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Transcriptome Profile Analysis of Twisted Leaf Disease Response in Susceptible Sugarcane with Narenga porphyrocoma Genetic Background

Transcriptome Profile Analysis of Twisted Leaf Disease Response in Susceptible Sugarcane with... Sugarcane is an important industrial crop with a high sugar yield that has become a leading energy crop worldwide. It is widely cultivated in tropical and subtropical regions. Various diseases beset the cultivation of sugarcane. The molecular study of disease resistance in sugarcane is limited by its complicated genome. In our study, RNA-seq was employed to detect the mechanism of twisted leaf disease tolerance in modern cultivar sugarcane, which derived from Narenga porphyrocoma. We completed high- throughput transcriptomic sequencing of 12 samples, including three stages of a susceptible (NSBC1_T “H3–8”) and an unsusceptible cultivar (NSBC1_CK “H-19”) with two biological repeats, respectively. Using the Saccharum spontaneum ge- nome as reference, the average mapping ratio of the clean data was over 70%. Among the differentially expressed genes between H3–8andH3–19, we focused on the analysis of hormone pathways and resistance (R) genes. The results showed that twisted leaf disease triggers hormone networks and around 40% of R genes conditioned lower expression in the susceptible cultivar. One of the possible reasons for H3–8 being susceptible to twisted leaf disease might be the null/retarded response of R genes, especially in pre-onset stage (46% down-regulated) of pathogens infection. . . . Keywords Hormones R genes RNA-seq Sugarcane Jinju Wei, Zhihui Xiu and Huiping Ou equally contributed to this work. Communicated by: Rui Xia Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12042-019-09231-5) contains supplementary material, which is available to authorized users. * Hongwei Tan Ronghua Zhang hwtan@gxaas.net zhangronghua@gxaas.net * Xihui Liu Hui Zhou liuxihui@gxaas.net zhoukuai@126.com Jinju Wei Yiyun Gui jjwei@gxaas.net guiyiyun@gxaas.net Zhihui Xiu Haibi Li xiuzhihui@genomics.cn 350245462@qq.com Huiping Ou Yangrui Li ouhuiping2006@163.com liyr@gxaas.net Junhui Chen Rongzhong Yang chenjunhui@genomics.cn rozyang@126.com Huayan Jiang Dongliang Huang jianghuayan@genomics.cn hdl666@163.com Xiaoqiu Zhang zhangxiaoqiuxhd@163.com Extended author information available on the last page of the article 294 Tropical Plant Biol. (2019) 12:293–303 Abbreviations background blocks the sequencing of the whole sugarcane NR Non-redundant protein sequences in NCBI genome. Studies on biological traits, such as biomass yield, COG/ Clusters of orthologous group sugar accumulation, and stress tolerance, have focused on KOG transcriptome analysis (Kido et al. 2012; Fracasso et al. KEGG Kyoto encyclopedia of genes and genomes 2016; Huang et al. 2016). The transcriptome reveals specific databases transcripts produced under biotic stress (e.g., smut) and abiotic TGICL TIGR gene indices clustering tools stress (e.g., drought) conditions in sugarcane (Iskandar et al. Blast Basic Local Alignment Search Tool 2011; Mattielllo et al. 2015). With the Saccharum spontaneum genome, which is rela- tive high quality, as reference (Zhang et al. 2018), we Introduction employed transcriptome profiling to analyze the genes and pathways involved in twist leaf disease in modern sugarcane Sugarcane belongs to the Poaceae family, which includes cultivar. Twisted leaf disease, caused by Phoma sp., which is maize, wheat, rice, sorghum, and many types of grass (Li one of the largest fungal genera, was first reported in Guangxi, 2010). Cultivated sugarcane is an important industrial crop China, in 2014, when more than 5% of sugarcane was infected with a high sugar yield and has become a major energy crop in the field. Twisted leaf disease is somewhat similar to worldwide (Grivet and Arruda 2001;Yanget al. 2010). It is Pokkah Boeng disease (caused by Fusarium moniliforme cultivated in ~26 million hectares in tropical and subtropical Sheldon). The symptoms begin with yellowing on the midribs regions of the world, producing up to 1.8 billion metric tons of and leaf margins, then spread to the entire leaf, along with crushable stems (Zhang et al. 2018). Sugarcane provides twisting and curling of the crown leaves (Lin et al. 2014). around 80% of the world’s sugar, with the secondary produc- The modern sugarcane cultivar used in this study was gener- tion as raw materials for pulp, ethanol and bioplastics (Lam ated from the BC generation offspring of Narenga et al. 2009;Liand Yang 2015). Modern sugarcane cultivars porphyrocoma via crossing and backcrossing with cultivated are interspecific hybrids which were generated from the cross sugarcane varieties, so that it harbored the Narenga between Saccharum officinarum and Saccharum spontaneum, porphyrocom genetic background. The RNA-seq (two biolog- followed by backcrossing into Saccharum officinarum to se- ical repeats) comparison between un-susceptible and suscep- lect sugar-poor relative traits (Roah 1972; Branes and Sartoris tible cultivars in different stages of infection provided a refer- 1936). In China, modern sugarcane is mainly distributed in the ence for understanding the mechanism of twisted leaf disease. provinces of Guangxi, Yunnan, Guangdong, and Hainan. Analysis revealed that twisted leaf disease triggered the hor- Some cultivars were also improved disease resistance by mone network. R genes expression profile showed ~40% R crossing with Saccharum barberi Jesweit or/and Narenga genes expressed lower in susceptible cultivar. The porphyrocoma (Gao et al. 2012; Liu et al. 2012a, b). null/retarded response of R genes might be one of the possible Sugarcane diseases, such as smut, white leaf, and wilt/top reasons for H3–8 being susceptible to twisted leaf disease. rot/Pokkah Boeng, are critical limitations of production, caus- ing serious losses in yield and quality among susceptible cul- tivars (Hameed et al. 2015; Paulo et al. 2016;Suetal. 2016). Results Traditionally, the resistant traits of Saccharum spontaneum, Saccharum barberi Jesweit or/and Narenga porphyrocoma RNA Sequencing and Mapping to the Reference were introduced into Saccharum officinarum by hybridization Genome to improve the disease resistance of cultivated sugarcane (Gao et al. 2012; Liu et al. 2012a, b). With the development of Compared to the un-susceptible cultivar, the susceptible culti- breeding approaches, molecular breeding with precise ge- var displayed twisted leaves after infection with the disease nome information accelerates the collection of disease resis- (Fig. 1a). To obtain the gene expression profile associated tance genes in one cultivar. However, applying this to sugar- with twisted leaf disease in susceptible sugarcane, twelve se- cane is difficult due to its high polyploidy and complex ge- quencing cDNA libraries were constructed. They contained a nome, with ploidy levels ranging from 5× to 16×, and chro- susceptible genotype (NSBC _T) and un-susceptible geno- mosome numbers from 2n = 40–128, with some even as high type (NSBC _CK) at three stages (pre-onset, early, and as 200 (Sreenivasan et al. 1987; Liu et al. 2012a, b). The serious symptoms) (Fig. 1b) with two biological repeats (2 estimated polyploid genome size of sugarcane ranges from cultivars ×3 stages ×2 biological repeats). As shown in 3.36 to 12.64 Gb, and the monoploid genome size ranges from Table 1, a total of 258,301,460 and 279,952,436 raw reads 760 to 985 Mb (D’hont et al. 1996; Zhang et al. 2012), which were generated from the NSBC _T and NSBC _CK library 1 1 is larger than the rice (400 MB) and the sorghum (760 MB) by sequencing on the Illumina Hiseq 2000 platform, respec- genomes (Soderlund et al. 2011). Such complex genetic tively. After filtering low-quality reads, unknown nucleotides, Tropical Plant Biol. (2019) 12:293–303 295 Fig. 1 The susceptible sugarcane clone H3–8 under twisted leaf disease stress. a Twisted leaf disease of H3–8 was observed in the field. b Phenotype of three stages of Phoma sp. infection. From top to bottom: pre-onset, early, and serious symptom stages and the contained adapters, 251,482,172 and 273,757,964 protein coding genes, and 15,469 transcripts were classified clean reads were left in the NSBC _T and NSBC _CK librar- into long non-coding RNAs. Details of each sample were 1 1 ies, respectively. Using the genome of Saccharum exhibited in Table S1 and Fig. S1. spontaneum as reference (Zhang et al. 2018), we mapped the Based on the Pearson correlation coefficient of genes ex- clean reads to the reference via HISAT (hierarchical indexing pression of each sample, we obtained the correlation heatmap for spliced alignment of transcripts) (Kim et al. 2015). All of all samples (Fig. 2a). The samples from the same cultivar samples conditioned over 70% mapping ratio (average conditioned high correlation Pearson value. Of course, two 76.74%). The details were summarized in Table 2. biological repeats of each stage clustered together in the Cluster Dendrogram (Fig. 2b). That means two biological re- Gene Expression Statistics peats of each stage were consistent and satisfied for further analysis. To elucidate gene expression profile of un-susceptible and susceptible cultivars, we annotated the aligned reads accord- ing to the reference genome. The result showed that there were Table 2 Genome mapping summary of all samples 91,386 genes expressed that included 65,391 known genes and 25,995 novel genes. In the 96,101 annotated transcripts, Sample Total Clean Reads Total Mapping Ratio 53,481 transcripts with novel alternative splicing subtypes en- code known proteins, 27,151 transcripts were defined as novel NSBC1_CK1–1 49,273,558 76.43% NSBC1_CK1–2 51,677,702 77.54% NSBC1_CK2–1 47,765,358 71.71% NSBC1_CK2–2 44,108,084 75.97% Table 1 Overview of the transcriptome sequencing and de novo assembly results NSBC1_CK3–1 42,718,254 77.39% NSBC1_CK3–2 44,409,480 76.30% Description NSBC1_T NSBC1_CK NSBC1_T1–1 44,375,910 77.42% Raw reads 258,301,460 279,952,436 NSBC1_T1–2 42,446,806 78.30% Q20 percentage 55.56% 55.61% NSBC1_T2–1 23,302,262 78.14% Clean reads 251,482,172 273,757,964 NSBC1_T2–2 23,302,264 77.40% Total reads 525,240,136 NSBC1_T3–1 42,487,034 76.63% Total nucleotides (nt) 65,780,017,250 NSBC1_T3–2 41,604,074 77.65% 296 Tropical Plant Biol. (2019) 12:293–303 Fig. 2 Correlation analysis of all samples. a Heatmap was constructed by the Pearson correlation coefficient of genes expression. b Cluster analysis of all samples based on genes expression quantity Differentially Expressed Genes (DEGs) part, and organelle conditioned most enriched DEGs in the in Un-Susceptible and Susceptible Cultivars cellular component. In the term of molecular function, DEGs were mainly distributed in catalytic activity and bind- The significance of the gene expression difference was deter- ing. Most enriched GO term are corresponding to various mined according to the threshold of FDR (False Discovery aspects of activated metabolism (e.g. “metabolic process” Rate) < 0.05 and |log FC (Fold Change) | >1. In the compar- and “catalytic activity”) and stress response (e.g. “biological ison to the un-susceptible clone H3–19, the genes expression regulation” and “organelle”). We also performed a BLAST of susceptible clone H3–8 showed 8408 up-regulated and (The Basic Local Alignment Search Tool) (E-value 7834 down-regulated in pre-onset stage, 7334 up-regulated <0.00001) analysis of the DEGs against the KEGG (Kyoto and5558down-regulatedinearlystage,and24,456up- encyclopedia of genes and genomes) database. The DEGs regulated and 10,468 down-regulated in serious symptom were mainly enriched in metabolic pathways, biosynthesis of stage. (Fig. 3). The Gene Ontology (GO) analysis classified secondary metabolites, and plant-pathogen interaction (Fig. DEGs into three main categories: biological function, cellular S2). These are consistent with the sugarcane activities in re- component, and molecular function (Fig. 4). DEGs mainly sponse to Phoma sp. infection. According to these findings, accumulated in the cellular process, metabolic process, and we analyzed specific pathways and genes related to disease biological regulation in the biological function. The cell, cell defense. Fig. 3 DEG analysis in the susceptible cultivars. Numbers of up- and down-regulated genes identified in the pre-onset, early, and serious symptom stages in susceptible genotypes Tropical Plant Biol. (2019) 12:293–303 297 Fig. 4 Gene ontology (GO) annotation for DEGs in three stages Ingenuity Pathways Analysis response to biotic and abiotic stress (Bari and Jones 2009). The DEGs between the susceptible and un-susceptible culti- Plant hormones are the first reactors under environmental vars provided us an opportunity to study how the hormone stress (Verma et al. 2016). Among the major hormones, pathways response to twisted leaf disease. We analyzed the abscisic acid (ABA), ethylene (ET), jasmonates (JA), and expression patterns of genes involved in hormone biogenesis salicylic acid (SA) play essential roles in regulating plant in three different stage of Phoma sp. infection (Fig. 5; 298 Tropical Plant Biol. (2019) 12:293–303 Fig. 5 Ingenuity analysis of DEGs in hormone pathways during fungi infection process. Fold change (log ) in hormone biosynthesis genes at different stages of the infection process relative to the resistance condition Table S1). As the main response hormones in biotic stress, the secondary plant reflections (Robert-Seilaniantz et al. 2011). biosynthesis of SA, JA, and ET up-regulated in the susceptible These results show that twisted leaf disease triggered the re- cultivar (genes in green, brown, and blue lines). The SA, action of the hormones network. which is generally involved in the defense against biotrophic Also, we analyzed the specific biosynthesis genes in SA, and hemi-biotrophic pathogens (Loake and Grant 2007), was JA and ET pathway, respectively. In details, the phenylalanine enriched with high expression (~4 folds up-regulated) of ammonia-lyase, an enzyme of phenylalanine ammonium ly- LOC_Os02g41630-D10and Sspon.04G0008070-3C (Fig. 5, ase (PAL) pathway in SA synthesis (Yokoo et al. 2018), was green lines). JA and ET, which involve in the defense of increased in expression at the pre-onset of Phoma sp. infec- necrotrophic pathogens and herbivorous insects (Gamalero tion. The isochorismate synthase 1, which is involved in the and Glick 2012; Wasternack and Hause 2013), accumulated synthesis of SA (Yokoo et al. 2018), showed lower expression through up-regulating biosynthesis genes (Fig. 5,brown level in three stages of the susceptible cultivar (Table S1). In lines). ABA, auxin (IAA), and cytokinins (CK) biosynthesis the JA pathway, 12-oxophytodienoate reductase, which catal- genes also exhibited differential expressions in the susceptible yses the reduction of 10, 11-double bonds of 12- cultivar, which resulted from hormone pathways crosstalk and oxophytodienoate to 3-oxo-2-(2′-pentenyl)-cyclopentane-1- Tropical Plant Biol. (2019) 12:293–303 299 octanoic acid (OPC-8:0) (Tani et al. 2008), conditioned rela- expressed lower at pre-onset and kept increasing in the later tive higher expression in the susceptible cultivar (Table S1). stages. That means R genes with such expression patterns in The aminotransferase, a gene family including VAS1 which the susceptible cultivar were retarded in the defense of Phoma participates in auxin and ethylene biosynthesis (Zhang et al. sp. infection. Some R genes like Sspon.02G0020920-2B, 2012), varied in expression at different stages of the twisted Sspon.02G0041050-1B, and Sspon.01G0037110-1B, condi- leaf disease. These findings supported that the infection of tioned higher expression or/and irregular expression patterns Phoma sp. induced the response of the hormones network. in three stages. These R genes could respond to Phoma sp. infection in the susceptible cultivar. Also, the sequence anal- ysis of these representative genes, including alternative splic- Evaluation of Differentially Expressed ing and SNPs, didn’t offer clue for the structural mutation in Disease-Resistant Genes the susceptible cultivar (data not shown). The expression level analysis of R genes revealed that the null and retarded re- To defend against infection of pathogen, the expression of sponse of R genes in the Phoma sp. infection, especially at resistance (R) genes that encode proteins containing con- the pre-onset stage might be one of the reasons that the H3–8 served nucleotide binding site plus leucine-rich repeat (NBS- cultivar is susceptible to twisted leaf disease. LRR domain) are induced (Bakker et al. 2006). To elucidate the R genes involved in the twisted leaf disease, we annotated R genes in DEGs based on PRG (Pathogen receptor genes) database. (Table 3). There were 46%, 42%, and 26% R genes Discussion conditioned lower expression in pre-onset, early stage, and serious symptom of susceptible cultivar relative to the control, The transcriptome, which is influenced by external environ- respectively. That means a part of R genes in the susceptible mental conditions, can effectively reveal the response mecha- cultivar were null/retarded in the response of Phoma sp. nism of biotic and abiotic stress in plants (Wei et al. 2011). In infection. our study, the natural infected cultivar H3–8 was defined as From the R genes expression profile, we selected out rep- susceptible cultivar and the un-susceptible cultivar (H3–19) resentative of each class to exhibit their expression patterns in was used as control. With two biological repeats, we used three stages of Phoma sp. infection (Fig. 6). transcriptome profiling to detect the genes associated with Sspon.06G0034670-1D, Sspon.04G0004450-1A and twisted leaf disease and obtained 65,780,017,250 bp data from Sspon.02G0029950-1T dramatically decreased in three stages three stages of the twisted leaf disease. Using the genome of of Phoma sp. infection. Sspon.01G0011870-1A and Saccharum spontaneum L. as reference (Zhang et al. 2018), Sspon.01G0013680-3C also showed lower expression level the average mapping ratio of twelve samples was 76.74%. The in the three stages. R genes like these genes were null function annotation of aligned reads showed 91,386 genes expressed in the susceptible cultivar. Sspon.05G0016050-1A, (65,391 known and 25,995 novel), and 96,101 annotated tran- Sspon.08G0026530-1C and Sspon.03G0025750-3D scripts, including 53,481 novel alternative splicing subtypes Table 3 Classification of Class Count Representatives Description annotated R genes and representatives for ingenuity NL 543 Sspon.02G0020920-2B Disease resistance protein (NBS-LRR class) family analysis RLP 683 Sspon.02G0041050-1B Pyridoxal phosphate-dependent transferases superfamily protein CNL 242 Sspon.05G0016050-1A HOPZ-ACTIVATED RESISTANCE 1 N 309 Sspon.08G0026530-1C NB-ARC domain-containing disease resistance protein TNL 100 Sspon.06G0034670-1D TIR-NBS-TIR-TIR-WRKY type disease resistance protein RLK 7 BGI_novel_G009740 S-locus lectin protein kinase family protein RLK-GNK2 94 Sspon.01G0015790-1A cysteine-rich RLK (RECEPTOR-like protein kinase) CN 67 Sspon.04G0004450-1A LRR and NB-ARC domains-containing disease resistance protein Other 40 Sspon.01G0037110-1B NADPH HC toxin reductase-like protein T 17 Sspon.01G0011870-1A NAC domain containing protein Mlo-like 21 Sspon.01G0013680-3C Seven transmembrane MLO family protein L 5 Sspon.03G0025750-3D Protein phosphatase 1 regulatory subunit RPW8-NL 4 Sspon.02G0029950-1T ADR1-like 1 disease resistance protein 300 Tropical Plant Biol. (2019) 12:293–303 Fig. 6 Differential expression analysis of specific R genes in the susceptible cultivar. Genes were selected out as representative based the tendency of each R gene class. Fold change (log ) was achieved from the FPKM value of T and CK encode known proteins, 27,151 novel protein coding genes, down-regulated, especially in pre-onset stage. For example, and 15,469 long non-coding RNAs. With these data, we ana- belongs to LRR and NB-ARC domains-containing proteins lyzed the DEGs, including hormone pathways and R genes, of which members participate in disease resistance (Fischer between the susceptible cultivar and the control. Also, these et al. 2016). is predicted to encode RGA protein which con- data provided a foundation for the further analysis of the twist- tains NB-ARC domain and functions disease resistance ed leaf disease, such as specific gene characterization, resis- (Cesari et al. 2013). is descripted as CRKs (cysteine-rich pro- tant genes selection, and immune network description. tein kinase), which involves in the ROS signaling pathway The comparison between susceptible cultivar and the con- and plays important roles in the elimination of fungal growth trol identified 16,242, 12,892 and 34,924 DEGs in pre-onset, damage (Niina Idänheimo 2015). The low expression of these early stage and serious symptom stage, respectively (Fig. 3). R genes in the twisted leaf disease would drag down the rapid These DEGs are involved in critical biological activities that response of R genes defensive system. Besides ~40% down- are essential for disease resistance (Fig. 4). Ingenuity pathway regulated R genes, there were still a number of R genes con- analysis revealed hormone biosynthesis genes variations in ditioned normal expression level or even higher expression the susceptible cultivar (Fig. 5). The SA and ET pathways level in the susceptible cultivar. Unfortunately, these high were induced in the infection of Phoma sp. at the pre-onset expressed R genes were unable to eliminate the pathogens stage. Along with the spread of the disease, JA and ET syn- successfully. These increased expressions might be a result thesis increased and maintained high levels in the serious of universal reactions of sugarcane under pathogens stress symptom stages. As three major hormones involved in stress rather than the specific response of Phoma sp. infection. resistance, SA is a biotrophic and hemi-biotrophic pathogen- Such phenomenon was commonly reported in the studies of triggered signaling pathway, and JA combined with ET sig- other species (Heath 2000;van Loon 2015). naling pathways are induced by necrotrophic pathogens (Glazebrook 2005). Therefore, twisted leaf disease, caused by fungal pathogens, first triggers the SA pathway as response Materials and Methods to pathogens infection. The high level of JA and ET in serious symptoms might partially result from tissue damage in dis- Plant Materials and Sampling ease. Other hormones, including ABA, auxin, and CK, crosstalk with major disease response hormones (SA, JA, Narenga porphyrocoma, which is an important relative genus and ET) and construct a network to respond to disease stress of sugarcane, was collected from a barren mountainous area in (Verma et al. 2016). Guangxi province, China. We obtained the BC generation Plant disease resistance (R) genes are prevalent in all plant offspring of the Narenga porphyrocoma via crossing and species and harbor a conserved LRR domain (Meyers et al. backcrossing with sugarcane varieties several years. Among 1998). R genes are defined as gene-for-gene interaction and the BC offspring, H3–8 was susceptible to twisted leaf dis- specifically recognize an avirulence protein encoded by a ease at the sugarcane elongating stage, whereas H3–19 was pathogen with a hypersensitive response (Flor 1956). In the not. H3–8 was proven to affect stable twisted leaf disease R gene analysis of the susceptible cultivar, around 40% R occurrence after 3 years based on field and greenhouse genes, including those that play essential functions, were observations. Tropical Plant Biol. (2019) 12:293–303 301 Plantlets at the same growth stage were selected and Data Filtering and Genome Mapping planted in pots filled with a mixture of peat soil and washed sand, and grown in a greenhouse at the Sugarcane Research The raw reads were filtered according to the following criteria: Institute, Guangxi Academy of Agricultural Sciences. The (1) reads containing adaptor, (2) reads with unknown nucleo- natural infected plants were selected and defined as the sus- tides larger than 5%, and (3) low-quality reads (the rate of ceptible cultivator. The leaf samples of the susceptible sugar- reads with a quality value of ≤10 was morethan20%.The cane clone H3–8 were collected at three stages corresponding clean data was mapped to the genome of Saccharum to the pre-onset (named NSBC _T1), early (named spontaneum L. (Zhang et al. 2018) via HISAT (v2.0.4; −- NSBC _T2), and serious symptom (named NSBC _T3) phred64 –sensitive –no-discordant –no-mixed -I 1 -X 1000) 1 1 stages of the disease, respectively. The leaf samples of the (Kim et al. 2015). The transcripts were re-constructed via un-susceptible sugarcane clone H3–19 were simultaneously StringTie and predicted using Cuffcompare tool in Cufflink collected, labeled NSBC _CK1, NSBC _CK2, and (Pertea et al. 2003; Trapnell et al. 2010; Pertea et al. 2016). 1 1 NSBC _CK3, respectively. Two biological repeats of each sample were collected. All samples were immediately frozen Differential Gene Expression in liquid nitrogen and stored at −80 °C. The DEGs analysis was performed as the description of RNA Extraction and Quality Determination DEGseq (Wang et al. 2010) and corrected the P-values to Q-values based on Benjamini-Hochberg method The total RNA was extracted using TRIzol reagent (Benjamini and Hochberg 1995) and Storey-Tibshirani (Invitrogen, Carlsbad, CA, USA) and treated with RNase- method (Storey and Tibshirani 2003). GO (gene ontolo- free DNase I and RNA integrity number (RIN > 8.0). RNA gy) terms were assigned based on the best-hits BLASTx quality and quantity were verified using a NanoDrop 1000 resulted from NR alignments that were derived from spectrophotometer (Wilmington, DE, CA, USA) and an Blast2GO (v2.5.0) against GO database (release- Agilent 2100 Bioanalyzer (Santa Clara, CA, USA) prior to 20,120,801). The DEGs were aligned against KEGG the library construction. No sign of degradation was found. (Kyoto Encyclopedia of Genes and Genomes) by BLASTx package with threshold of E-value <=10–5. cDNA Library Construction and Sequencing Ingenuity Analysis of DEGs The poly(A) RNA was fragmented into approximately 300-nt fragments using RNA Fragmentation Reagents (Ambion, Thericehormonesproteinswereusedasreference to Austin, TX, USA). Using these short fragments as templates, identify the hormone genes in sugarcane (Cohen et al. the double-stranded cDNA was synthesized with random 2017). The longest transcript of genes was selected as primers (Invitrogen, Carlsbad, CA, USA). The end repair of representative for analysis. All predicted protein se- these cDNA fragments was subsequently performed with quences were aligned against the sugarcane gene models Klenow polymerase, T4 DNA polymerase, and T4 polynucle- by BLASTp (Identity>50%; Coverage>50%; E-value: otide kinase (NEB,Ipswich, MA, USA). Illumina adapters 1× 10 ), and defined as homologs of hormones biosyn- (containing primer sites for sequencing and flow cell surface thesis genes. Also, we aligned all predicted hormone annealing) were ligated to the end-repaired fragments using genes against the sugarcane genome via tBLASTn (E- T4 DNA ligase (Invitrogen, Carlsbad, CA, USA). The prod- value: 1E-10), and used GeneWise (v2.2.0) to identify ucts were enriched for the cDNA fragments using a Qiaquick the structure of candidate hormone genes. Among these Gel Extraction Kit (Qiagen, Duesseldorf, Germany) and am- genes, the ones with non-significant different expression plified with polymerase chain reaction (PCR) to prepare the in three stages were eliminated. The final candidates sequencing library. Agilent 2100 Bioanalyzer (Agilent, were aligned against KEGG database (version:84). The Beijing, China) was used to detect the quantity and quality log fold of each gene was obtained from the case- of the cDNA. Then, the paired-end RNA-seq libraries were control group. The patterns of the gene in each group prepared following Illumina’s protocols and sequenced on the were compared. Similarly, predicted proteins were Illumina HiSeq 2500 platform (Illumina, San Diego, CA, aligned against the PRG database using BLASTp. The USA).Sequencingwas performedinaflowcellonan results were filtered (E-value: 1e10), and the best hit of Illumina HiSeq 2500 sequencer using the TruSeq Paired- predicted protein was retained. The fpkm (Reads per End Cluster Kit v3 (Illumina PE-401-3001) and the TruSeq kilobase of exon per million reads mapped) of each SBS HS Kit v3200 cycles (Illumina FC-401-3001), generat- predicted resistant gene in the case sample was filtered ing 2 × 125 bp. out and a downstream analysis was performed. 302 Tropical Plant Biol. (2019) 12:293–303 Acknowledgments We thank Mick Rose in Primary Industry Department kinase (LRR-RLK) subfamily in angiosperms. Plant Physiol 170: of Australia for the assistance of writing paper. 1595–1610 Flor HH (1956) The complementary genic system in flax and flax rust. Adv Genet 8:29–54 Author Contributions Hongwei Tan, and Xihui Liu conceived and de- Fracasso A, Trindade LM, Amaducci S (2016) Drought stress tolerance signed the experiments. Jinju Wei and Huiping Ou performed the exper- strategies revealed by RNA-Seq in two sorghum genotypes with iments. Xiaoqiu Zhang, Ronghua Zhang, Hui Zhou, Yiyun Gui,Haibi Li, contrasting WUE. BMC Biol 16:115 Yangrui Li, Rongzhong Yang and Dongliang Huang performed cross and backcross of Narenga porphyrocoma, planted sugarcane and collected Gamalero E, Glick BR (2012) Ethylene and abiotic stress tolerance in samples. Zhihui Xiu, Junhui Chen and Huayan Jiang analyzed the data. plants. In: Environmental adaptations and stress tolerance of plants Zhihui Xiu and Xihui Liu wrote the manuscript. All authors have read and in the era of climate change. Springer, New York, pp 395–412 approved the final manuscript. Gao Y, Fang F, Liu XH et al (2012) Identification of progeny from crosses between sugarcane (Saccharum spp.) and intergeneric hybrid com- plex (Erianthus arundinaceus × Saccharum spontaneum)withmo- Funding This work was financially supported by the National Science lecular markers. J Plant Genet Res 13:912–916 Foundation of China (31760368, 31101195), Guangxi Fund (GKAA17202042–6), Fund of Guangxi Academy of Agricultural Glazebrook J (2005) Contrasting mechanisms of defense against Sciences (GNK2018YT02 and GNK2018YM01) and Fund of Modern biotrophic and necrotrophic pathogens. Annu Rev Phytopathol 43: Agriculture Technology (CARS-170105, gjnytxgxcxtd-03). 205–227 Grivet L, Arruda P (2001) Sugarcane genomics: depicting the complex ge- nome of an important tropical crop. 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Planta 227:517–526 Trapnell C, Williams BA, Pertea G, Mortazavi AM, Kwan G, van Baren Publisher’s Note Springer Nature remains neutral with regard to jurisdictional MJ, Salzberg SL, Wold B, Pachter L (2010) Transcript assembly and claims in published maps and institutional affiliations. Affiliations 1 2 1 2 2 1 1 Jinju Wei & Zhihui Xiu & Huiping Ou & Junhui Chen & Huayan Jiang & Xiaoqiu Zhang & Ronghua Zhang & 1 1 1 1 1 1 1 1 Hui Zhou & Yiyun Gui & Haibi Li & Yangrui Li & Rongzhong Yang & Dongliang Huang & Hongwei Tan & Xihui Liu Key Laboratory of Sugarcane Biotechnology and Genetic Research Institute, Guangxi Academy of Agricultural Sciences, Improvement (Guangxi), Guangxi Key Laboratory of Sugarcane Nanning 530007, China Genetic Improvement, Ministry of Agriculture, Sugarcane Research BGI Genomics, BGI-Shenzhen, Shenzhen 518083, China Center, Chinese Academy of Agricultural Sciences, Sugarcane http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Tropical Plant Biology Springer Journals

Transcriptome Profile Analysis of Twisted Leaf Disease Response in Susceptible Sugarcane with Narenga porphyrocoma Genetic Background

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Copyright © 2019 by The Author(s)
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Life Sciences; Plant Sciences; Plant Genetics and Genomics; Plant Breeding/Biotechnology; Plant Ecology; Transgenics
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1935-9756
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1935-9764
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10.1007/s12042-019-09231-5
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Abstract

Sugarcane is an important industrial crop with a high sugar yield that has become a leading energy crop worldwide. It is widely cultivated in tropical and subtropical regions. Various diseases beset the cultivation of sugarcane. The molecular study of disease resistance in sugarcane is limited by its complicated genome. In our study, RNA-seq was employed to detect the mechanism of twisted leaf disease tolerance in modern cultivar sugarcane, which derived from Narenga porphyrocoma. We completed high- throughput transcriptomic sequencing of 12 samples, including three stages of a susceptible (NSBC1_T “H3–8”) and an unsusceptible cultivar (NSBC1_CK “H-19”) with two biological repeats, respectively. Using the Saccharum spontaneum ge- nome as reference, the average mapping ratio of the clean data was over 70%. Among the differentially expressed genes between H3–8andH3–19, we focused on the analysis of hormone pathways and resistance (R) genes. The results showed that twisted leaf disease triggers hormone networks and around 40% of R genes conditioned lower expression in the susceptible cultivar. One of the possible reasons for H3–8 being susceptible to twisted leaf disease might be the null/retarded response of R genes, especially in pre-onset stage (46% down-regulated) of pathogens infection. . . . Keywords Hormones R genes RNA-seq Sugarcane Jinju Wei, Zhihui Xiu and Huiping Ou equally contributed to this work. Communicated by: Rui Xia Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12042-019-09231-5) contains supplementary material, which is available to authorized users. * Hongwei Tan Ronghua Zhang hwtan@gxaas.net zhangronghua@gxaas.net * Xihui Liu Hui Zhou liuxihui@gxaas.net zhoukuai@126.com Jinju Wei Yiyun Gui jjwei@gxaas.net guiyiyun@gxaas.net Zhihui Xiu Haibi Li xiuzhihui@genomics.cn 350245462@qq.com Huiping Ou Yangrui Li ouhuiping2006@163.com liyr@gxaas.net Junhui Chen Rongzhong Yang chenjunhui@genomics.cn rozyang@126.com Huayan Jiang Dongliang Huang jianghuayan@genomics.cn hdl666@163.com Xiaoqiu Zhang zhangxiaoqiuxhd@163.com Extended author information available on the last page of the article 294 Tropical Plant Biol. (2019) 12:293–303 Abbreviations background blocks the sequencing of the whole sugarcane NR Non-redundant protein sequences in NCBI genome. Studies on biological traits, such as biomass yield, COG/ Clusters of orthologous group sugar accumulation, and stress tolerance, have focused on KOG transcriptome analysis (Kido et al. 2012; Fracasso et al. KEGG Kyoto encyclopedia of genes and genomes 2016; Huang et al. 2016). The transcriptome reveals specific databases transcripts produced under biotic stress (e.g., smut) and abiotic TGICL TIGR gene indices clustering tools stress (e.g., drought) conditions in sugarcane (Iskandar et al. Blast Basic Local Alignment Search Tool 2011; Mattielllo et al. 2015). With the Saccharum spontaneum genome, which is rela- tive high quality, as reference (Zhang et al. 2018), we Introduction employed transcriptome profiling to analyze the genes and pathways involved in twist leaf disease in modern sugarcane Sugarcane belongs to the Poaceae family, which includes cultivar. Twisted leaf disease, caused by Phoma sp., which is maize, wheat, rice, sorghum, and many types of grass (Li one of the largest fungal genera, was first reported in Guangxi, 2010). Cultivated sugarcane is an important industrial crop China, in 2014, when more than 5% of sugarcane was infected with a high sugar yield and has become a major energy crop in the field. Twisted leaf disease is somewhat similar to worldwide (Grivet and Arruda 2001;Yanget al. 2010). It is Pokkah Boeng disease (caused by Fusarium moniliforme cultivated in ~26 million hectares in tropical and subtropical Sheldon). The symptoms begin with yellowing on the midribs regions of the world, producing up to 1.8 billion metric tons of and leaf margins, then spread to the entire leaf, along with crushable stems (Zhang et al. 2018). Sugarcane provides twisting and curling of the crown leaves (Lin et al. 2014). around 80% of the world’s sugar, with the secondary produc- The modern sugarcane cultivar used in this study was gener- tion as raw materials for pulp, ethanol and bioplastics (Lam ated from the BC generation offspring of Narenga et al. 2009;Liand Yang 2015). Modern sugarcane cultivars porphyrocoma via crossing and backcrossing with cultivated are interspecific hybrids which were generated from the cross sugarcane varieties, so that it harbored the Narenga between Saccharum officinarum and Saccharum spontaneum, porphyrocom genetic background. The RNA-seq (two biolog- followed by backcrossing into Saccharum officinarum to se- ical repeats) comparison between un-susceptible and suscep- lect sugar-poor relative traits (Roah 1972; Branes and Sartoris tible cultivars in different stages of infection provided a refer- 1936). In China, modern sugarcane is mainly distributed in the ence for understanding the mechanism of twisted leaf disease. provinces of Guangxi, Yunnan, Guangdong, and Hainan. Analysis revealed that twisted leaf disease triggered the hor- Some cultivars were also improved disease resistance by mone network. R genes expression profile showed ~40% R crossing with Saccharum barberi Jesweit or/and Narenga genes expressed lower in susceptible cultivar. The porphyrocoma (Gao et al. 2012; Liu et al. 2012a, b). null/retarded response of R genes might be one of the possible Sugarcane diseases, such as smut, white leaf, and wilt/top reasons for H3–8 being susceptible to twisted leaf disease. rot/Pokkah Boeng, are critical limitations of production, caus- ing serious losses in yield and quality among susceptible cul- tivars (Hameed et al. 2015; Paulo et al. 2016;Suetal. 2016). Results Traditionally, the resistant traits of Saccharum spontaneum, Saccharum barberi Jesweit or/and Narenga porphyrocoma RNA Sequencing and Mapping to the Reference were introduced into Saccharum officinarum by hybridization Genome to improve the disease resistance of cultivated sugarcane (Gao et al. 2012; Liu et al. 2012a, b). With the development of Compared to the un-susceptible cultivar, the susceptible culti- breeding approaches, molecular breeding with precise ge- var displayed twisted leaves after infection with the disease nome information accelerates the collection of disease resis- (Fig. 1a). To obtain the gene expression profile associated tance genes in one cultivar. However, applying this to sugar- with twisted leaf disease in susceptible sugarcane, twelve se- cane is difficult due to its high polyploidy and complex ge- quencing cDNA libraries were constructed. They contained a nome, with ploidy levels ranging from 5× to 16×, and chro- susceptible genotype (NSBC _T) and un-susceptible geno- mosome numbers from 2n = 40–128, with some even as high type (NSBC _CK) at three stages (pre-onset, early, and as 200 (Sreenivasan et al. 1987; Liu et al. 2012a, b). The serious symptoms) (Fig. 1b) with two biological repeats (2 estimated polyploid genome size of sugarcane ranges from cultivars ×3 stages ×2 biological repeats). As shown in 3.36 to 12.64 Gb, and the monoploid genome size ranges from Table 1, a total of 258,301,460 and 279,952,436 raw reads 760 to 985 Mb (D’hont et al. 1996; Zhang et al. 2012), which were generated from the NSBC _T and NSBC _CK library 1 1 is larger than the rice (400 MB) and the sorghum (760 MB) by sequencing on the Illumina Hiseq 2000 platform, respec- genomes (Soderlund et al. 2011). Such complex genetic tively. After filtering low-quality reads, unknown nucleotides, Tropical Plant Biol. (2019) 12:293–303 295 Fig. 1 The susceptible sugarcane clone H3–8 under twisted leaf disease stress. a Twisted leaf disease of H3–8 was observed in the field. b Phenotype of three stages of Phoma sp. infection. From top to bottom: pre-onset, early, and serious symptom stages and the contained adapters, 251,482,172 and 273,757,964 protein coding genes, and 15,469 transcripts were classified clean reads were left in the NSBC _T and NSBC _CK librar- into long non-coding RNAs. Details of each sample were 1 1 ies, respectively. Using the genome of Saccharum exhibited in Table S1 and Fig. S1. spontaneum as reference (Zhang et al. 2018), we mapped the Based on the Pearson correlation coefficient of genes ex- clean reads to the reference via HISAT (hierarchical indexing pression of each sample, we obtained the correlation heatmap for spliced alignment of transcripts) (Kim et al. 2015). All of all samples (Fig. 2a). The samples from the same cultivar samples conditioned over 70% mapping ratio (average conditioned high correlation Pearson value. Of course, two 76.74%). The details were summarized in Table 2. biological repeats of each stage clustered together in the Cluster Dendrogram (Fig. 2b). That means two biological re- Gene Expression Statistics peats of each stage were consistent and satisfied for further analysis. To elucidate gene expression profile of un-susceptible and susceptible cultivars, we annotated the aligned reads accord- ing to the reference genome. The result showed that there were Table 2 Genome mapping summary of all samples 91,386 genes expressed that included 65,391 known genes and 25,995 novel genes. In the 96,101 annotated transcripts, Sample Total Clean Reads Total Mapping Ratio 53,481 transcripts with novel alternative splicing subtypes en- code known proteins, 27,151 transcripts were defined as novel NSBC1_CK1–1 49,273,558 76.43% NSBC1_CK1–2 51,677,702 77.54% NSBC1_CK2–1 47,765,358 71.71% NSBC1_CK2–2 44,108,084 75.97% Table 1 Overview of the transcriptome sequencing and de novo assembly results NSBC1_CK3–1 42,718,254 77.39% NSBC1_CK3–2 44,409,480 76.30% Description NSBC1_T NSBC1_CK NSBC1_T1–1 44,375,910 77.42% Raw reads 258,301,460 279,952,436 NSBC1_T1–2 42,446,806 78.30% Q20 percentage 55.56% 55.61% NSBC1_T2–1 23,302,262 78.14% Clean reads 251,482,172 273,757,964 NSBC1_T2–2 23,302,264 77.40% Total reads 525,240,136 NSBC1_T3–1 42,487,034 76.63% Total nucleotides (nt) 65,780,017,250 NSBC1_T3–2 41,604,074 77.65% 296 Tropical Plant Biol. (2019) 12:293–303 Fig. 2 Correlation analysis of all samples. a Heatmap was constructed by the Pearson correlation coefficient of genes expression. b Cluster analysis of all samples based on genes expression quantity Differentially Expressed Genes (DEGs) part, and organelle conditioned most enriched DEGs in the in Un-Susceptible and Susceptible Cultivars cellular component. In the term of molecular function, DEGs were mainly distributed in catalytic activity and bind- The significance of the gene expression difference was deter- ing. Most enriched GO term are corresponding to various mined according to the threshold of FDR (False Discovery aspects of activated metabolism (e.g. “metabolic process” Rate) < 0.05 and |log FC (Fold Change) | >1. In the compar- and “catalytic activity”) and stress response (e.g. “biological ison to the un-susceptible clone H3–19, the genes expression regulation” and “organelle”). We also performed a BLAST of susceptible clone H3–8 showed 8408 up-regulated and (The Basic Local Alignment Search Tool) (E-value 7834 down-regulated in pre-onset stage, 7334 up-regulated <0.00001) analysis of the DEGs against the KEGG (Kyoto and5558down-regulatedinearlystage,and24,456up- encyclopedia of genes and genomes) database. The DEGs regulated and 10,468 down-regulated in serious symptom were mainly enriched in metabolic pathways, biosynthesis of stage. (Fig. 3). The Gene Ontology (GO) analysis classified secondary metabolites, and plant-pathogen interaction (Fig. DEGs into three main categories: biological function, cellular S2). These are consistent with the sugarcane activities in re- component, and molecular function (Fig. 4). DEGs mainly sponse to Phoma sp. infection. According to these findings, accumulated in the cellular process, metabolic process, and we analyzed specific pathways and genes related to disease biological regulation in the biological function. The cell, cell defense. Fig. 3 DEG analysis in the susceptible cultivars. Numbers of up- and down-regulated genes identified in the pre-onset, early, and serious symptom stages in susceptible genotypes Tropical Plant Biol. (2019) 12:293–303 297 Fig. 4 Gene ontology (GO) annotation for DEGs in three stages Ingenuity Pathways Analysis response to biotic and abiotic stress (Bari and Jones 2009). The DEGs between the susceptible and un-susceptible culti- Plant hormones are the first reactors under environmental vars provided us an opportunity to study how the hormone stress (Verma et al. 2016). Among the major hormones, pathways response to twisted leaf disease. We analyzed the abscisic acid (ABA), ethylene (ET), jasmonates (JA), and expression patterns of genes involved in hormone biogenesis salicylic acid (SA) play essential roles in regulating plant in three different stage of Phoma sp. infection (Fig. 5; 298 Tropical Plant Biol. (2019) 12:293–303 Fig. 5 Ingenuity analysis of DEGs in hormone pathways during fungi infection process. Fold change (log ) in hormone biosynthesis genes at different stages of the infection process relative to the resistance condition Table S1). As the main response hormones in biotic stress, the secondary plant reflections (Robert-Seilaniantz et al. 2011). biosynthesis of SA, JA, and ET up-regulated in the susceptible These results show that twisted leaf disease triggered the re- cultivar (genes in green, brown, and blue lines). The SA, action of the hormones network. which is generally involved in the defense against biotrophic Also, we analyzed the specific biosynthesis genes in SA, and hemi-biotrophic pathogens (Loake and Grant 2007), was JA and ET pathway, respectively. In details, the phenylalanine enriched with high expression (~4 folds up-regulated) of ammonia-lyase, an enzyme of phenylalanine ammonium ly- LOC_Os02g41630-D10and Sspon.04G0008070-3C (Fig. 5, ase (PAL) pathway in SA synthesis (Yokoo et al. 2018), was green lines). JA and ET, which involve in the defense of increased in expression at the pre-onset of Phoma sp. infec- necrotrophic pathogens and herbivorous insects (Gamalero tion. The isochorismate synthase 1, which is involved in the and Glick 2012; Wasternack and Hause 2013), accumulated synthesis of SA (Yokoo et al. 2018), showed lower expression through up-regulating biosynthesis genes (Fig. 5,brown level in three stages of the susceptible cultivar (Table S1). In lines). ABA, auxin (IAA), and cytokinins (CK) biosynthesis the JA pathway, 12-oxophytodienoate reductase, which catal- genes also exhibited differential expressions in the susceptible yses the reduction of 10, 11-double bonds of 12- cultivar, which resulted from hormone pathways crosstalk and oxophytodienoate to 3-oxo-2-(2′-pentenyl)-cyclopentane-1- Tropical Plant Biol. (2019) 12:293–303 299 octanoic acid (OPC-8:0) (Tani et al. 2008), conditioned rela- expressed lower at pre-onset and kept increasing in the later tive higher expression in the susceptible cultivar (Table S1). stages. That means R genes with such expression patterns in The aminotransferase, a gene family including VAS1 which the susceptible cultivar were retarded in the defense of Phoma participates in auxin and ethylene biosynthesis (Zhang et al. sp. infection. Some R genes like Sspon.02G0020920-2B, 2012), varied in expression at different stages of the twisted Sspon.02G0041050-1B, and Sspon.01G0037110-1B, condi- leaf disease. These findings supported that the infection of tioned higher expression or/and irregular expression patterns Phoma sp. induced the response of the hormones network. in three stages. These R genes could respond to Phoma sp. infection in the susceptible cultivar. Also, the sequence anal- ysis of these representative genes, including alternative splic- Evaluation of Differentially Expressed ing and SNPs, didn’t offer clue for the structural mutation in Disease-Resistant Genes the susceptible cultivar (data not shown). The expression level analysis of R genes revealed that the null and retarded re- To defend against infection of pathogen, the expression of sponse of R genes in the Phoma sp. infection, especially at resistance (R) genes that encode proteins containing con- the pre-onset stage might be one of the reasons that the H3–8 served nucleotide binding site plus leucine-rich repeat (NBS- cultivar is susceptible to twisted leaf disease. LRR domain) are induced (Bakker et al. 2006). To elucidate the R genes involved in the twisted leaf disease, we annotated R genes in DEGs based on PRG (Pathogen receptor genes) database. (Table 3). There were 46%, 42%, and 26% R genes Discussion conditioned lower expression in pre-onset, early stage, and serious symptom of susceptible cultivar relative to the control, The transcriptome, which is influenced by external environ- respectively. That means a part of R genes in the susceptible mental conditions, can effectively reveal the response mecha- cultivar were null/retarded in the response of Phoma sp. nism of biotic and abiotic stress in plants (Wei et al. 2011). In infection. our study, the natural infected cultivar H3–8 was defined as From the R genes expression profile, we selected out rep- susceptible cultivar and the un-susceptible cultivar (H3–19) resentative of each class to exhibit their expression patterns in was used as control. With two biological repeats, we used three stages of Phoma sp. infection (Fig. 6). transcriptome profiling to detect the genes associated with Sspon.06G0034670-1D, Sspon.04G0004450-1A and twisted leaf disease and obtained 65,780,017,250 bp data from Sspon.02G0029950-1T dramatically decreased in three stages three stages of the twisted leaf disease. Using the genome of of Phoma sp. infection. Sspon.01G0011870-1A and Saccharum spontaneum L. as reference (Zhang et al. 2018), Sspon.01G0013680-3C also showed lower expression level the average mapping ratio of twelve samples was 76.74%. The in the three stages. R genes like these genes were null function annotation of aligned reads showed 91,386 genes expressed in the susceptible cultivar. Sspon.05G0016050-1A, (65,391 known and 25,995 novel), and 96,101 annotated tran- Sspon.08G0026530-1C and Sspon.03G0025750-3D scripts, including 53,481 novel alternative splicing subtypes Table 3 Classification of Class Count Representatives Description annotated R genes and representatives for ingenuity NL 543 Sspon.02G0020920-2B Disease resistance protein (NBS-LRR class) family analysis RLP 683 Sspon.02G0041050-1B Pyridoxal phosphate-dependent transferases superfamily protein CNL 242 Sspon.05G0016050-1A HOPZ-ACTIVATED RESISTANCE 1 N 309 Sspon.08G0026530-1C NB-ARC domain-containing disease resistance protein TNL 100 Sspon.06G0034670-1D TIR-NBS-TIR-TIR-WRKY type disease resistance protein RLK 7 BGI_novel_G009740 S-locus lectin protein kinase family protein RLK-GNK2 94 Sspon.01G0015790-1A cysteine-rich RLK (RECEPTOR-like protein kinase) CN 67 Sspon.04G0004450-1A LRR and NB-ARC domains-containing disease resistance protein Other 40 Sspon.01G0037110-1B NADPH HC toxin reductase-like protein T 17 Sspon.01G0011870-1A NAC domain containing protein Mlo-like 21 Sspon.01G0013680-3C Seven transmembrane MLO family protein L 5 Sspon.03G0025750-3D Protein phosphatase 1 regulatory subunit RPW8-NL 4 Sspon.02G0029950-1T ADR1-like 1 disease resistance protein 300 Tropical Plant Biol. (2019) 12:293–303 Fig. 6 Differential expression analysis of specific R genes in the susceptible cultivar. Genes were selected out as representative based the tendency of each R gene class. Fold change (log ) was achieved from the FPKM value of T and CK encode known proteins, 27,151 novel protein coding genes, down-regulated, especially in pre-onset stage. For example, and 15,469 long non-coding RNAs. With these data, we ana- belongs to LRR and NB-ARC domains-containing proteins lyzed the DEGs, including hormone pathways and R genes, of which members participate in disease resistance (Fischer between the susceptible cultivar and the control. Also, these et al. 2016). is predicted to encode RGA protein which con- data provided a foundation for the further analysis of the twist- tains NB-ARC domain and functions disease resistance ed leaf disease, such as specific gene characterization, resis- (Cesari et al. 2013). is descripted as CRKs (cysteine-rich pro- tant genes selection, and immune network description. tein kinase), which involves in the ROS signaling pathway The comparison between susceptible cultivar and the con- and plays important roles in the elimination of fungal growth trol identified 16,242, 12,892 and 34,924 DEGs in pre-onset, damage (Niina Idänheimo 2015). The low expression of these early stage and serious symptom stage, respectively (Fig. 3). R genes in the twisted leaf disease would drag down the rapid These DEGs are involved in critical biological activities that response of R genes defensive system. Besides ~40% down- are essential for disease resistance (Fig. 4). Ingenuity pathway regulated R genes, there were still a number of R genes con- analysis revealed hormone biosynthesis genes variations in ditioned normal expression level or even higher expression the susceptible cultivar (Fig. 5). The SA and ET pathways level in the susceptible cultivar. Unfortunately, these high were induced in the infection of Phoma sp. at the pre-onset expressed R genes were unable to eliminate the pathogens stage. Along with the spread of the disease, JA and ET syn- successfully. These increased expressions might be a result thesis increased and maintained high levels in the serious of universal reactions of sugarcane under pathogens stress symptom stages. As three major hormones involved in stress rather than the specific response of Phoma sp. infection. resistance, SA is a biotrophic and hemi-biotrophic pathogen- Such phenomenon was commonly reported in the studies of triggered signaling pathway, and JA combined with ET sig- other species (Heath 2000;van Loon 2015). naling pathways are induced by necrotrophic pathogens (Glazebrook 2005). Therefore, twisted leaf disease, caused by fungal pathogens, first triggers the SA pathway as response Materials and Methods to pathogens infection. The high level of JA and ET in serious symptoms might partially result from tissue damage in dis- Plant Materials and Sampling ease. Other hormones, including ABA, auxin, and CK, crosstalk with major disease response hormones (SA, JA, Narenga porphyrocoma, which is an important relative genus and ET) and construct a network to respond to disease stress of sugarcane, was collected from a barren mountainous area in (Verma et al. 2016). Guangxi province, China. We obtained the BC generation Plant disease resistance (R) genes are prevalent in all plant offspring of the Narenga porphyrocoma via crossing and species and harbor a conserved LRR domain (Meyers et al. backcrossing with sugarcane varieties several years. Among 1998). R genes are defined as gene-for-gene interaction and the BC offspring, H3–8 was susceptible to twisted leaf dis- specifically recognize an avirulence protein encoded by a ease at the sugarcane elongating stage, whereas H3–19 was pathogen with a hypersensitive response (Flor 1956). In the not. H3–8 was proven to affect stable twisted leaf disease R gene analysis of the susceptible cultivar, around 40% R occurrence after 3 years based on field and greenhouse genes, including those that play essential functions, were observations. Tropical Plant Biol. (2019) 12:293–303 301 Plantlets at the same growth stage were selected and Data Filtering and Genome Mapping planted in pots filled with a mixture of peat soil and washed sand, and grown in a greenhouse at the Sugarcane Research The raw reads were filtered according to the following criteria: Institute, Guangxi Academy of Agricultural Sciences. The (1) reads containing adaptor, (2) reads with unknown nucleo- natural infected plants were selected and defined as the sus- tides larger than 5%, and (3) low-quality reads (the rate of ceptible cultivator. The leaf samples of the susceptible sugar- reads with a quality value of ≤10 was morethan20%.The cane clone H3–8 were collected at three stages corresponding clean data was mapped to the genome of Saccharum to the pre-onset (named NSBC _T1), early (named spontaneum L. (Zhang et al. 2018) via HISAT (v2.0.4; −- NSBC _T2), and serious symptom (named NSBC _T3) phred64 –sensitive –no-discordant –no-mixed -I 1 -X 1000) 1 1 stages of the disease, respectively. The leaf samples of the (Kim et al. 2015). The transcripts were re-constructed via un-susceptible sugarcane clone H3–19 were simultaneously StringTie and predicted using Cuffcompare tool in Cufflink collected, labeled NSBC _CK1, NSBC _CK2, and (Pertea et al. 2003; Trapnell et al. 2010; Pertea et al. 2016). 1 1 NSBC _CK3, respectively. Two biological repeats of each sample were collected. All samples were immediately frozen Differential Gene Expression in liquid nitrogen and stored at −80 °C. The DEGs analysis was performed as the description of RNA Extraction and Quality Determination DEGseq (Wang et al. 2010) and corrected the P-values to Q-values based on Benjamini-Hochberg method The total RNA was extracted using TRIzol reagent (Benjamini and Hochberg 1995) and Storey-Tibshirani (Invitrogen, Carlsbad, CA, USA) and treated with RNase- method (Storey and Tibshirani 2003). GO (gene ontolo- free DNase I and RNA integrity number (RIN > 8.0). RNA gy) terms were assigned based on the best-hits BLASTx quality and quantity were verified using a NanoDrop 1000 resulted from NR alignments that were derived from spectrophotometer (Wilmington, DE, CA, USA) and an Blast2GO (v2.5.0) against GO database (release- Agilent 2100 Bioanalyzer (Santa Clara, CA, USA) prior to 20,120,801). The DEGs were aligned against KEGG the library construction. No sign of degradation was found. (Kyoto Encyclopedia of Genes and Genomes) by BLASTx package with threshold of E-value <=10–5. cDNA Library Construction and Sequencing Ingenuity Analysis of DEGs The poly(A) RNA was fragmented into approximately 300-nt fragments using RNA Fragmentation Reagents (Ambion, Thericehormonesproteinswereusedasreference to Austin, TX, USA). Using these short fragments as templates, identify the hormone genes in sugarcane (Cohen et al. the double-stranded cDNA was synthesized with random 2017). The longest transcript of genes was selected as primers (Invitrogen, Carlsbad, CA, USA). The end repair of representative for analysis. All predicted protein se- these cDNA fragments was subsequently performed with quences were aligned against the sugarcane gene models Klenow polymerase, T4 DNA polymerase, and T4 polynucle- by BLASTp (Identity>50%; Coverage>50%; E-value: otide kinase (NEB,Ipswich, MA, USA). Illumina adapters 1× 10 ), and defined as homologs of hormones biosyn- (containing primer sites for sequencing and flow cell surface thesis genes. Also, we aligned all predicted hormone annealing) were ligated to the end-repaired fragments using genes against the sugarcane genome via tBLASTn (E- T4 DNA ligase (Invitrogen, Carlsbad, CA, USA). The prod- value: 1E-10), and used GeneWise (v2.2.0) to identify ucts were enriched for the cDNA fragments using a Qiaquick the structure of candidate hormone genes. Among these Gel Extraction Kit (Qiagen, Duesseldorf, Germany) and am- genes, the ones with non-significant different expression plified with polymerase chain reaction (PCR) to prepare the in three stages were eliminated. The final candidates sequencing library. Agilent 2100 Bioanalyzer (Agilent, were aligned against KEGG database (version:84). The Beijing, China) was used to detect the quantity and quality log fold of each gene was obtained from the case- of the cDNA. Then, the paired-end RNA-seq libraries were control group. The patterns of the gene in each group prepared following Illumina’s protocols and sequenced on the were compared. Similarly, predicted proteins were Illumina HiSeq 2500 platform (Illumina, San Diego, CA, aligned against the PRG database using BLASTp. The USA).Sequencingwas performedinaflowcellonan results were filtered (E-value: 1e10), and the best hit of Illumina HiSeq 2500 sequencer using the TruSeq Paired- predicted protein was retained. The fpkm (Reads per End Cluster Kit v3 (Illumina PE-401-3001) and the TruSeq kilobase of exon per million reads mapped) of each SBS HS Kit v3200 cycles (Illumina FC-401-3001), generat- predicted resistant gene in the case sample was filtered ing 2 × 125 bp. out and a downstream analysis was performed. 302 Tropical Plant Biol. (2019) 12:293–303 Acknowledgments We thank Mick Rose in Primary Industry Department kinase (LRR-RLK) subfamily in angiosperms. Plant Physiol 170: of Australia for the assistance of writing paper. 1595–1610 Flor HH (1956) The complementary genic system in flax and flax rust. Adv Genet 8:29–54 Author Contributions Hongwei Tan, and Xihui Liu conceived and de- Fracasso A, Trindade LM, Amaducci S (2016) Drought stress tolerance signed the experiments. Jinju Wei and Huiping Ou performed the exper- strategies revealed by RNA-Seq in two sorghum genotypes with iments. Xiaoqiu Zhang, Ronghua Zhang, Hui Zhou, Yiyun Gui,Haibi Li, contrasting WUE. BMC Biol 16:115 Yangrui Li, Rongzhong Yang and Dongliang Huang performed cross and backcross of Narenga porphyrocoma, planted sugarcane and collected Gamalero E, Glick BR (2012) Ethylene and abiotic stress tolerance in samples. Zhihui Xiu, Junhui Chen and Huayan Jiang analyzed the data. plants. In: Environmental adaptations and stress tolerance of plants Zhihui Xiu and Xihui Liu wrote the manuscript. All authors have read and in the era of climate change. Springer, New York, pp 395–412 approved the final manuscript. Gao Y, Fang F, Liu XH et al (2012) Identification of progeny from crosses between sugarcane (Saccharum spp.) and intergeneric hybrid com- plex (Erianthus arundinaceus × Saccharum spontaneum)withmo- Funding This work was financially supported by the National Science lecular markers. J Plant Genet Res 13:912–916 Foundation of China (31760368, 31101195), Guangxi Fund (GKAA17202042–6), Fund of Guangxi Academy of Agricultural Glazebrook J (2005) Contrasting mechanisms of defense against Sciences (GNK2018YT02 and GNK2018YM01) and Fund of Modern biotrophic and necrotrophic pathogens. Annu Rev Phytopathol 43: Agriculture Technology (CARS-170105, gjnytxgxcxtd-03). 205–227 Grivet L, Arruda P (2001) Sugarcane genomics: depicting the complex ge- nome of an important tropical crop. Curr Opin Plant Biol 5:122–127 Compliance with Ethical Standards Hameed U, Pan YB, Iqbal J (2015) Genetic analysis of resistance gene analogues from a sugarcane cultivar resistant to red rot disease. J Conflict of Interest The authors declare no conflict of interest. Phytopathol 163:755–763 The data that support the findings of this study have been deposited in Heath MC (2000) Nonhost resistance and nonspecific plant defenses. the CNSA (https://db.cngb.org/cnsa/) of CNGBdb with accession code Curr Opin Plant Biol 3:315–319 CNP0000220, and NCBI with accession code SAMN05428728 to Huang DL, Gao YJ, Gui YY, Chen ZL, Qin CX et al (2016) SAMN05428739. Transcriptome of high sucrose sugarcane variety GT35. Sugar Tech 18:520–528 Open Access This article is distributed under the terms of the Creative Idänheimo N (2015) The role of cysteine-rich receptor-like protein ki- Commons Attribution 4.0 International License (http:// nases in ROS signaling in Arabidopsis thaliana. 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Planta 227:517–526 Trapnell C, Williams BA, Pertea G, Mortazavi AM, Kwan G, van Baren Publisher’s Note Springer Nature remains neutral with regard to jurisdictional MJ, Salzberg SL, Wold B, Pachter L (2010) Transcript assembly and claims in published maps and institutional affiliations. Affiliations 1 2 1 2 2 1 1 Jinju Wei & Zhihui Xiu & Huiping Ou & Junhui Chen & Huayan Jiang & Xiaoqiu Zhang & Ronghua Zhang & 1 1 1 1 1 1 1 1 Hui Zhou & Yiyun Gui & Haibi Li & Yangrui Li & Rongzhong Yang & Dongliang Huang & Hongwei Tan & Xihui Liu Key Laboratory of Sugarcane Biotechnology and Genetic Research Institute, Guangxi Academy of Agricultural Sciences, Improvement (Guangxi), Guangxi Key Laboratory of Sugarcane Nanning 530007, China Genetic Improvement, Ministry of Agriculture, Sugarcane Research BGI Genomics, BGI-Shenzhen, Shenzhen 518083, China Center, Chinese Academy of Agricultural Sciences, Sugarcane

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Tropical Plant BiologySpringer Journals

Published: Jun 15, 2019

References