Extreme specificity of NCR gene expression in Medicago truncatula

BMC Genomics

Benoît Alunni³  Peter Mergaert⁵  Eva Kondorosi⁶  Michael K Udvardi¹  Mohamed Mars⁴  Pascal Ratet⁵  Willem Van de Velde²  Catalina I Pislariu¹  Marianna Nagymihaly⁶  Ibtissem Guefrachi⁴ 

[1] Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401, USA;Present address: Ablynx, Technologiepark 21, 9052 Zwijnaarde, Belgium;Département de Biologie, Université Paris Sud 11, 91400 Orsay, France;Research Unit Biodiversity & Valorization of Arid Areas, Bioressources (BVBAA), Faculty of Sciences, Gabès University, Erriadh-Zrig, 6072 Gabès, Tunisia;Institut des Sciences du Végétal, Centre National de la Recherche Scientifique UPR2355, 91198 Gif-sur-Yvette, France;Institute of Biochemistry, Hungarian Academy of Sciences, Biological Research Centre, 6726 Szeged, Hungary

关键词: Transcriptome compendium;    Gene expression;    Defensin;    NCR;    Sinorhizobium meliloti;    Medicago truncatula;    Bacteroid;    Nodulation;    Legume nitrogen fixation;    Symbiosis;   

Others  :  1141240
DOI  :  10.1186/1471-2164-15-712

 received in 2014-05-19, accepted in 2014-08-12,  发布年份 2014

【 摘 要 】

Background

Legumes form root nodules to house nitrogen fixing bacteria of the rhizobium family. The rhizobia are located intracellularly in the symbiotic nodule cells. In the legume Medicago truncatula these cells produce high amounts of Nodule-specific Cysteine-Rich (NCR) peptides which induce differentiation of the rhizobia into enlarged, polyploid and non-cultivable bacterial cells. NCRs are similar to innate immunity antimicrobial peptides. The NCR gene family is extremely large in Medicago with about 600 genes.

Results

Here we used the Medicago truncatula Gene Expression Atlas (MtGEA) and other published microarray data to analyze the expression of 334 NCR genes in 267 different experimental conditions. We find that all but five of these genes are expressed in nodules but in no other plant organ or in response to any other biotic interaction or abiotic stress tested. During symbiosis, none of the genes are induced by Nod factors. The NCR genes are activated in successive waves during nodule organogenesis, correlated with bacterial infection of the nodule cells and with a specific spatial localization of their transcripts from the apical to the proximal nodule zones. However, NCR expression is not associated with nodule senescence. According to their Shannon entropy, a measure expressing tissue specificity of gene expression, the NCR genes are among the most specifically expressed genes in M. truncatula. Moreover, when activated in nodules, their expression level is among the highest of all genes.

Conclusions

Together, these data show that the NCR gene expression is subject to an extreme tight regulation and is only activated during nodule organogenesis in the polyploid symbiotic cells.

【 授权许可】

   
2014 Guefrachi et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Cebolla A, Vinardell JM, Kiss E, Olah B, Roudier F, Kondorosi A, Kondorosi E: The mitotic inhibitor ccs52 is required for endoreduplication and ploidy-dependent cell enlargement in plants. EMBO J 1999, 18:4476-4484.
• [2]Vinardell JM, Fedorova E, Cebolla A, Kevei Z, Horvath G, Tarayre S, Roudier F, Mergaert P, Kondorosi A, Kondorosi E: Endoreduplication mediated by the APC activator CCS52A is required for symbiotic cell differentiation in Medicago truncatula nodules. Plant Cell 2003, 15:2093-2105.
• [3]Wildermuth MC: Modulation of host nuclear ploidy: a common plant biotroph mechanism. Curr Opin Plant Biol 2010, 13:449-458.
• [4]Koga R, Meng XY, Tsuchida T, Fukatsu T: Cellular mechanism for selective vertical transmission of an obligate insect symbiont at the bacteriocyte-embryo interface. Proc Natl Acad Sci U S A 2012, 109:E1230-E1237.
• [5]Kereszt A, Mergaert P, Kondorosi E: Bacteroid development in legume nodules: evolution of mutual benefit or of sacrificial victims? Mol Plant-Microbe Interact 2011, 24:1300-1309.
• [6]Mergaert P, Uchiumi T, Alunni B, Evanno G, Cheron A, Catrice O, Mausset AE, Barloy-Hubler F, Galibert F, Kondorosi A, Kondorosi E: Eukaryotic control on bacterial cell cycle and differentiation in Rhizobium-legume symbiosis. Proc Natl Acad Sci U S A 2006, 103:5230-5235.
• [7]Kondorosi E, Mergaert P, Kereszt A: A paradigm for endosymbiotic life: cell differentiation of Rhizobium bacteria provoked by host plant factors. Annu Rev Microbiol 2013, 67:611-628.
• [8]Van de Velde W, Zehirov G, Szatmari A, Debreczeny M, Ishihara H, Kevei Z, Farkas A, Mikulass K, Nagy A, Tiricz H, Satiat-Jeunemaître B, Alunni B, Bourge M, Kucho K, Abe M, Kereszt A, Maorti G, Uchiumi T, Kondorosi E, Mergaert P: Nodule specific peptides govern terminal differentiation of bacteria in symbiosis. Science 2010, 327:1122-1126.
• [9]Tiricz H, Szucs A, Farkas A, Pap B, Lima RM, Maróti G, Kondorosi E, Kereszt A: Antimicrobial nodule-specific cysteine-rich peptides induce membrane depolarization-associated changes in the transcriptome of Sinorhizobium meliloti. Appl Environ Microbiol 2013, 79:6737-6746.
• [10]Farkas A, Maróti G, Dürgő H, Györgypál Z, Lima RM, Medzihradszky KF, Kereszt A, Mergaert P, Kondorosi É: The Medicago truncatula symbiotic peptide NCR247 contributes to bacteroid differentiation through multiple mechanisms. Proc Natl Acad Sci U S A 2014, 111:5183-5188.
• [11]Penterman J, Abo RP, De Nisco NJ, Arnold MFF, Longhi R, Zanda M, Walker GC: Host plant peptides elicit a transcriptional response to control the Sinorhizobium meliloti cell cycle during symbiosis. Proc Natl Acad Sci U S A 2014, 111:3561-3566.
• [12]Haag AF, Baloban M, Sani M, Kerscher B, Pierre O, Farkas A, Longhi R, Boncompagni E, Hérouart D, Dall’Angelo S, Kondorosi E, Zanda M, Mergaert P, Ferguson GP: Protection of Sinorhizobium against host cysteine-rich antimicrobial peptides is critical for symbiosis. PLoS Biol 2011, 9:e1001169.
• [13]Haag AF, Kerscher B, Dall’Angelo S, Sani M, Longhi R, Baloban M, Wilson HM, Mergaert P, Zanda M, Ferguson GP: Role of cysteine residues and disulfide bonds on the activity of a legume root nodule-specific, cysteine-rich peptide. J Biol Chem 2012, 287:10791-10798.
• [14]Mergaert P, Nikovics K, Kelemen Z, Maunoury N, Vaubert D, Kondorosi A, Kondorosi E: A novel family in Medicago truncatula consisting of more than 300 nodule-specific genes coding for small, secreted polypeptides with conserved cysteine motifs. Plant Physiol 2003, 132:161-173.
• [15]Zhou P, Silverstein KA, Gao L, Walton JD, Nallu S, Guhlin J, Young ND: Detecting small plant peptides using SPADA (Small Peptide Alignment Discovery Application). BMC Bioinformatics 2013, 14:335. BioMed Central Full Text
• [16]Alunni B, Kevei Z, Redondo-Nieto M, Kondorosi A, Mergaert P, Kondorosi E: Genomic organization and evolutionary insights on GRPs and NCRs, two large nodule-specific gene families in Medicago truncatula. Mol Plant-Microbe Interact 2007, 20:1138-1148.
• [17]Young ND, Debellé F, Oldroyd GE, Geurts R, Cannon SB, Udvardi MK, Benedito VA, Mayer KF, Gouzy J, Schoof H, Van de Peer Y, Proost S, Cook DR, Meyers BC, Spannagl M, Cheung F, De Mita S, Krishnakumar V, Gundlach H, Zhou S, Mudge J, Bharti AK, Murray JD, Naoumkina MA, Rosen B, Silverstein KA, Tang H, Rombauts S, Zhao PX, Zhou P, et al.: The Medicago genome provides insight into the evolution of rhizobial symbioses. Nature 2011, 480:520-524.
• [18]Nallu S, Silverstein KAT, Samac DA, Bucciarelli B, Vance CP, VandenBosch KA: Regulatory patterns of a large family of defensin-like genes expressed in nodules of Medicago truncatula. PLoS One 2013, 8:e60355.
• [19]Maunoury N, Redondo-Nieto M, Bourcy M, Van de Velde W, Alunni B, Laporte P, Durand P, Agier N, Marissa L, Vaubert D, Delacroix H, Duc G, Ratet P, Aggerbeck L, Kondorosi E, Mergaert P: Differentiation of symbiotic cells and endosymbionts are coupled to two transcriptome-switches in Medicago truncatula nodulation. PLoS One 2010, 5:e9519.
• [20]Benedito VA, Torres-Jerez I, Murray JD, Andriankaja A, Allen S, Kakar K, Wandrey M, Verdier J, Zuber H, Ott T, Moreau S, Niebel A, Frickey T, Weiller G, He J, Dai X, Zhao PX, Tang Y, Udvardi MK: A gene expression atlas of the model legume Medicago truncatula. Plant J 2008, 55:504-513.
• [21]He J, Benedito VA, Wang M, Murray JD, Zhao PX, Tang Y, Udvardi MK: The Medicago truncatula gene expression atlas web server. BMC Bioinformatics 2009, 10:441. BioMed Central Full Text
• [22]Jayaraman D, Valdés-López O, Kaspar CW, Ané JM: Response of Medicago truncatula seedlings to colonization by Salmonella enterica and Escherichia coli O157:H7. PLoS One 2014, 9:e87970.
• [23]Limpens E, Moling S, Hooiveld G, Pereira PA, Bisseling T, Becker JD, Küster H: Cell- and tissue-specific transcriptome analyses of Medicago truncatula root nodules. PLoS One 2013, 8:e64377.
• [24]Gaude N, Bortfeld S, Duensing N, Lohse M, Krajinski F: Arbuscule-containing and non-colonized cortical cells of mycorrhizal roots undergo extensive and specific reprogramming during arbuscular mycorrhizal development. Plant J 2012, 69:510-528.
• [25]Czaja LF, Hogekamp C, Lamm P, Maillet F, Martinez EA, Samain E, Dénarié J, Küster H, Hohnjec N: Transcriptional responses toward diffusible signals from symbiotic microbes reveal MtNFP- and MtDMI3-dependent reprogramming of host gene expression by arbuscular mycorrhizal fungal lipochitooligosaccharides. Plant Physiol 2012, 159:1671-1685.
• [26]Downie A: Legume nodulation. Curr Biol 2014, 24:R184-R190.
• [27]Maillet F, Poinsot V, André O, Puech-Pagès V, Haouy A, Gueunier M, Crome L, Giraudet D, Formey D, Niebel A, Martinez EA, Driguez H, Bécard G, Dénarié J: Fungal lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza. Nature 2011, 469:58-63.
• [28]Van de Velde W, Pérez-Guerra JC, De Keyser A, De Rycke R, Maunoury N, Mergaert P, Kondorosi E, Holsters M, Goormachtig S: Aging in legume symbiosis: a molecular view on nodule senescence in Medicago truncatula. Plant Physiol 2006, 141:711-720.
• [29]Seabra AR, Pereira PA, Becker JD, Carvalho HG: Inhibition of glutamine synthetase by phosphinothricin leads to transcriptome reprograming in root nodules of Medicago truncatula. Mol Plant-Microbe Interact 2012, 25:976-992.
• [30]Cabeza R, Koester B, Liese R, Lingner A, Baumgarten V, Dirks J, Salinas-Riester G, Pommerenke C, Dittert K, Schulze J: A RNA-Seq transcriptome analysis reveals novel insights into molecular aspects of the nitrate Impact on nodule activity of Medicago truncatula. Plant Physiol 2014, 164:400-411.
• [31]Tesfaye M, Silverstein KAT, Nallu S, Wang L, Botanga CJ, Gomez SK, Costa LM, Harrison MJ, Samac DA, Glazebrook J, Katagiri F, Gutierrez-Marcos JF, VandenBosch KA: Spatio-temporal expression patterns of Arabidopsis thaliana and Medicago truncatula defensin-like genes. PLoS One 2013, 8:e58992.
• [32]Imin N, Goffard N, Nizamidin M, Rolfe BG: Genome-wide transcriptional analysis of super-embryogenic Medicago truncatula explant cultures. BMC Plant Biol 2008, 8:110. BioMed Central Full Text
• [33]Rightmyer AP, Long S: Pseudonodule formation by wild-type and symbiotic mutant Medicago truncatula in response to auxin transport inhibitors. Mol Plant-Microbe Interact 2011, 11:1372-1384.
• [34]Maróti G, Kereszt A, Kondorosi E, Mergaert P: Natural roles of antimicrobial peptides in microbes, plants and animals. Res Microbiol 2011, 162:363-374.
• [35]Sels J, Mathys J, De Coninck BM, Cammue BP, De Bolle MF: Plant pathogenesis-related (PR) proteins: a focus on PR peptides. Plant Physiol Biochem 2008, 46:941-950.
• [36]De Coninck BM, Sels J, Venmans E, Thys W, Goderis IJ, Carron D, Delauré SL, Cammue BP, De Bolle MF, Mathys J: Arabidopsis thaliana plant defensin AtPDF1.1 is involved in the plant response to biotic stress. New Phytol 2010, 87:1075-1088.
• [37]De Coninck B, Carron D, Tavormina P, Willem L, Craik DJ, Vos C, Thevissen K, Mathys J, Cammue BP: Mining the genome of Arabidopsis thaliana as a basis for the identification of novel bioactive peptides involved in oxidative stress tolerance. J Exp Bot 2013, 64:5297-5307.
• [38]Uppalapati SR, Marek SM, Lee HK, Nakashima J, Tang Y, Sledge MK, Dixon RA, Mysore KS: Global gene expression profiling during Medicago truncatula-Phymatotrichopsis omnivora interaction reveals a role for jasmonic acid, ethylene, and the flavonoid pathway in disease development. Mol Plant-Microbe Interact 2009, 22:7-17.
• [39]Mah KM, Uppalapati SR, Tang YH, Allen S, Shuai B: Gene expression profiling of Macrophomina phaseolina infected Medicago truncatula roots reveals a role for auxin in plant tolerance against the charcoal rot pathogen. Physiol Mol Plant Pathol 2012, 79:21-30.
• [40]Hogekamp C, Arndt D, Pereira PA, Becker JD, Hohnjec N, Küster H: Laser microdissection unravels cell-type-specific transcription in arbuscular mycorrhizal roots, including CAAT-box transcription factor gene expression correlating with fungal contact and spread. Plant Physiol 2011, 157:2023-2043.
• [41]Rey T, Nars A, Bonhomme M, Bottin A, Huguet S, Balzergue S, Jardinaud MF, Bono JJ, Cullimore J, Dumas B, Gough C, Jacquet C: NFP, a LysM protein controlling Nod factor perception, also intervenes in Medicago truncatula resistance to pathogens. New Phytol 2013, 198:875-886.
• [42]Damiani I, Baldacci-Cresp F, Hopkins J, Andrio E, Balzergue S, Lecomte P, Puppo A, Abad P, Favery B, Hérouart D: Plant genes involved in harbouring symbiotic rhizobia or pathogenic nematodes. New Phytol 2012, 194:511-522.
• [43]Naoumkina M, Vaghchhipawala S, Tang Y, Ben Y, Powell RJ, Dixon RA: Different mechanisms for phytoalexin induction by pathogen and wound signals in Medicago truncatula. Proc Natl Acad Sci U S A 2007, 104:17909-17915.
• [44]Li D, Su Z, Dong J, Wang T: An expression database for roots of the model legume Medicago truncatula under salt stress. BMC Genomics 2009, 10:517. BioMed Central Full Text
• [45]Zhang JY, Cruz DE Carvalho MH, Torres-Jerez I, Kang Y, Allen SN, Huhman DV, Tang Y, Murray J, Sumner LW, Udvardi MK: Global reprogramming of transcription and metabolism in Medicago truncatula during progressive drought and after re-watering. Plant Cell Environin press. doi:10.1111/pce.12328
• [46]Hann DR, Rathjen JP: Early events in the pathogenicity of Pseudomonas syringae on Nicotiana benthamiana. Plant J 2007, 49:607-618.
• [47]Schug J, Schuller WP, Kappen C, Salbaum JM, Bucan M, Stoeckert CJ Jr: Promoter features related to tissue specificity as measured by Shannon entropy. Genome Biol 2005, 6:R33. BioMed Central Full Text
• [48]Zhang X, Yazaki J, Sundaresan A, Cokus S, Chan SW, Chen H, Henderson IR, Shinn P, Pellegrini M, Jacobsen SE, Ecker JR: Genome-wide high-resolution mapping and functional analysis of DNA methylation in Arabidopsis. Cell 2006, 126:1189-1201.
• [49]Kevei Z, Vinardell JM, Kiss GB, Kondorosi A, Kondorosi E: Glycine-rich proteins encoded by a nodule-specific gene family are implicated in different stages of symbiotic nodule development in Medicago spp. Mol Plant-Microbe Interact 2002, 15:922-931.
• [50]Laporte P, Satiat-Jeunemaître B, Velasco I, Csorba T, Van de Velde W, Campalans A, Burgyan J, Arevalo-Rodriguez M, Crespi M: A novel RNA-binding peptide regulates the establishment of the Medicago truncatula–Sinorhizobium meliloti nitrogen-fixing symbiosis. Plant J 2010, 62:24-38.
• [51]Liu J, Miller SS, Graham M, Bucciarelli B, Catalano CM, Sherrier DJ, Samac DA, Ivashuta S, Fedorova M, Matsumoto P, Gantt JS, Vance CP: Recruitment of novel calcium-binding proteins for root nodule symbiosis in Medicago truncatula. Plant Physiol 2006, 141:167-177.
• [52]Bourcy M, Brocard L, Pislariu CI, Cosson V, Mergaert P, Tadege M, Mysore KS, Udvardi MK, Gourion B, Ratet P: Medicago truncatula DNF2 is a PI-PLC-XD-containing protein required for bacteroid persistence and prevention of nodule senescence and defense-like reactions. New Phytol 2013, 197:1250-1261.
• [53]Moreau S, Verdenaud M, Ott T, Letort S, de Billy F, Niebel A, Gouzy J, De Carvalho-Niebel F, Gamas P: Transcriptional reprogramming during root nodule development in Medicago truncatula. PLoS One 2011, 6:e16463.
• [54]Roux B, Rodde N, Jardinaud MF, Timmers T, Sauviac L, Cottret L, Carrère S, Sallet E, Courcelle E, Moreau S, Debellé F, Capela D, de Carvalho-Niebel F, Gouzy J, Bruand C, Gamas P: An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing. Plant J 2014, 77:817-837.
• [55]Horváth B, Yeun LH, Domonkos A, Halász G, Gobbato E, Ayaydin F, Míró K, Hirsch S, Sun J, Tadege M, Ratet P, Mysore K, Ané JM, Oldroyd GED, Kaló P: Medicago truncatula IPD3 is a member of the common symbiotic signaling pathway required for rhizobial and mycorrhizal symbioses. Mol Plant-Microbe Interact 2011, 24:1345-1358.
• [56]Ovchinnikova E, Journet EP, Chabaud M, Cosson V, Ratet P, Duc G, Fedorova E, Liu W, Op den Camp R, Zhukov V, Tikhonovich I, Borisov A, Bisseling T, Limpens E: IPD3 controls the intracellular accommodation of rhizobia in pea and Medicago. Mol Plant-Microbe Interact 2011, 24:1333-1344.
• [57]Vernié T, Moreau S, de Billy F, Plet J, Combier JP, Rogers C, Oldroyd G, Frugier F, Niebel A, Gamas P: EFD Is an ERF transcription factor involved in the control of nodule number and differentiation in Medicago truncatula. Plant Cell 2008, 20:2696-2713.
• [58]Singh S, Katzer K, Lambert J, Cerri M, Parniske M: CYCLOPS, a DNA-binding transcriptional activator, orchestrates symbiotic root nodule development. Cell Host Microbe 2014, 15:139-152.
• [59]Limpens E, Bisseling T: CYCLOPS: a new vision on Rhizobium-induced nodule organogenesis. Cell Host Microbe 2014, 15:127-129.
• [60]Messinese E, Mun JH, Yeun LH, Jayaraman D, Rougé P, Barre A, Lougnon G, Schornack S, Bono JJ, Cook DR, Ané JM: A novel nuclear protein interacts with the symbiotic DMI3 calcium- and calmodulin-dependent protein kinase of Medicago truncatula. Mol Plant-Microbe Interact 2007, 20:912-921.
• [61]Lelandais-Brière C, Naya L, Sallet E, Calenge F, Frugier F, Hartmann C, Gouzy J, Crespi M: Genome-wide Medicago truncatula small RNA analysis revealed novel microRNAs and isoforms differentially regulated in roots and nodules. Plant Cell 2009, 21:2780-2796.
• [62]Li F, Pignatta D, Bendix C, Brunkard JO, Cohn MM, Tung J, Sun H, Kumar P, Baker B: MicroRNA regulation of plant innate immune receptors. Proc Natl Acad Sci U S A 2012, 109:1790-1795.
• [63]Zhou C, Han L, Pislariu C, Nakashima J, Fu C, Jiang Q, Quan L, Blancaflor E, Tang Y, Bouton JH, Udvardi M, Xia G, Wang ZY: From model to crop: functional analysis of a STAY-GREEN gene in the model legume Medicago truncatula and effective use of the gene for alfalfa improvement. Plant Physiol 2011, 157:1483-1496.
• [64]Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, Speed TP: Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 2003, 4:249-264.
• [65]Ratet P, Wen J, Cosson V, Tadege M, Mysore KS: Tnt1 Induced Mutations in Medicago: Characterisation and Applications. In The Handbook of Plant Mutation Screening, Mining of Natural and Induced Alleles. Edited by Meksem K, Kahl G. KGaA,Weinheim: Wiley-VCH Verlag GmbH & Co; 2010:83-99.
• [66]Karimi M, De Meyer B, Hilson P: Modular cloning in plant cells. Trends Plant Sci 2005, 10:103-105.
• [67]Cosson V, Durand P, D’Erfurth I, Kondorosi A, Ratet P: Medicago truncatula transformation using leaf explants. Methods Mol Biol 2006, 343:115-127.
• [68]Mondy S, Lenglet A, Cosson V, Pelletier S, Pateyron S, Gilard F, Scholte M, Brocard L, Couzigou J-M, Tcherkez G, Péan M, Ratet P: GOLLUM [FeFe]-hydrogenase-like proteins are essential for plant development in normoxic conditions and modulate energy metabolism. Plant Cell Env 2013, 37:54-69.
• [69]Nick G, de Lajudie P, Eardly BD, Suomalainen S, Paulin L, Zhang X, Gillis M, Lindström K: Sinorhizobium arboris sp. nov. and Sinorhizobium kostiense sp. nov., isolated from leguminous trees in Sudan and Kenya. Int J Syst Evol Microbiol 1999, 49:1359-1368.
• [70]Vanstraelen M, Baloban M, Da Ines O, Cultrone A, Lammens T, Boudolf V, Brown SC, De Veylder L, Mergaert P, Kondorosi E: APC/C-CCS52A complexes control meristem maintenance in the Arabidopsis root. Proc Natl Acad Sci U S A 2009, 106:11806-11811.
• [71]Amselem J, Cuomo CA, van Kan JAL, Viaud M, Benito EP, Couloux A, Coutinho PM, de Vries RP, Dyer PS, Fillinger S, Fournier E, Gout L, Hahn M, Kohn L, Lapalu N, Plummer KM, Pradier JM, Quévillon E, Sharon A, Simon A, ten Have A, Tudzynski B, Tudzynski P, Wincker P, Andrew M, Anthouard V, Beever RE, Beffa R, Benoit I, Bouzid O, et al.: Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea. PLoS Genet 2011, 7:e1002230.