients and other vital molecules and at the same time, does not Fan et al. (2012) who compared resistant rough lemon and prevent the spread of CaLas. Killiny et al. (2022) suggested that the susceptible sweet orange following CaLas infection. They increased ROS could be one of the factors affecting callose reported that cell wall-related pathways were upregulated in deposition as reported for CaLas-infected citrus trees. rough lemon during the late stage of infection, while there was The vascular cambium increases stem diameter via periclinal downregulation in the susceptible sweet orange. Thus, the rough divisions and the circumference by anticlinal divisions, resulting in lemon trees generated healthy new growth following infection the development of secondary phloem and xylem (Chaffey, 1999). despite older leaves having some blotchy mottle, while the Proteolytic enzymes, including serine, Cysteine and threonine growth was inhibited in sweet orange trees. proteases, have been implicated in the regulation of vascular Frontiers in Plant Science 18 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 FIGURE 9 Schematic diagram elucidating the potential mechanism of CaLas tolerance in the Finger lime, and susceptibility in ‘Valencia’ sweet orange. The figure was created in BioRender.com. Frontiers in Plant Science 19 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 tissue differentiation. Beers and Zhao (2001) screened seven Conclusions protease genes, including members of the serine, Cysteine, and aspartic acid protease families, and reported that the expression of To date, there are no effective practical strategies to mitigate three genes (XCP1, XCP2, and XSP1) was xylem specific. XCP1 and citrus greening disease (HLB). Understanding the mechanisms XCP2 are predicted to encode papain-like Cys proteases, and XSP1 against CaLas can contribute to the development of effective is predicted to encode a subtilisin-like SP. Clark et al. (2018) approaches for combatting HLB. This report provides insights evaluated six candidates of Cys protease and reported that the C. into different mechanisms of HLB tolerance in finger lime, an sinensis protein annotated as “xylem Cys protease 1”, a member of HLB tolerant citrus species. Finger lime trees protect themselves the PLCP family, was confirmed by yeast two-hybrid (Y2H) assays from CaLas infection and disease symptoms through the control of as interacting with CaLas effectors (SDE1). Papain-like Cys ROS-overproduction by enhancing redox control factors such as proteases are important regulators involved in numerous plant glutathione-S-transferases (GSTs), catalase, thioredoxin and growth biological processes, including programmed cell death (PCD) and hormones to mitigate HLB symptoms. Primary recognition of ROS leaf senescence. It has been demonstrated that activation of cysteine leads to stronger activation of defense responses against CaLas proteases induced programmed cell death (PCD) pathway of plant infection. The over-accumulation of Cys proteases and their cells (Minami and Fukuda, 1995; Ye and Varner, 1996). inhibitors in ‘Valencia’ linked with the degradation of citrus Particularly, two xylem-specificPLCPs (AtXCP1 and AtXCP2) defense proteins such as the cysteine rich proteins. Finger lime were found to be expressed at a high level in xylem during the did not exhibit any imbalance in the expression of the Cys proteases PCD process (Liu et al., 2018). We did not find any significant and their inhibitors. Several defense-related-factors were expression of many of the xylem Cys protease 1 genes in our finger upregulated in the finger lime such as R-proteins, Cys-rich lime transcriptome data; however, these genes were upregulated in secretory proteins or PR proteins and hormone signaling-related the infected ‘Valencia’ sweet orange. Cys proteases and their genes. Additionally, we observed that ‘Valencia’ sweet orange inhibitors were highly expressed in ‘Valencia’ sweet orange field phloem constitutively produced more callose and expressed more samples. Under stress conditions, the activity of PLCPs and their phloem proteins than finger lime following infection. Taken regulatory factors can be disrupted, resulting in unknown together, this study provides evidence that HLB can be managed consequences at the cellular level (Liu et al., 2018). We suggest by mitigating ROS and control of the over-accumulation of cysteine that the overexpression of proteases and inhibitors in ‘Valencia’ protease related genes that induce cell death (Figure 9). sweet orange reflects an imbalance in metabolism. CaLas infection can alter the function of the transcription factors responsible for the Cys proteases, which led to an abundance of Cys proteases in Data availability statement ‘Valencia’ cells. To maintain a relatively balanced intercellular level of proteases, the activities of the inhibitors that can directly control The datasets presented in this study can be found in online Cys protease activity are also induced in the cells. The inhibition repositories. The names of the repository/repositories and ratio of Cys proteases revealed the overproduction of Cys proteases accession number(s) can be found below: https://www.ncbi. in ‘Valencia’ sweet orange leaves. Yu and Killiny, (2018) reported nlm.nih.gov/, PRJNA755969. the presence of proteases and endoglucanase in the saliva of D. citri, potentially linked with the ability of the insects to degrade/inhibit citrus defense proteins. We hypothesized that the overaccumulation Author Contributions of cysteine proteases causes degradation of defense proteins and/or induces PCD and are linked to the enhanced susceptibility of KW, LM, DS and MD - wrote the manuscript. LM - gene expression analysis. KW - transcriptome data analysis. DS – ‘Valencia’ trees to CaLas. SPs appear to be widespread in the xylem sap of different species (Buhtz et al., 2004). SPs have Microscopy. SW – callose density estimation. LM and WQ – RNA extraction. LM - statistical analysis. AL and JG – resources. various functions in plant cells, including functions in the pathogen response (Tornero et al., 1997; Jordá et al., 1999). MD – designed the study, obtained funding, and supervised the project. All authors contributed to the article and approved the Specific citrus SPs were detected, suggesting that protease enzymatic activity is finely tuned during CaLas infection (Franco submitted version. et al., 2020). The overexpression of XSP1 in finger lime suggests that there is specific mechanism underlying the defense against CaLas infection in finger lime tissues. Since the role of several genes Funding reported in this study have not been elucidated before, further comprehensive studies are needed to understand in detail their role This work was supported by the Florida State legislative in the finger lime HLB defense pathways. funding for the UF/IFAS Citrus Initiative. Frontiers in Plant Science 20 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 Acknowledgments Publisher’s note We thank E. Nielsen, C. Hardy, H Rubio, J. Thomson and K. All claims expressed in this article are solely those of the Plant for aiding in various aspects of the work. Microscopy work authors and do not necessarily represent those of their affiliated was carried out at the Microscopy Core Facility of the Citrus organizations, or those of the publisher, the editors and the Research and Education Center. We thank Novogene reviewers. Any product that may be evaluated in this article, or Corporation Inc. for library construction and RNA sequencing. claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Supplementary material Conflict of interest The Supplementary Material for this article can be found The authors declare that the research was conducted in the online at: https://www.frontiersin.org/articles/10.3389/ absence of any commercial or financial relationships that could fpls.2022.1019295/full#supplementary-material be construed as a potential conflict of interest. References Albrecht, U., and Bowman, K. D. (2008). Gene expression in Citrus sinensis (L.) kinase in Arabidopsis. Plant Mol. Biol. 53, 61–74. doi: 10.1023/b: osbeck following infection with the bacterial pathogen candidatus liberibacter plan.0000009265.72567.58 asiaticus causing huanglongbing in Florida. Plant Sci. 175, 291–306. doi: 10.1016/ Clark, K., Franco, J. Y., Schwizer, S., Pang, Z., Hawara, E., Liebrand, T. W. H., j.plantsci.2008.05.001 et al. (2018). An effector from the huanglongbing-associated pathogen targets citrus Alves, M. N., Lopes, S. A., Raiol-Junior, L. L., Wulff, N. A., Girardi, E. A., proteases. Nat. Commun. 9, 1718. doi: 10.1038/s41467-018-04140-9 Ollitrault, P., et al. (2020). Resistance to 'Candidatus liberibacter asiaticus,' the Curtolo, M., de Souza Pacheco, I., Boava, L. P., Takita, M. A., Granato, L. M., huanglongbing associated bacterium, in sexually and/or graft-compatible citrus Galdeano, D. M., et al. (2020). Wide-ranging transcriptomic analysis of poncirus relatives. Front. Plant Sci. 11. doi: 10.3389/fpls.2020.617664 trifoliata, citrus sunki, citrus sinensis and contrasting hybrids reveals HLB tolerance Andrews, S. (2010) FASTQC. a quality control tool for high throughput sequence mechanisms. Sci. Rep. 10, 1–14. doi: 10.1038/s41598-020-77840-2 data. Available at: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ Czernic, P., Visser, B., Sun, W., Savouré, A., Deslandes, L., Marco, Y., et al. (Accessed 6 December 2021). (1999). Characterization of an arabidopsis thaliana receptor-like protein kinase Aritua, V., Achor, D., Gmitter, F. G., Albrigo, G., and Wang, N. (2013). gene activated by oxidative stress and pathogen attack. Plant J. 18, 321–327. doi: Transcriptional and microscopic analyses of citrus stem and root responses to 10.1046/j.1365-313X.1999.00447.x Candidatus liberibacter asiaticus infection. PloS One 8, e73742. doi: 10.1371/ Deng, B., Wang, W., Deng, L., Yao, S., Ming, J., and Zeng, K. (2018). journal.pone.0073742 Comparative RNA-seq analysis of citrus fruit in response to infection with three Baindara, P., Kapoor, A., Korpole, S., and Grover, V. (2017). Cysteine-rich low major postharvest fungi. Postharvest Biol. Technol. 146, 134–146. doi: 10.1016/ molecular weight antimicrobial peptides from brevibacillus and related genera for j.postharvbio.2018.08.012 biotechnological applications. World J. Microbiol. Biotechnol. 33, 124. doi: 10.1007/ Dobin, A., Davis, C. A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., et al. s11274-017-2291-9 (2013). STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21. Barrett, A. J. (1980). Fluorimetric assays for cathepsin b and cathepsin h with doi: 10.1093/bioinformatics/bts635 methylcoumarylamide substrates. Biochem. J. 187, 909–912. doi: 10.1042/ Dou, D., and Zhou, J. M. (2012). Phytopathogen effectors subverting host bj1870909 immunity: different foes, similar battleground. Cell Host Microbe 12, 484–495. Beers, E. P., and Zhao, C. (2001). “Arabidopsis as a model for investigating gene doi: 10.1016/j.chom.2012.09.003 activity and function in vascular tissues,” in Progress in biotechnology. Eds. N. Duan, Y., Zhou, L., Hall, D. G., Li, W., Doddapaneni, H., Lin, H., et al. (2009). Morohoshi and A. Komamine (Amsterdam: Elsevier), 43–52. Complete genome sequence of citrus huanglongbing bacterium, ‘Candidatus Benavente-Garcıa,́ O., and Castillo, J. (2008). Update on uses and properties of liberibacter asiaticus’ obtained through metagenomics. Mol. Plant-Microbe citrus flavonoids: new findings in anticancer, cardiovascular, and anti- Interact. 22, 1011–1020. doi: 10.1094/mpmi-22-8-1011 inflammatory activity. J. Agric. Food Chem. 56, 6185–6205. doi: 10.1021/jf8006568 Du, L., and Chen, Z. (2000). Identification of genes encoding receptor-like Benjamini, Y., Krieger, A. M., and Yekutieli, D. (2006). Adaptive linear step-up protein kinases as possible targets of pathogen-and salicylic acid-induced WRKY procedures that control the false discovery rate. Biometrika 93, 491–507. DNA-binding proteins in arabidopsis. Plant J. 24, 837–847. doi: 10.1046/j.1365- doi: 10.1093/biomet/93.3.491 313x.2000.00923.x Bolger, A. M., Lohse, M., and Usadel, B. (2014). Trimmomatic: a flexible Fan, J., Chen, C., Yu, Q., Khalaf, A., Achor, D. S., Brlansky, R. H., et al. (2012). trimmer for illumina sequence data. Bioinformatics 30, 2114–2120. doi: 10.1093/ Comparative transcriptional and anatomical analyses of tolerant rough lemon and bioinformatics/btu170 susceptible sweet orange in response to ‘Candidatus liberibacter asiaticus’ infection. Mol. Plant-Microbe Interact. 25, 1396–1407. doi: 10.1094/MPMI-06-12-0150-R Bové, J. M. (2006). Huanglongbing: a destructive, newly-emerging, century-old disease of citrus. J. Plant Pathol. 88, 7–37. Felisberto, P. A., Girardi, E. A., Peña, L., Felisberto, G., Beattie, G. A. C., and Lopes, S. A. (2019). Unsuitability of indigenous south American rutaceae as Buhtz, A., Kolasa, A., Arlt, K., Walz, C., and Kehr, J. (2004). Xylem sap protein potential hosts of Diaphorina citri. Pest Manage. Sci. 75, 1911–1920. composition is conserved among different plant species. Planta 219, 610–618. doi: 10.1002/ps.5304 doi: 10.1007/s00425-004-1259-9 Feng, F., and Zhou, J. M. (2012). Plant–bacterial pathogen interactions mediated Chaffey, N. (1999). Wood formation in forest trees: from Arabidopsis to Zinnia. by type III effectors. Curr. Opin. Plant Biol. 15, 469–476. doi: 10.1016/ Trends Plant Sci. 4, 203–204. doi: 10.1016/s1360-1385(99)01417-x j.pbi.2012.03.004 Chen, Z. (2001). A superfamily of proteins with novel cysteine-rich repeats. Ferguson, K., da Cruz, M. A., Ferrarezi, R., Dorado, C., Bai, J., and Cameron, R. Plant Physiol. 126, 473–476. doi: 10.1104/pp.126.2.473 G. (2021). Impact of huanglongbing (HLB) on grapefruit pectin yield and quality Chen, K., Du, L., and Chen, Z. (2003). Sensitization of defense responses and during grapefruit maturation. Food Hydrocoll. 113, 106553. doi: 10.1016/ activation of programmed cell death by a pathogen-induced receptor-like protein j.foodhyd.2020.106553 Frontiers in Plant Science 21 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 Ferrara, T., Schneider, V. K., Kishi, L. T., Carmona, A. K., Alves, M. F. M., Lindgreen, S. (2012). AdapterRemoval: easy cleaning of next-generation Belasque-Júnior, J., et al. (2015). Characterization of a recombinant cathepsin b- sequencing reads. BMC Res. Notes 5, 337. doi: 10.1186/1756-0500-5-337 like cysteine peptidase from Diaphorina citri kuwayama (Hemiptera: Liviidae): a Liu, H., Hu, M., Wang, Q., Cheng, L., and Zhang, Z. (2018). Role of papain-like putative target for control of citrus huanglongbing. PloS One 10, e0145132. cysteine proteases in plant development. Front. Plant Sci. 9. doi: 10.3389/ doi: 10.1371/journal.pone.0145132 fpls.2018.01717 Folimonova, S. Y., Robertson, C. J., Garnsey, S. M., Gowda, S., and Dawson, W. Livak, K. J., and Schmittgen, T. D. (2001). Analysis of relative gene expression O. (2009). Examination of the responses of different genotypes of citrus to data using real-time quantitative PCR and the 2–DDCT method. Methods 25, 402– huanglongbing (citrus greening) under different conditions. Phytopathology 99, 408. doi: 10.1006/meth.2001.1262 1346–1354. doi: 10.1094/phyto-99-12-1346 Love, M. I., Huber, W., and Anders, S. (2014). Moderated estimation of fold Forster, P. I., and Smith, M. W. (2010). Citrus wakonai PI forst. & MW change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550. Sm.(Rutaceae), a new species from goodenough island, Papua new Guinea. doi: 10.1186/s13059-014-0550-8 Austrobaileya 8, 133–138. Ma, W., Pang, Z., Huang, X., Xu, J., Pandey, S. S., Li, J., et al. (2022). Citrus Franco, J. Y., Thapa, S. P., Pang, Z., Gurung, F. B., Liebrand, T. W. H., Stevens, huanglongbing is a pathogen-triggered immune disease that can be mitigated with D. M., et al. (2020). Citrus vascular proteomics highlights the role of peroxidases antioxidants and gibberellin. Nat. Commun. 13, 1–13. doi: 10.1038/s41467-022- and serine proteases during huanglongbing disease progression. Mol. Cell. Proteom. 28189-9 19, 1936–1952. doi: 10.1074/mcp.RA120.002075 Marın,́ F. R., Soler-Rivas, C., Benavente-Garcıa,́ O., Castillo, J., and Pérez- Ghosh, D., Motghare, M., and Gowda, S. (2018). Citrus greening: overview of the Alvarez, J. A. (2007). By-products from different citrus processes as a source of most severe disease of citrus. Adv. Agric. Res. Technol. J. 2, 83–100. customized functional fibres. Food Chem. 100, 736–741. doi: 10.1016/ Graham, J., Gottwald, T., and Setamou, M. (2020). Status of huanglongbing j.foodchem.2005.04.040 (HLB) outbreaks in Florida, California and Texas. Trop. Plant Pathol. 45, 265–278. Martinelli, F., and Dandekar, A. M. (2017). Genetic mechanisms of the devious doi: 10.1007/s40858-020-00335-y intruder candidatus liberibacter in citrus. Front. Plant Sci. 8, 904. doi: 10.3389/ Halbert, S. (2005). Citrus greening/Huanglongbingx (Gainesville, Florida: Pest fpls.2017.00904 Alert, Florida Department of Agriculture and Consumer Services, Division of Plant Matsushima, N., and Miyashita, H. (2012). Leucine-rich repeat (LRR) domains Industry). containing intervening motifs in plants. Biomolecules 2, 288–311. doi: 10.3390/ Huang, C. Y., Araujo, K., Sánchez, J. N., Kund, G., Trumble, J., Roper, C., et al. biom2020288 (2021b). A stable antimicrobial peptide with dual functions of treating and Mattos-Jr, D., Kadyampakeni, D. M., da Silva, J. R., Vashisth, T., and Boaretto, R. preventing citrus huanglongbing. Proc. Natl. Acad. Sci. U.S.A. 118, e2019628118. M. (2020). Reciprocal effects of huanglongbing infection and nutritional status of doi: 10.1073/pnas.2019628118 citrus trees: a review. Trop. Plant Pathol. 45, 586–596. doi: 10.1007/s40858-020- Huang, C. Y., Niu, D., Kund, G., Jones, M., Albrecht, U., Nguyen, L., et al. 00389-y (2021a). Identification of citrus immune regulators involved in defence against Miles, G. P., Stover, E., Ramadugu, C., Keremane, M. L., and Lee, R. F. (2017). huanglongbing using a new functional screening system. Plant Biotechnol. J. 19, Apparent tolerance to huanglongbing in citrus and citrus-related germplasm. 757–766. doi: 10.1111/pbi.13502 HortScience 52, 31–39. doi: 10.21273/hortsci11374-16 Hu, Y., Zhong, X., Liu, X., Lou, B., Zhou, C., and Wang, X. (2017). Comparative Milne, A. E., Teiken, C., Deledalle, F., van den Bosch, F., Gottwald, T., and transcriptome analysis unveils the tolerance mechanisms of Citrus hystrix in McRoberts, N. (2018). Growers' risk perception and trust in control options for response to 'Candidatus liberibacter asiaticus' infection. PloS One 12, e0189229. huanglongbing citrus-disease in Florida and California. Crop Prot. 114, 177–186. doi: 10.1371/journal.pone.0189229 doi: 10.1016/j.cropro.2018.08.028 Jones, J. D. G., and Dangl, J. L. (2006). The plant immune system. Nature 444, Minami, A., and Fukuda, H. (1995). Transient and specific expression of a 323–329. doi: 10.1038/nature05286 cysteine endopeptidase associated with autolysis during differentiation of zinnia Jordá, L., Coego, A., Conejero, V., and Vera, P. (1999). A genomic cluster mesophyll cells into tracheary elements. Plant Cell Physiol. 36, 1599–1606. containing four differentially regulated subtilisin-like processing protease genes is doi: 10.1093/oxfordjournals.pcp.a078926 in tomato plants. J. Biol. Chem. 274, 2360–2365. doi: 10.1074/jbc.274.4.2360 Mou, S., Meng, Q., Gao, F., Zhang, T., He, W., Guan, D., et al. (2021). A cysteine- Jose,J., Ghantasala,S., andRoy Choudhury, S. (2020).Arabidopsis rich receptor-like protein kinase CaCKR5 modulates immune response against transmembrane receptor-like kinases (RLKs): a bridge between extracellular Ralstonia solanacearum infection in pepper. BMC Plant Biol. 21, 382. doi: 10.1186/ signal and intracellular regulatory machinery. Int. J. Mol. Sci. 21, 4000. s12870-021-03150-y doi: 10.3390/ijms21114000 Musetti, R., Paolacci, A., Ciaffi, M., Tanzarella, O. A., Polizzotto, R., Tubaro, F., Killiny, N., Jones, S. E., and Gonzalez-Blanco, P. (2022). Silencing of d- et al. (2010). Phloem cytochemical modification and gene expression following the aminolevulinic acid dehydratase via virus induced gene silencing promotes recovery of apple plants from apple proliferation disease. Phytopathology 100, 390– callose deposition in plant phloem. Plant Signaling Behav., 1:2024733. doi: 399. doi: 10.1094/phyto-100-4-0390 10.1080/15592324.2021.2024733 Nehela, Y., and Killiny, N. (2020). Revisiting the complex pathosystem of Killiny, N., Jones, S. E., Nehela, Y., Hijaz, F., Dutt, M., Gmitter, F. G., et al. huanglongbing: Deciphering the role of citrus metabolites in symptom (2018). All roads lead to Rome: Towards understanding different avenues of development. Metabolites 10, 409. doi: 10.3390/metabo10100409 tolerance to huanglongbing in citrus cultivars. Plant Physiol. Biochem. 129, 1–10. Ngou, B. P. M., Ahn, H. K., Ding, P., and Jones, J. D. G. (2021). Mutual doi: 10.1016/j.plaphy.2018.05.005 potentiation of plant immunity by cell-surface and intracellular receptors. Nature Kim, J.-S., Sagaram, U. S., Burns, J. K., Li, J. L., and Wang, N. (2009). Response of 592, 110–115. doi: 10.1038/s41586-021-03315-7 sweet orange (Citrus sinensis)to ‘Candidatus liberibacter asiaticus’ infection: Ohtake, Y., Takahashi, T., and Komeda, Y. (2000). Salicylic acid induces the microscopy and microarray analyses. Phytopathology 99, 50–57. doi: 10.1094/ expression of a number of receptor-like kinase genes in arabidopsis thaliana. Plant PHYTO-99-1-0050 Cell Physiol. 41, 1038–1044. doi: 10.1093/pcp/pcd028 Koh, E. J., Zhou, L., Williams, D. S., Park, J., Ding, N., Duan, Y. P., et al. (2012). Pagliaccia, D., Shi, J., Pang, Z., Hawara, E., Clark, K., Thapa, S. P., et al. (2017). A Callose deposition in the phloem plasmodesmata and inhibition of phloem pathogen secreted protein as a detection marker for citrus huanglongbing. Front. transportincitrusleavesinfected with “Candidatus liberibacter asiaticus”. Microbiol. 8. doi: 10.3389/fmicb.2017.02041 Protoplasma 249, 687–697. doi: 10.1007/s00709-011-0312-3 Peng, Z., Bredeson, J. V., Wu, G. A., Shu, S., Rawat, N., Du, D., et al. (2020). A Kramer, J., Simnitt, S., and Weber, C. (2022) Fruit and tree nuts outlook chromosome-scale reference genome of trifoliate orange (Poncirus trifoliata) (Washington, DC: USDA ERS). Available at: https://www.ers.usda.gov/webdocs/ provides insights into disease resistance, cold tolerance and genome evolution in outlooks/103650/fts-374.pdf (Accessed 30 June 2022). Citrus. Plant J. 104, 1215–1232. doi: 10.1111/tpj.14993 Leszczuk, A., Koziol, A., Szczuka, E., and Zdunek, A. (2019). Analysis of AGP Pushpanathan, M., Gunasekaran, P., and Rajendhran, J. (2013). Antimicrobial contribution to the dynamic assembly and mechanical properties of cell wall during peptides: versatile biological properties, Antimicrobial peptides: versatile Biol. pollen tube growth. Plant Sci 281, 9–18. properties. Int. J. Pept. 2013, 675391. doi: 10.1155/2013/675391 Liao, Y., Smyth, G. K., and Shi, W. (2014). featureCounts: an efficient general Qiu, W., Soares, J., Pang, Z., Huang, Y., Sun, Z., Wang, N., et al. (2020). Potential purpose program for assigning sequence reads to genomic features. Bioinformatics mechanisms of AtNPR1 mediated resistance against huanglongbing (HLB) in 30, 923–930. doi: 10.1093/bioinformatics/btt656 Citrus. int. J. Mol. Sci. 21, 2009. doi: 10.3390/ijms21062009 Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., et al. (2009). Quezada, E.-H., Garcıa,́ G.-X., Arthikala, M.-K., Melappa, G., Lara, M., and The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079. Nanjareddy, K. (2019). Cysteine-rich receptor-like kinase gene family identification doi: 10.1093/bioinformatics/btp352 in the phaseolus genome and comparative analysis of their expression profiles Frontiers in Plant Science 22 frontiersin.org Weber et al. 10.3389/fpls.2022.1019295 specific to mycorrhizal and rhizobial symbiosis. Genes 10, 59. doi: 10.3390/ Van Loon, L. C. (1997). Induced resistance in plants and the role of genes10010059 pathogenesis-related proteins. Eur. J. Plant Pathol. 103, 753–765. doi: 10.1023/ a:1008638109140 Ramadugu, C., Keremane, M. L., Halbert, S. E., Duan, Y. P., Roose, M. L., Stover, E., et al. (2016). Long-term field evaluation reveals huanglongbing resistance in Wang, Y., Liu, X. J., Chen, J. B., Cao, J. P., Li, X., and Sun, C. D. (2022). Citrus Citrus relatives. Plant Dis. 100, 1858–1869. doi: 10.1094/pdis-03-16-0271-re flavonoids and their antioxidant evaluation. Crit. Rev. Food Sci. Nutr. 62, 3833– 3854. doi: 10.1080/10408398.2020.1870035 Rawat, N., Kiran, S.P., Du,D., Gmitter, F. G.,and Deng,Z.(2015). Comprehensive meta-analysis, co-expression, and miRNA nested network Wang, Z., Yin, Y., Hu, H., Yuan, Q., Peng, G., and Xia, Y. (2006). Development analysis identifies gene candidates in citrus against huanglongbing disease. BMC and application of molecular-based diagnosis for 'Candidatus liberibacter asiaticus', Plant Biol. 15, 1–21. doi: 10.1186/s12870-015-0568-4 the causal pathogen of citrus huanglongbing. Plant Pathol. 55, 630–638. doi: 10.1111/j.1365-3059.2006.01438.x Rayapuram, C., Jensen, M. K., Maiser, F., Shanir, J. V., Hornshøj, H., Rung, J. H., et al. (2012). Regulation of basal resistance by a powdery mildew-induced cysteine- Welker, S., and Levy, A. (2022). Comparing machine learning and binary rich receptor-like protein kinase in barley. Mol. Plant Pathol. 13, 135–147. thresholding methods for quantification of callose deposits in the citrus phloem. doi: 10.1111/j.1364-3703.2011.00736.x Plants 11, 624. doi: 10.3390/plants11050624 R Core Team (2013). R: A language and environment for statistical computing Welker, S., Pierre, M., Santiago, J. P., Dutt, M., Vincent, C., and Levy, A. (2022). (Vienna: R Foundation for Statistical Computing). Phloem transport limitation in huanglongbing -affected sweet orange is dependent on phloem-limited bacteria and callose. Tree Physiol. 42, 379–390. doi: 10.1093/ Rozman-Pungerčar, J., Kopitar-Jerala, N., Bogyo, M., Turk, D., Vasiljeva, O., treephys/tpab134 Stefe, I., et al. (2003). Inhibition of papain-like cysteine proteases and legumain by caspase-specific inhibitors: when reaction mechanism is more important than Wen, Q., Xie, Z., Wu, L., He, Y., Chen, S., and Zou, X. (2018). Clone and specificity. Cell Death Differ. 10, 881–888. doi: 10.1038/sj.cdd.4401247 expression analysis of the citrus phloem protein 2 gene CsPP2B15 responding to huanglongbing infection in citrus. Acta Hortic. Sin. 45, 2347–2357. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., et al. (2012). Fiji: an open-source platform for biological-image analysis. Nat. Wu, H., Hu, Y., Fu, S., Zhou, C., and Wang, X. (2020). Coordination of multiple Methods 9, 676–682. doi: 10.1038/nmeth.2019 regulation pathways contributes to the tolerance of a wild citrus species (Citrus ichangensis ‘2586’) against huanglongbing. Physiol. Mol. Plant Pathol. 109, 101457. Silverstein, K. A. T., Moskal, W. A., Wu, H. C., Underwood, B. A., Graham, M. doi: 10.1016/j.pmpp.2019.101457 A., Town, C. D., et al. (2007). Small cysteine-rich peptides resembling antimicrobial peptides have been under-predicted in plants. Plant J. 51, 262–280. doi: 10.1111/ Wu, G. A., Prochnik, S., Jenkins, J., Salse, J., Hellsten, U., Murat, F., et al. (2014). j.1365-313x.2007.03136.x Sequencing of diverse mandarin, pummelo and orange genomes reveals complex history of admixture during citrus domestication. Nat. Biotechnol. 32, 656–662. Slavokhotova, A. A., Shelenkov, A. A., Korostyleva, T. V., Rogozhin, E. A., doi: 10.1038/nbt.2906 Melnikova, N. V., Kudryavtseva, A. V., et al. (2017). Defense peptide repertoire of Stellaria media predicted by high throughput next generation sequencing. Wu, G. A., Terol, J., Ibanez, V., López-Garcıa,́ A., Pérez-Román, E., Borredá, C., Biochimie 135, 15–27. doi: 10.1016/j.biochi.2016.12.017 et al. (2018). Genomics of the origin and evolution of Citrus. Nature 554, 311–316. doi: 10.1038/nature25447 Sugio, A., MacLean, A. M., Kingdom, H. N., Grieve, V. M., Manimekalai, R., and Hogenhout, S. A. (2011). Diverse targets of phytoplasma effectors: from plant Yang, C., and Ancona, V. (2022). An overview of the mechanisms against development to defense against insects. Annu. Rev. Phytopathol. 49, 175–195. "Candidatus liberibacter asiaticus": virulence targets, citrus defenses, and doi: 10.1146/annurev-phyto-072910-095323 microbiome. Front. Microbiol. 13. doi: 10.3389/fmicb.2022.850588 ̌ ̌ Supek, F., Bosnjak, ̌ M., Skunca, N., and Smuc, T. (2011). REVIGO summarizes Ye, Z. H., and Varner, J. E. (1996). Induction of cysteine and serine proteases and visualizes long lists of gene ontology terms. PloS One 6, e21800. doi: 10.1371/ during xylogenesis in zinnia elegans. Plant Mol. Biol. 30, 1233–1246. doi: 10.1007/ journal.pone.0021800 BF00019555 Tchoupé, J. R., Moreau, T., Gauthier, F., and Bieth, J. G. (1991). Photometric or Yu, X., and Killiny, N. (2018). The secreted salivary proteome of Asian citrus psyllid fluorometric assay of cathepsin b, l and h and papain using substrates with an diaphorina citri. Physiol. Entomology 43 (4), 324–333. doi: 10.1111/phen.12263 aminotrifluoromethylcoumarin leaving group. Biochim. Biophys. Acta Protein Zambon, F. T., Kadyampakeni, D. M., and Grosser, J. W. (2019). Ground Struct. Mol. Enzymol. 1076, 149–151. doi: 10.1016/0167-4838(91)90232-o application of overdoses of manganese have a therapeutic effect on sweet orange Thimm, O., Bläsing, O., Gibon, Y., Nagel, A., Meyer, S., Krüger, P., et al. (2004). trees infected with candidatus liberibacter asiaticus. HortScience 54, 1077–1086. Mapman: a user-driven tool to display genomics data sets onto diagrams of doi: 10.21273/HORTSCI13635-18 metabolic pathways and other biological processes. Plant J. 37, 914–939. Zhang, X., Han, X., Shi, R., Yang, G., Qi, L., Wang, R., et al. (2013). Arabidopsis doi: 10.1111/j.1365-313x.2004.02016.x cysteine-rich receptor-like kinase 45 positively regulates disease resistance to Tian, T., Liu, Y., Yan, H., You, Q., Yi, X., Du, Z., et al. (2017). agriGO v2.0: a GO Pseudomonas syringae. plant physiol. Biochem. 73, 383–391. doi: 10.1016/ analysis toolkit for the agricultural community 2017 update. Nucleic Acids Res. 45, j.plaphy.2013.10.024 W122–W129. doi: 10.1093/nar/gkx382 Zhang, Q., Li, W., Yang, J., Xu, J., Meng, Y., and Shan, W. (2020a). Two Tornero, P., Conejero, V., and Vera, P. (1997). Identification of a new pathogen- Phytophthora parasitica cysteine protease genes, PpCys44 and PpCys45, trigger cell induced member of the subtilisin-like processing protease family from plants. J. death in various Nicotiana spp. and act as virulence factors. Mol. Plant Pathol. 21, Biol. Chem. 272, 14412–14419. doi: 10.1074/jbc.272.22.14412 541–554. doi: 10.1111/mpp.12915 Tran, T.-T., Clark, K., Ma, W., and Mulchandani, A. (2020). Detection of a Zhang, X. H., Pizzo, N., Abutineh, M., Jin, X.-L., Naylon, S., Meredith, T. L., et al. secreted protein biomarker for citrus huanglongbing using a single-walled carbon (2020b). Molecular and cellular analysis of orange plants infected with nanotubes-based chemiresistive biosensor. Biosens. Bioelectron. 147, 111766. huanglongbing (citrus greening disease). Plant Growth Regul. 92, 333–343. doi: 10.1016/j.bios.2019.111766 doi: 10.1007/s10725-020-00642-z Usadel, B., Nagel, A., Steinhauser, D., Gibon, Y., Bläsing, O. E., Redestig, H., et al. Zou, X., Bai, X., Wen, Q., Xie, Z., Wu, L., Peng, A., et al. (2019). Comparative (2006). PageMan: an interactive ontology tool to generate, display, and annotate analysis of tolerant and susceptible citrus reveals the role of methyl salicylate overview graphs for profiling experiments. BMC Bioinform. 7, 535. doi: 10.1186/ signaling in the response to huanglongbing. J. Plant Growth Regul. 38, 1516–1528. 1471-2105-7-535 doi: 10.1007/s00344-019-09953-6 Frontiers in Plant Science 23 frontiersin.org

Journal

Frontiers in Plant SciencePubmed Central

Published: Oct 21, 2022

There are no references for this article.