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Tilling and Fox-Hunting: New Methods for Functional Analysis of Genes

Tilling and Fox-Hunting: New Methods for Functional Analysis of Genes ADVANCES IN CELL BIOLOGY VOL. 3, ISSUE 1/2011 (1­16) Krystyna RYBKA Department of Plant Biochemistry and Physiology, Plant Breeding and Acclimatization Institute DOI: 10.2478/v10052-011-0001-6 Summary: Theoretical and experimental bases of TILLING and FOX-hunting, new tools for precise identification of genes in functional studies are reviewed. TILLING (Targeting Induced Local Lesions IN Genomes) is a technique combining chemical mutagenesis with its sensitive and precise detection. The method involves PCR amplification of DNA samples pooled following extraction from a collection of chemically-treated organisms and a subsequent screening for mutations using Cel1 endonuclease, detecting mismatches in heteroduplexes [52]. FOX-hunting (Full-length cDNA Over-eXpressing gene hunting system) is a new method of plant gene overexpression, which enables a quick gene isolation and sequencing, paralelly with functional studies. Key words: TILLING, FOX-hunting, functional analysis of genes, mutagenesis INTRODUCTION The beginning of the twenty-first century has been called post-genomic era as more than 180 complete genome sequences started from the sequence of the phage X174 in 1977 (5,368 bp), Haemophilus influenzae in 1995, the fruit fly and Arabidopsis in year 2000, a man in 2001, rice in 2002, and in 2007 the first complete genome, 6 billion nucleotides, of one man [22] have been known. Knowledge of genomes sequences is not eqiuvalent with the knowledge about gene number and function. For example, the rice genome which is the smallest among cereals at 430 Mbp, and thus assumed to be a model for plant genome sutdies, was in silico predicted to encode approximately 50 000 potential genes, mostly of unknown function. For more than a decade, comprehensive data describing changes in gene expression profiles (mainly quantitative) have been collected. In 1996, the biotech company, Affymetrix, produced the first commercial DNA microarray. Identification of differentially expressed mRNAs using microarray technology generates enormous amounts of data. For example, a microarray experiment using cDNA derived from drought tolerant and sensitive rice varieties identified approximately 16 000 genes affected by drought stress, two thirds of which have no known function [5]. Similarly SAGE (Serial Analysis of Gene Expression) developed in the mid-nineties, and MPSSE (Massively Parallel Signature Sequencing) developed in the year 2000, which allow the mass sequencing of cDNAs fragments, generate large data sets [21, 24]. To sort and organize the large amount of experimental data which is still growing, there is a need for rapid expansion of knowledge about gene function, as well as new tools for construction and handling of multi-parameter databases [25, 33 and 34]. For many years functional analyses of genes were carried out according to principles of classical genetics, from the phenotype to the gene (TopDown/Forward Genetics). The development of molecular biology techniques has allowed the opposite way: from a mutation to a phenotype (Bottom-up/Reverse Genetics) (Table 1). Functional analysis of genes sped up with the development in the U.S. of TILLING platform (Targeting Induced Local Lesions IN Genomes) in the mid 90's, which allows massive and rapid identification of point mutations basing on DNA amplification and specific digestion [36]. The rate of accumulated functional data has also increased due to the development of the FOX-hunting method (Full-length cDNA Over-Expressing gene hunting system) in Japan. This method, patented in 2001 and made available to a broader range of researchers in 2008, allows for quick gene isolation and functional identification under conditions of gene over expression [17]. The dynamic developments of gene functional analysis has therefore provided the inspiration for overview of a conceptual framework of TILLING and FOX-hunting techniques on the background of the classical forward genetic screen. FUNCTIONAL ANALYSIS OF GENES ACCORDING TO THE PRINCIPLES OF CLASSICAL GENETICS In accordance to the principles of a classical forward genetics screen, functional analysis of genes involves identifying a gene which encodes a specific protein. Positional cloning of the gene is the example of such an approach. This method requires finding the molecular markers closely linked to the trait of interest. Depending on the nature of the trait, different methods of mapping are applied: qualitative or quantitative (QTL or associative, based on an analysis of Linkage Disequilibrium (LD)). For plants, mapping is accomplished based on segregating populations: BC2 or F2 (Back Crossed or regularly fertilized 2nd generation of offspring), doubled haploid lines (DH), recombinant lines (RILs) as well as near-isogenic lines (NILS), aneuploids or/and substitution lines [15]. Molecular markers which strongly co-segregate with the trait, are placed on the physical maps generated from contigs of clones selected from genomic libraries. After the region of DNA located between the flanking markers is sequenced, selected DNA fragments are isolated and cloned into transformation vectors. Changes in the phenotype of mutant plant gained by transformation, which are consistent with the expected result, confirm the biological function of the gene. Lack of expected phenotypic changes however is uninformative as it may be the result of gene silencing in transformants [27]. An example of such studies might be a search for a gene Pi-ta2 encoding resistance to Pyricularia grisea in rice [31]. In wheat genome several genes has been identified by positional cloning. While majority of these are genes of resistance to stresses and response to vernalization, on chromosome 5AL a regulatory locus involved in the domestication has been identified. This locus encodes genes of high gluten content in grain, as well as locus of homologous chromosomes conjugation during meiosis (Table 2). A more efficient approach to functional gene analysis is to create, identify and then analyze mutants. Mutagenesis was introduced into breeding in the early thirties, when the possibility of mutant induction by X-ray was discovered. The development of molecular biology techniques allowed for use of this approach to find genes and to understand their functions without knowledge of phenotype and /or protein products. TABLE 1. Comparison of methods of gene functional analysis: Top-down vs. Bottom-up approaches [30, modified] TABLE 2. Wheat genes identified from positional cloning projects [12, modified] Gen/QTL Lr1 Lr10 Lr21 Chromosome localization 5DL 1AS 1DS Trait Leaf rust resistance Leaf rust resistance Leaf rust resistance University/ Group lider University of Zurich/ B. Keller University of Zurich/ B. Keller Kansas State University/ B. S. Gill Kansas State University/ B. S. Gill University of Zurich/ B. Keller Qfhs.Ndsu-3bs 3BS Fusarium head blight resistance Powdery mildew resistance Resistance to stripe rust Pm3b 1AS Yr5 2BL USDA-ARS, Wheat Genetics/ K. G Campbell CSIRO Plant Industry, Australia/ E. S. Lagudah North Dakota State University/ J. D. Faris University of Adelaide/ P. Langridge University of California, Davis/ J. Dubcovsky University of California, Davis/ J. Dubcovsky University of California, Davis/ J. Dubcovsky University of California, Davis/ J. Dubcovsky University of California, Davis/ J. Dubcovsky Kansas State University/ B. S. Gill University of California, Davis/ J. Dubcovsky John Innes Centre, Colney, Norwich/ G. Moore Sr2 3BS Stem rust resistance Tsn1 5BL Host-selective toxin Ptr ToxA Boron tolerance 7BL Fr2 5BL Frost resistance VRN1 5A VRN2 5A VRN-B3 7BS EPS-1 1AL Flowering time 5AL Free threshing character GPC-B1 6BS High grain protein content Chromosome pairing locus Ph1 1A/5B FUNCTIONAL ANALYSIS OF GENES ACCORDING TO THE PRINCIPLES OF MOLECULAR GENETICS Methods for induction of mutants Physical mutagenesis Physical mutagenic agents include mostly different types of radiation: gamma (source: radioactive isotopes), X (sources: X-ray tubes, synchrotrons), ultraviolet and electrons with high and low kinetic energy (source: accelerators) [3]. Radiation energy alters the nature of chemical and short-distance physical bonds existing between atoms in bio-polymers, leading to the destruction of proper ones by the formation of invalid, which finally results in disturbances of DNA metabolism and mutation formation. The phenomenon of X-ray-induced mutagenesis was first described for the barley genome during interwar period. Comprehensive programs for diversity generation by physical mutagenesis in collections of crop plants were undertaken in the '60s. The cooperation of FAO (Food & Agriculture Organization) and IAEA (International Atomic Energy Agency) in the peaceful uses of atomic energy has resulted in many breeding programs worldwide. Since then, 2.570 mutants were registered throughout the world, including the 1020 mutants of major cereal crops: 439 mutants of rice, 305 mutants of barley, 204 mutants of wheat and 71 mutants of maize [26]. Most of those mutations were induced by -rays (30%) and to a lesser extent by the X-rays (3%). Two thirds of these mutants were generated in China. Notably, for the spectacular achievements of physical mutagenesis belongs barley variety well yielding on the attitude above 5 000 m above sea level in Andes in Peru. To natural phenomenon generated by mutagenesis belongs also the variety of rice that can grow in water of high salinity, in the Mekong Delta in southern Vietnam [38]. In Poland, significant achievement of -ray mutagenesis was self-ending mutant of horse bean (V. faba var. equina) [1]. Chemical mutagenesis Chemical mutagenesis (mostly point mutation) occurs under the influence of conformational changes in one pair of nucleotides in the complementary chains of DNA. Chemicals that cause conformational changes include analogues of nucleotide bases (5-bromouracil or 2-aminopurine); hydroxyl agents (hydroxylamine); alkyl reagents (ethyl methanesulfonate or dimethylnitrosamine); deamination reagents (nitrous acid or sodium sulfite). Intercalation of aromatic compounds molecules, such as proflavine or ethidium bromide, into the DNA helix may also lead to disruptions in DNA replication, repair, or recombination. The likelihood of generating a dominant point mutation in the cereals is less than half permil [18]. Transparent example of the use of chemical mutagenesis are stidies of Finkelstein group, which used A. thaliana mutants insensitive to abscisic acid and explained many aspects of signal transduction on the hormone-dependent path [9]. An important achievement of Polish breeders was obtaining of winter oilseed rape forms characterized by increased content of oleic acid and reduced content of linolenic acid in the seeds of mutagenesed plant in comparison to the double-improved varieties [32]. TABLE 3. Existing and proposed projects of gene functional analysis in grass species based on chemical mutagenesis and TILLING analytical system [37, modified] Cereal Crop BARLEY cv. Optic Project DIStilling (SCRI) Mutagen Web- page address http://germinate.scri.sari.a c.uk/barley/mutants/ cv. Barke GABI-TILL TILLMore Risø National Labs, KVL Denmark Maize TILLING Project, Purdue cv. Morex www.gabi-till.de/project/ ipk/barley.html www.gabi-till.de/project/ipk/barley.html www.distagenomics.unibo .it/TILLMore/ www.pgrc.ipkgatersleben.de/barleynet http://genome.purdue.edu/ maizetilling/ www.croptailor.com/Enge lsk/engindex.htm www.tilling.ucdavis.edu/ index.php/Rice_Tilling cv. Lux MAIZE OAT CropTailor AB RICE RiceTILL (UC Davis) or MNU + NaN3 MNU (ssp. japonica) WHEAT T. aestivum T. monococcum Mishima Arcadia Biosciences Rothamstead Research (RRes) http://www.rothamsted.bb src.ac.uk/ppi/staff/hcj.html www.rothamsted.ac.uk/cpi/ optiwheat/indexcontent.html T. durum OPTIWHEAT SORGO SWITCHGRASS BRACHYPODIUM USDA, Lubbock, TX Purdue TILLING Project Risø National Labs, KVL Denmark NaN3 http://genome.purdue.edu/ www.risoe.dk/rispubl/BIO /biopdf/ris-r-510.pdf. For over half a century, chemical mutagenesis has been an important method for generating mutants, and in the last decade, due to the development of TILLING platform, new comprehensive research projects have been established (Table 3). Insertional mutagenesis Insertional mutants are obtained by transformation of wild plants with naturally occurring retrotransposons or T-DNAs [20, 35]. Mutants generated from these methods typically arise from gene silencing (called: knock out or loss-offunction) due to insertion into the coding sequence [40]. Naturally occurring insertional mutants were first analyzed in maize due to the presence of active transposon elements [23]. This discovery led Barbara McClintock to be awarded a Nobel Prize (1948). Cloning of Ac and Ds transposons of maize [7] enabled the transformation of species whose genomes have not active transposon system. The first gene isolated using transposon tagging, was the tomato Cf-9 gene required for resistance to the fungus Cladosporium fulvum [16]. Such a strategy of functional gene analysis dominated for nearly two decades in laboratories worldwide, so it's not surprising that the number of generated, in this way, mutant plant is more than 290 000 [11]. Factors limiting the use of insertional mutagenesis include the inability to analyze the genes when multiple copies are inserted and also the inability to study genes expressed in early stages of plant development due to embryo lethality. In addition, these mutants, in contrast to the T-DNA mutants, are unstable, which is a direct result of the properties of transposons. T-DNA is integrated into the genome of a plant with an average of 1.5 copies in the genome of Arabidopsis or rice [8]. Table 4 summarizes the available research collections of insertion mutants in rice. In general, transformations with either transposons or T-DNA lead mainly to recessive mutant. Therefore, selecting a suitable mutant for further study requires a number of crosses and analysis of the phenotypes of many offsprings; in case of Cf-9 gene approximately 160 000 mutants of tomato individuals were studied [16]. Increased transformation efficiency coupled with simplifications in syst of plant regeneration and mutant identification resulted in development of the FOX-hunting system. This system does not require in vitro cultures, as embryos in young inflorescences, dipped in a solution of Agrobacterium tumefaciens, are transformed with a T-DNA binary vector [4]. T-DNA binary vectors used in these experiments carry a full-length cDNA of the gene of studied organism [14]. This promotes the generation of mutants characterized by ectopic gene expression which is also referred to as the gain-of-function (Fig. 1). In recent years, RNAi technology has become a powerful tool for functional analysis of genes, however, as it is based on post-translation gene silencing [27, 29] it is not the object of the present article. TABLE 4. Rice mutant resources [19, modified] Institution total Dongjin, Hwayoung T-DNA ET/AT Tos17 T-DNA ET Tos17 T-DNA AT 30 000 18 382 31 000 TRIM http://trim.sinica.edu.tw 45 000 14 137 100 000 13 745 http://urgi.versailles.inra.f r/OryzaTagLine 17 414 11 488 (03.`09) 150 000 84 680 400 000 RISD http://an6.postech.ac. 58 943 Genotype MMutagen Mutated loci classified available Web site Leader/e-mail address G. An genean@postech.ac. kr/pfg E. Guiderdoni guiderdoni@cirad.fr POSTECH South Korea CIRADINRAIRD-CNRS, Genoplante France Tainung 67 Nipponbare IPMB, Academia Sinica Taiwan Zhonghua 11 Zhonghua 15 Nipponbare T-DNA ET 113 262 16 158 26 000 (12.`08) 14 197 1 101 97 500 8 840 8 840 11 000 1 009 Zhonghua 11 T-DNA ET T-DNA 1 009 Y.C. Hsing bohsing@gate.sinica. ed.tw Q. Zhang qifazh@mail.hzau.ed u.cn Huazhong Agricultural University China RMD http://rmd.ncpgr.cn SIPP China Nipponbare Zhonghua 11 http://ship.plantsignal.cn/ home.do http://www.pi.csiro. au/fgrttpub F. Fu ship@sibs.ac.cn P. Wu clspwu@zju.edu.cn Zhejiang University China TABLE 4. Rice mutant resources [19, modified] Institution total Nipponbare Tos17 500 000 34 844 34 844 http://tos.nias.affrc.go.jp Genotype Mutagen Mutated loci classified available Web site Leader/e-mail address H. Hirochika hirohiko@nias.affrc. go.jp V. Sundaresan sundar@ucdavis.edu NIAS Japan UC Davis USA Nipponbare Ac-Ds GT Spm/dSpm Ac-Ds GT 30 000 4 820 4 820 KRDD http://www. niab.go.kr/RDS 20 000 Ds 4 735 4 630 dSpm 9 036 9 469 http://www-plb.ucdavis. edu/ Labs/sundar Gyeongsang National University; South Korea 20 000 3 500 2 000 Nipponbare Ac-Ds GT Dongjin Byeo C.-D. Han cdhan@nongae.gsnu. ac.kr R. Srinivasan sri@tll.org.sg Temasek Lifesciences, Singapore Nipponbare Ac-Ds ET Ac-Ds GT/ET 16 000 25 000 1 380 1 300 EU-OSTID France Nipponbare http://orygenesdb. cirad.fr E. Guiderdoni guiderdoni@cirad.fr http://www.genomics.zju. edu.cn/ricetdna CSIRO Plant Industry Australia 611 ~50% nonfertile N.M. Upadhyaya narayana.upadhyay a@ csiro.au FIGURE 1. The binary vector pBIG2113SF used in the FOX-hunting system: LB/RB ­ T-DNA border sequences; EI ­ two tandem repeats of sequence strengthening the transcription (5-upstream sequence of CaMV 35S promoter -419 to -90 bp; P35S ­ P35S, CaMV 35S promoter -90 to -1 bp; ­ 5'-upstream sequence of TMV, which increases translation effectiveness of inserted gen; NOS-T ­ polyadenylation signal of nopaline synthase gen from Ti plasmid; Hyg ­ hygromycin resistance gene; GS4, GS6 ­ sequences of PCR starters to GS4 and GS6 primers used to recover the cDNAs; SfiI(A), SfiI(B) ­ restriction sites for SfiI endonuclease; RAFL cDNA ­ RIKEN Arabidopsis Full-Length cDNA METHODS OF EXPLORATION AND ANALYSIS OF MUTANTS Brief description of the TILLING-method [36] TILLING technique combines traditional chemical mutagenesis with the identification of point mutantion using SNPs (Single Nucleotide Polymorphism). The critical step in this procedure is in obtaining a sufficiently large population of mutant seed using standard chemical mutagenesis. Following mutagenesis, M1 plants are self-fertilized and the M2 seeds are collected and sown out. DNA from the M2 plants is isolated and used in multiplex PCR reactions. In order to identify mutations in the gene of interest, gene-specific primers are used to amplify M2 DNA. PCR products are then denaturated and left to cool which causes heteroduplex of unpaired basepairs in the mutation place. By digesting these PCR products with endonuclease Cel1, mismatched bases arising from mutations are identifiable after electrophoresis on sequencing gel (Fig. 2). The classic method of SNP identification bases on multiplying, and then sequencing of selected genes for each individual plant from studied population. The TILLING method of mutant identification is faster, because instead of costand time-consuming sequencing, restriction enzyme digests and standard DNA electrophoresis is used. In addition as PCR amplification is multiplexed, and the result of proper analysis accurately indicates the mutant, which not only allows the direct typing for usage in functional studies, but also, in case of plants, allows the immediate use of the mutant in breeding programs. Brief description of the FOX- hunting [14, 17] This concept was developed by a team of RIKKEN (Japan) and published under the name Fox-hunting. The strategy has been developed for A. thaliana and it is a modification of Arabidopsis transformation method by binary vectors that are transferred into plant cells by A. tumefaciens. Essential for the effectiveness of the transformation are sequences of enhancers and transcription terminators, placed between the left and right T-DNA border sequences (LB andRB respectively). Typically, in a FOX-hunting system the binary vectors contains between border sequences, two tandem repeats of transcription amplifier (sequences -490 to -90 bp preceding the promoter of 35S of cauliflower mosaic virus CaMV); the 35S promoter (sequence -90 to -1 bp of the CaMV virus), leader sequence from the tobacco mosaic virus (which increases the efficiency of translation of the introduced gene), a cassette containing the gene of interest followed by a transcription terminator of the nopaline synthase gene (nos) from the Ti plasmid and finally a gene for resistance to the antibiotic Hygromycin B- a mutant selection gene, between the border sequences. The gene of interest is inserted into the cassette flanked by the GS4 and GS6 primer sequences which facilitate gene identification in the mutant and contain the recognition sequence for SfiI. FIGURE 2. Schematic diagram representing the TILLING technique. Equal amounts of mutant DNAs as well as the DNA of the wild plant are mixed and amplified in PCR reaction with gene specific primers. Digestion of that DNA by Cel1 endonuclease, specific to unpaired bases of the double-helix DNA, enables mutant identification after the electrophoresis on sequencing gel [10, 13, modified]. In order to generate a library in a binary vector, single copies of each gene collected in cDNA libraries constructed in vectors LambdaZAP and Lambda FLC1-B are used . In order to standardize of the library, four bacterial genes that cause known phenotypic changes and plants sterility were used. These genes have been cloned at the cleavage site of the restriction enzyme SfiI. A mixture of genomic and bacterial cDNA was prepared in such concentration that in mutants bacterial gene expression should be seen with a frequency of 1 / 7500 clones. The mixture was digested with SfiI, and then ligated with a modified binary vector pBIG2113N in which the XbaI restriction enzyme site include SfiI adapters, to enable directional cloning in the "sense" orientation. Escherichia coli DH10B was then transformed by electroporation. While preparing a library is expensive and time-consuming, transformation of A. thaliana is relatively straight forward. Once a library has been generated in A. tumefaciens, the Agrobacterium is resuspended in a 100-200 ml solution containing 5% sucrose and 0, 05% surfactant Silwet L-77. Inflorescences of A. thaliana are immersed in this solution for 2-3 seconds, with gentle stirring, so as to cover them with a layer of a suspension of A. tumefaciens. After transformation plants are grown in phytotron chamber under elevated relative humidity followed by cultivatation in a standard manner. Seeds harvested from transformed plants are germinated on agar supplemented with Hygromycin B for selection of transgenic plants. Over 15 000 fertile transformants were obtained using this method, which accounts for 77% of the cDNA library. To determine the transformation efficiency 24 lines FOX were tested for the presence of the Hygromycin B resistance using Southern hybridization. All lines tested contained at least one copy of the the Hygromycin B resistance gene with the average number of integrated vectors in the genome of a single line of FOX was 2.6. The average length of DNA inserts in the population of FOX lines was about 30% less than the average length of inserts in cDNA library in A. tumefaciens and ranged from 0.2 to 4.6 kb, with a median 11,4 kb. A small number of transformants with inserts smaller or larger than the insert in the library in binary vector were detected. The number of transformants with phenotype induced by the bacterial genes was higher than expected, which was explained by differences in multiplication speed of A. tumefaciens cells that carried plant or bacterial library. Currently, several FOX-hunting projects for cereal genomes are introduced and implemented throughout the world (Table 5). Advantages of the FOX-hunting system include the small percentage of cosuppressed genes due to full-length cDNA clones and libraries in the standard binary vectors; a reduction in the number of basic metabolism gene (housekeeping genes) in the standard libraries the facilitation in phenotype analysis which is caused by a short life cycle of A. thaliana, and easy isolation and sequencing of genes. The disadvantage of this system is reduction of phenotypic analysis only to genes used for construction of the library in A. tumefaciens. TABLE 5. FOX-hunting projects in studies of cereal genomes FOX lines [literature] A. thaliana [14, 17] Genome Project aim / details Project Leader rice searches for resistance genes, especially for high temperature; number of mutants: 23 000 Ichikawa Y. youichi@psc.riken.jp rice [28] rice use of the ubiquitin promoter; acquisition and phenotypic analysis of mutants number of mutants: total- 12 000, with single insertion: 8322 Ichikawa Y. youichi@psc.riken.jp A. thaliana [6] Bruguiera Gymnorhiza searches for resistance genes against abiotic stresses Tada Y. tadayui@bs.teu.ac.jp wheat [39] wheat project at the initial stage, small number of stable mutants were obtained Steber C. jzale@utk.edu SUMMARY TILLING and FOX-hunting techniques alow for acceleration of gene functional analysis. Programs that use them can also be an additional source of diversified material used by breeders. TILLING system has been developed to study chemically induced mutants. It enables for immediate and direct introduction of mutants into breeding programs. FOX-hunting system gives new oportunities for functional analysis of genes, generating valuable mutants overexpressing particular genes. ACKNOWLEDGMENTS I would like to express my gratitude to Dr Anna Goc (Nicolaus Copernicus University, Toru) for her insightful discussion and comments. This work was supported by a grant IHAR/1-1-01-4-05 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Advances in Cell Biology de Gruyter

Tilling and Fox-Hunting: New Methods for Functional Analysis of Genes

Rybka, Krystyna
Advances in Cell Biology , Volume 3 (1) – Feb 1, 2011
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ADVANCES IN CELL BIOLOGY VOL. 3, ISSUE 1/2011 (1­16) Krystyna RYBKA Department of Plant Biochemistry and Physiology, Plant Breeding and Acclimatization Institute DOI: 10.2478/v10052-011-0001-6 Summary: Theoretical and experimental bases of TILLING and FOX-hunting, new tools for precise identification of genes in functional studies are reviewed. TILLING (Targeting Induced Local Lesions IN Genomes) is a technique combining chemical mutagenesis with its sensitive and precise detection. The method involves PCR amplification of DNA samples pooled following extraction from a collection of chemically-treated organisms and a subsequent screening for mutations using Cel1 endonuclease, detecting mismatches in heteroduplexes [52]. FOX-hunting (Full-length cDNA Over-eXpressing gene hunting system) is a new method of plant gene overexpression, which enables a quick gene isolation and sequencing, paralelly with functional studies. Key words: TILLING, FOX-hunting, functional analysis of genes, mutagenesis INTRODUCTION The beginning of the twenty-first century has been called post-genomic era as more than 180 complete genome sequences started from the sequence of the phage X174 in 1977 (5,368 bp), Haemophilus influenzae in 1995, the fruit fly and Arabidopsis in year 2000, a man in 2001, rice in 2002, and in 2007 the first complete genome, 6 billion nucleotides, of one man [22] have been known. Knowledge of genomes sequences is not eqiuvalent with the knowledge about gene number and function. For example, the rice genome which is the smallest among cereals at 430 Mbp, and thus assumed to be a model for plant genome sutdies, was in silico predicted to encode approximately 50 000 potential genes, mostly of unknown function. For more than a decade, comprehensive data describing changes in gene expression profiles (mainly quantitative) have been collected. In 1996, the biotech company, Affymetrix, produced the first commercial DNA microarray. Identification of differentially expressed mRNAs using microarray technology generates enormous amounts of data. For example, a microarray experiment using cDNA derived from drought tolerant and sensitive rice varieties identified approximately 16 000 genes affected by drought stress, two thirds of which have no known function [5]. Similarly SAGE (Serial Analysis of Gene Expression) developed in the mid-nineties, and MPSSE (Massively Parallel Signature Sequencing) developed in the year 2000, which allow the mass sequencing of cDNAs fragments, generate large data sets [21, 24]. To sort and organize the large amount of experimental data which is still growing, there is a need for rapid expansion of knowledge about gene function, as well as new tools for construction and handling of multi-parameter databases [25, 33 and 34]. For many years functional analyses of genes were carried out according to principles of classical genetics, from the phenotype to the gene (TopDown/Forward Genetics). The development of molecular biology techniques has allowed the opposite way: from a mutation to a phenotype (Bottom-up/Reverse Genetics) (Table 1). Functional analysis of genes sped up with the development in the U.S. of TILLING platform (Targeting Induced Local Lesions IN Genomes) in the mid 90's, which allows massive and rapid identification of point mutations basing on DNA amplification and specific digestion [36]. The rate of accumulated functional data has also increased due to the development of the FOX-hunting method (Full-length cDNA Over-Expressing gene hunting system) in Japan. This method, patented in 2001 and made available to a broader range of researchers in 2008, allows for quick gene isolation and functional identification under conditions of gene over expression [17]. The dynamic developments of gene functional analysis has therefore provided the inspiration for overview of a conceptual framework of TILLING and FOX-hunting techniques on the background of the classical forward genetic screen. FUNCTIONAL ANALYSIS OF GENES ACCORDING TO THE PRINCIPLES OF CLASSICAL GENETICS In accordance to the principles of a classical forward genetics screen, functional analysis of genes involves identifying a gene which encodes a specific protein. Positional cloning of the gene is the example of such an approach. This method requires finding the molecular markers closely linked to the trait of interest. Depending on the nature of the trait, different methods of mapping are applied: qualitative or quantitative (QTL or associative, based on an analysis of Linkage Disequilibrium (LD)). For plants, mapping is accomplished based on segregating populations: BC2 or F2 (Back Crossed or regularly fertilized 2nd generation of offspring), doubled haploid lines (DH), recombinant lines (RILs) as well as near-isogenic lines (NILS), aneuploids or/and substitution lines [15]. Molecular markers which strongly co-segregate with the trait, are placed on the physical maps generated from contigs of clones selected from genomic libraries. After the region of DNA located between the flanking markers is sequenced, selected DNA fragments are isolated and cloned into transformation vectors. Changes in the phenotype of mutant plant gained by transformation, which are consistent with the expected result, confirm the biological function of the gene. Lack of expected phenotypic changes however is uninformative as it may be the result of gene silencing in transformants [27]. An example of such studies might be a search for a gene Pi-ta2 encoding resistance to Pyricularia grisea in rice [31]. In wheat genome several genes has been identified by positional cloning. While majority of these are genes of resistance to stresses and response to vernalization, on chromosome 5AL a regulatory locus involved in the domestication has been identified. This locus encodes genes of high gluten content in grain, as well as locus of homologous chromosomes conjugation during meiosis (Table 2). A more efficient approach to functional gene analysis is to create, identify and then analyze mutants. Mutagenesis was introduced into breeding in the early thirties, when the possibility of mutant induction by X-ray was discovered. The development of molecular biology techniques allowed for use of this approach to find genes and to understand their functions without knowledge of phenotype and /or protein products. TABLE 1. Comparison of methods of gene functional analysis: Top-down vs. Bottom-up approaches [30, modified] TABLE 2. Wheat genes identified from positional cloning projects [12, modified] Gen/QTL Lr1 Lr10 Lr21 Chromosome localization 5DL 1AS 1DS Trait Leaf rust resistance Leaf rust resistance Leaf rust resistance University/ Group lider University of Zurich/ B. Keller University of Zurich/ B. Keller Kansas State University/ B. S. Gill Kansas State University/ B. S. Gill University of Zurich/ B. Keller Qfhs.Ndsu-3bs 3BS Fusarium head blight resistance Powdery mildew resistance Resistance to stripe rust Pm3b 1AS Yr5 2BL USDA-ARS, Wheat Genetics/ K. G Campbell CSIRO Plant Industry, Australia/ E. S. Lagudah North Dakota State University/ J. D. Faris University of Adelaide/ P. Langridge University of California, Davis/ J. Dubcovsky University of California, Davis/ J. Dubcovsky University of California, Davis/ J. Dubcovsky University of California, Davis/ J. Dubcovsky University of California, Davis/ J. Dubcovsky Kansas State University/ B. S. Gill University of California, Davis/ J. Dubcovsky John Innes Centre, Colney, Norwich/ G. Moore Sr2 3BS Stem rust resistance Tsn1 5BL Host-selective toxin Ptr ToxA Boron tolerance 7BL Fr2 5BL Frost resistance VRN1 5A VRN2 5A VRN-B3 7BS EPS-1 1AL Flowering time 5AL Free threshing character GPC-B1 6BS High grain protein content Chromosome pairing locus Ph1 1A/5B FUNCTIONAL ANALYSIS OF GENES ACCORDING TO THE PRINCIPLES OF MOLECULAR GENETICS Methods for induction of mutants Physical mutagenesis Physical mutagenic agents include mostly different types of radiation: gamma (source: radioactive isotopes), X (sources: X-ray tubes, synchrotrons), ultraviolet and electrons with high and low kinetic energy (source: accelerators) [3]. Radiation energy alters the nature of chemical and short-distance physical bonds existing between atoms in bio-polymers, leading to the destruction of proper ones by the formation of invalid, which finally results in disturbances of DNA metabolism and mutation formation. The phenomenon of X-ray-induced mutagenesis was first described for the barley genome during interwar period. Comprehensive programs for diversity generation by physical mutagenesis in collections of crop plants were undertaken in the '60s. The cooperation of FAO (Food & Agriculture Organization) and IAEA (International Atomic Energy Agency) in the peaceful uses of atomic energy has resulted in many breeding programs worldwide. Since then, 2.570 mutants were registered throughout the world, including the 1020 mutants of major cereal crops: 439 mutants of rice, 305 mutants of barley, 204 mutants of wheat and 71 mutants of maize [26]. Most of those mutations were induced by -rays (30%) and to a lesser extent by the X-rays (3%). Two thirds of these mutants were generated in China. Notably, for the spectacular achievements of physical mutagenesis belongs barley variety well yielding on the attitude above 5 000 m above sea level in Andes in Peru. To natural phenomenon generated by mutagenesis belongs also the variety of rice that can grow in water of high salinity, in the Mekong Delta in southern Vietnam [38]. In Poland, significant achievement of -ray mutagenesis was self-ending mutant of horse bean (V. faba var. equina) [1]. Chemical mutagenesis Chemical mutagenesis (mostly point mutation) occurs under the influence of conformational changes in one pair of nucleotides in the complementary chains of DNA. Chemicals that cause conformational changes include analogues of nucleotide bases (5-bromouracil or 2-aminopurine); hydroxyl agents (hydroxylamine); alkyl reagents (ethyl methanesulfonate or dimethylnitrosamine); deamination reagents (nitrous acid or sodium sulfite). Intercalation of aromatic compounds molecules, such as proflavine or ethidium bromide, into the DNA helix may also lead to disruptions in DNA replication, repair, or recombination. The likelihood of generating a dominant point mutation in the cereals is less than half permil [18]. Transparent example of the use of chemical mutagenesis are stidies of Finkelstein group, which used A. thaliana mutants insensitive to abscisic acid and explained many aspects of signal transduction on the hormone-dependent path [9]. An important achievement of Polish breeders was obtaining of winter oilseed rape forms characterized by increased content of oleic acid and reduced content of linolenic acid in the seeds of mutagenesed plant in comparison to the double-improved varieties [32]. TABLE 3. Existing and proposed projects of gene functional analysis in grass species based on chemical mutagenesis and TILLING analytical system [37, modified] Cereal Crop BARLEY cv. Optic Project DIStilling (SCRI) Mutagen Web- page address http://germinate.scri.sari.a c.uk/barley/mutants/ cv. Barke GABI-TILL TILLMore Risø National Labs, KVL Denmark Maize TILLING Project, Purdue cv. Morex www.gabi-till.de/project/ ipk/barley.html www.gabi-till.de/project/ipk/barley.html www.distagenomics.unibo .it/TILLMore/ www.pgrc.ipkgatersleben.de/barleynet http://genome.purdue.edu/ maizetilling/ www.croptailor.com/Enge lsk/engindex.htm www.tilling.ucdavis.edu/ index.php/Rice_Tilling cv. Lux MAIZE OAT CropTailor AB RICE RiceTILL (UC Davis) or MNU + NaN3 MNU (ssp. japonica) WHEAT T. aestivum T. monococcum Mishima Arcadia Biosciences Rothamstead Research (RRes) http://www.rothamsted.bb src.ac.uk/ppi/staff/hcj.html www.rothamsted.ac.uk/cpi/ optiwheat/indexcontent.html T. durum OPTIWHEAT SORGO SWITCHGRASS BRACHYPODIUM USDA, Lubbock, TX Purdue TILLING Project Risø National Labs, KVL Denmark NaN3 http://genome.purdue.edu/ www.risoe.dk/rispubl/BIO /biopdf/ris-r-510.pdf. For over half a century, chemical mutagenesis has been an important method for generating mutants, and in the last decade, due to the development of TILLING platform, new comprehensive research projects have been established (Table 3). Insertional mutagenesis Insertional mutants are obtained by transformation of wild plants with naturally occurring retrotransposons or T-DNAs [20, 35]. Mutants generated from these methods typically arise from gene silencing (called: knock out or loss-offunction) due to insertion into the coding sequence [40]. Naturally occurring insertional mutants were first analyzed in maize due to the presence of active transposon elements [23]. This discovery led Barbara McClintock to be awarded a Nobel Prize (1948). Cloning of Ac and Ds transposons of maize [7] enabled the transformation of species whose genomes have not active transposon system. The first gene isolated using transposon tagging, was the tomato Cf-9 gene required for resistance to the fungus Cladosporium fulvum [16]. Such a strategy of functional gene analysis dominated for nearly two decades in laboratories worldwide, so it's not surprising that the number of generated, in this way, mutant plant is more than 290 000 [11]. Factors limiting the use of insertional mutagenesis include the inability to analyze the genes when multiple copies are inserted and also the inability to study genes expressed in early stages of plant development due to embryo lethality. In addition, these mutants, in contrast to the T-DNA mutants, are unstable, which is a direct result of the properties of transposons. T-DNA is integrated into the genome of a plant with an average of 1.5 copies in the genome of Arabidopsis or rice [8]. Table 4 summarizes the available research collections of insertion mutants in rice. In general, transformations with either transposons or T-DNA lead mainly to recessive mutant. Therefore, selecting a suitable mutant for further study requires a number of crosses and analysis of the phenotypes of many offsprings; in case of Cf-9 gene approximately 160 000 mutants of tomato individuals were studied [16]. Increased transformation efficiency coupled with simplifications in syst of plant regeneration and mutant identification resulted in development of the FOX-hunting system. This system does not require in vitro cultures, as embryos in young inflorescences, dipped in a solution of Agrobacterium tumefaciens, are transformed with a T-DNA binary vector [4]. T-DNA binary vectors used in these experiments carry a full-length cDNA of the gene of studied organism [14]. This promotes the generation of mutants characterized by ectopic gene expression which is also referred to as the gain-of-function (Fig. 1). In recent years, RNAi technology has become a powerful tool for functional analysis of genes, however, as it is based on post-translation gene silencing [27, 29] it is not the object of the present article. TABLE 4. Rice mutant resources [19, modified] Institution total Dongjin, Hwayoung T-DNA ET/AT Tos17 T-DNA ET Tos17 T-DNA AT 30 000 18 382 31 000 TRIM http://trim.sinica.edu.tw 45 000 14 137 100 000 13 745 http://urgi.versailles.inra.f r/OryzaTagLine 17 414 11 488 (03.`09) 150 000 84 680 400 000 RISD http://an6.postech.ac. 58 943 Genotype MMutagen Mutated loci classified available Web site Leader/e-mail address G. An genean@postech.ac. kr/pfg E. Guiderdoni guiderdoni@cirad.fr POSTECH South Korea CIRADINRAIRD-CNRS, Genoplante France Tainung 67 Nipponbare IPMB, Academia Sinica Taiwan Zhonghua 11 Zhonghua 15 Nipponbare T-DNA ET 113 262 16 158 26 000 (12.`08) 14 197 1 101 97 500 8 840 8 840 11 000 1 009 Zhonghua 11 T-DNA ET T-DNA 1 009 Y.C. Hsing bohsing@gate.sinica. ed.tw Q. Zhang qifazh@mail.hzau.ed u.cn Huazhong Agricultural University China RMD http://rmd.ncpgr.cn SIPP China Nipponbare Zhonghua 11 http://ship.plantsignal.cn/ home.do http://www.pi.csiro. au/fgrttpub F. Fu ship@sibs.ac.cn P. Wu clspwu@zju.edu.cn Zhejiang University China TABLE 4. Rice mutant resources [19, modified] Institution total Nipponbare Tos17 500 000 34 844 34 844 http://tos.nias.affrc.go.jp Genotype Mutagen Mutated loci classified available Web site Leader/e-mail address H. Hirochika hirohiko@nias.affrc. go.jp V. Sundaresan sundar@ucdavis.edu NIAS Japan UC Davis USA Nipponbare Ac-Ds GT Spm/dSpm Ac-Ds GT 30 000 4 820 4 820 KRDD http://www. niab.go.kr/RDS 20 000 Ds 4 735 4 630 dSpm 9 036 9 469 http://www-plb.ucdavis. edu/ Labs/sundar Gyeongsang National University; South Korea 20 000 3 500 2 000 Nipponbare Ac-Ds GT Dongjin Byeo C.-D. Han cdhan@nongae.gsnu. ac.kr R. Srinivasan sri@tll.org.sg Temasek Lifesciences, Singapore Nipponbare Ac-Ds ET Ac-Ds GT/ET 16 000 25 000 1 380 1 300 EU-OSTID France Nipponbare http://orygenesdb. cirad.fr E. Guiderdoni guiderdoni@cirad.fr http://www.genomics.zju. edu.cn/ricetdna CSIRO Plant Industry Australia 611 ~50% nonfertile N.M. Upadhyaya narayana.upadhyay a@ csiro.au FIGURE 1. The binary vector pBIG2113SF used in the FOX-hunting system: LB/RB ­ T-DNA border sequences; EI ­ two tandem repeats of sequence strengthening the transcription (5-upstream sequence of CaMV 35S promoter -419 to -90 bp; P35S ­ P35S, CaMV 35S promoter -90 to -1 bp; ­ 5'-upstream sequence of TMV, which increases translation effectiveness of inserted gen; NOS-T ­ polyadenylation signal of nopaline synthase gen from Ti plasmid; Hyg ­ hygromycin resistance gene; GS4, GS6 ­ sequences of PCR starters to GS4 and GS6 primers used to recover the cDNAs; SfiI(A), SfiI(B) ­ restriction sites for SfiI endonuclease; RAFL cDNA ­ RIKEN Arabidopsis Full-Length cDNA METHODS OF EXPLORATION AND ANALYSIS OF MUTANTS Brief description of the TILLING-method [36] TILLING technique combines traditional chemical mutagenesis with the identification of point mutantion using SNPs (Single Nucleotide Polymorphism). The critical step in this procedure is in obtaining a sufficiently large population of mutant seed using standard chemical mutagenesis. Following mutagenesis, M1 plants are self-fertilized and the M2 seeds are collected and sown out. DNA from the M2 plants is isolated and used in multiplex PCR reactions. In order to identify mutations in the gene of interest, gene-specific primers are used to amplify M2 DNA. PCR products are then denaturated and left to cool which causes heteroduplex of unpaired basepairs in the mutation place. By digesting these PCR products with endonuclease Cel1, mismatched bases arising from mutations are identifiable after electrophoresis on sequencing gel (Fig. 2). The classic method of SNP identification bases on multiplying, and then sequencing of selected genes for each individual plant from studied population. The TILLING method of mutant identification is faster, because instead of costand time-consuming sequencing, restriction enzyme digests and standard DNA electrophoresis is used. In addition as PCR amplification is multiplexed, and the result of proper analysis accurately indicates the mutant, which not only allows the direct typing for usage in functional studies, but also, in case of plants, allows the immediate use of the mutant in breeding programs. Brief description of the FOX- hunting [14, 17] This concept was developed by a team of RIKKEN (Japan) and published under the name Fox-hunting. The strategy has been developed for A. thaliana and it is a modification of Arabidopsis transformation method by binary vectors that are transferred into plant cells by A. tumefaciens. Essential for the effectiveness of the transformation are sequences of enhancers and transcription terminators, placed between the left and right T-DNA border sequences (LB andRB respectively). Typically, in a FOX-hunting system the binary vectors contains between border sequences, two tandem repeats of transcription amplifier (sequences -490 to -90 bp preceding the promoter of 35S of cauliflower mosaic virus CaMV); the 35S promoter (sequence -90 to -1 bp of the CaMV virus), leader sequence from the tobacco mosaic virus (which increases the efficiency of translation of the introduced gene), a cassette containing the gene of interest followed by a transcription terminator of the nopaline synthase gene (nos) from the Ti plasmid and finally a gene for resistance to the antibiotic Hygromycin B- a mutant selection gene, between the border sequences. The gene of interest is inserted into the cassette flanked by the GS4 and GS6 primer sequences which facilitate gene identification in the mutant and contain the recognition sequence for SfiI. FIGURE 2. Schematic diagram representing the TILLING technique. Equal amounts of mutant DNAs as well as the DNA of the wild plant are mixed and amplified in PCR reaction with gene specific primers. Digestion of that DNA by Cel1 endonuclease, specific to unpaired bases of the double-helix DNA, enables mutant identification after the electrophoresis on sequencing gel [10, 13, modified]. In order to generate a library in a binary vector, single copies of each gene collected in cDNA libraries constructed in vectors LambdaZAP and Lambda FLC1-B are used . In order to standardize of the library, four bacterial genes that cause known phenotypic changes and plants sterility were used. These genes have been cloned at the cleavage site of the restriction enzyme SfiI. A mixture of genomic and bacterial cDNA was prepared in such concentration that in mutants bacterial gene expression should be seen with a frequency of 1 / 7500 clones. The mixture was digested with SfiI, and then ligated with a modified binary vector pBIG2113N in which the XbaI restriction enzyme site include SfiI adapters, to enable directional cloning in the "sense" orientation. Escherichia coli DH10B was then transformed by electroporation. While preparing a library is expensive and time-consuming, transformation of A. thaliana is relatively straight forward. Once a library has been generated in A. tumefaciens, the Agrobacterium is resuspended in a 100-200 ml solution containing 5% sucrose and 0, 05% surfactant Silwet L-77. Inflorescences of A. thaliana are immersed in this solution for 2-3 seconds, with gentle stirring, so as to cover them with a layer of a suspension of A. tumefaciens. After transformation plants are grown in phytotron chamber under elevated relative humidity followed by cultivatation in a standard manner. Seeds harvested from transformed plants are germinated on agar supplemented with Hygromycin B for selection of transgenic plants. Over 15 000 fertile transformants were obtained using this method, which accounts for 77% of the cDNA library. To determine the transformation efficiency 24 lines FOX were tested for the presence of the Hygromycin B resistance using Southern hybridization. All lines tested contained at least one copy of the the Hygromycin B resistance gene with the average number of integrated vectors in the genome of a single line of FOX was 2.6. The average length of DNA inserts in the population of FOX lines was about 30% less than the average length of inserts in cDNA library in A. tumefaciens and ranged from 0.2 to 4.6 kb, with a median 11,4 kb. A small number of transformants with inserts smaller or larger than the insert in the library in binary vector were detected. The number of transformants with phenotype induced by the bacterial genes was higher than expected, which was explained by differences in multiplication speed of A. tumefaciens cells that carried plant or bacterial library. Currently, several FOX-hunting projects for cereal genomes are introduced and implemented throughout the world (Table 5). Advantages of the FOX-hunting system include the small percentage of cosuppressed genes due to full-length cDNA clones and libraries in the standard binary vectors; a reduction in the number of basic metabolism gene (housekeeping genes) in the standard libraries the facilitation in phenotype analysis which is caused by a short life cycle of A. thaliana, and easy isolation and sequencing of genes. The disadvantage of this system is reduction of phenotypic analysis only to genes used for construction of the library in A. tumefaciens. TABLE 5. FOX-hunting projects in studies of cereal genomes FOX lines [literature] A. thaliana [14, 17] Genome Project aim / details Project Leader rice searches for resistance genes, especially for high temperature; number of mutants: 23 000 Ichikawa Y. youichi@psc.riken.jp rice [28] rice use of the ubiquitin promoter; acquisition and phenotypic analysis of mutants number of mutants: total- 12 000, with single insertion: 8322 Ichikawa Y. youichi@psc.riken.jp A. thaliana [6] Bruguiera Gymnorhiza searches for resistance genes against abiotic stresses Tada Y. tadayui@bs.teu.ac.jp wheat [39] wheat project at the initial stage, small number of stable mutants were obtained Steber C. jzale@utk.edu SUMMARY TILLING and FOX-hunting techniques alow for acceleration of gene functional analysis. Programs that use them can also be an additional source of diversified material used by breeders. TILLING system has been developed to study chemically induced mutants. It enables for immediate and direct introduction of mutants into breeding programs. FOX-hunting system gives new oportunities for functional analysis of genes, generating valuable mutants overexpressing particular genes. ACKNOWLEDGMENTS I would like to express my gratitude to Dr Anna Goc (Nicolaus Copernicus University, Toru) for her insightful discussion and comments. This work was supported by a grant IHAR/1-1-01-4-05

Journal

Advances in Cell Biology – de Gruyter

Published: Feb 1, 2011

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