Background
==========

White mold, caused by *Sclerotinia sclerotiorum* (Lib.) de Bary, is a devastating disease of over 400 reported dicotyledonous hosts \[[@B1]\]. The disease causes economically significant losses of many crop plants including pea (*Pisum sativum* L.) under the appropriate environmental conditions \[[@B2]\]. Currently, little is known about the genetic control of pathogenicity in the fungus and mechanisms of resistance in pea. Although hundreds of pea cultivars have been screened for white mold resistance in replicated greenhouse and laboratory tests \[[@B3]\], only partial resistance has been identified to date.

The identification of genes underlying *S*. *sclerotiorum* pathogenicity and resistance in pea would increase our knowledge of the pea-*S*. *sclerotiorum* interaction and facilitate the introgression of resistance into pea varieties. However, progress in these areas has been hampered by the lack of sequence information regarding the pea genome. Although other legume genomes, including the models *Medicago truncatula*, *Lotus japonicus* and economically important *Glycine max* (soybean) are available \[[@B4]\], *Pisum sativum* is still genome resource-poor in part due to the large genome size and large fraction of highly repetitive DNA \[[@B5]\].

The performance of Next-Generation sequencing (NGS) technologies continue to rise while costs continue to fall which enables researchers to conduct whole transcriptome sequencing (RNAseq) studies of interactions between plants and pathogenic fungi \[[@B6]\]. The application of NGS in plant-fungal interaction research promises to shorten the overall time of development of molecular genetic information necessary for functional and translational studies. However, RNAseq has rarely been used to study plant-pathogen interactions, particularly in non-model systems. One reason for this is the difficulty in distinguishing plant and fungal ESTs and even virus or viroid contamination, particularly when reference genomes are not available. Here we report novel transcriptome sequence information from the pea-*S*. *sclerotiorum* interaction obtained by 454 pyrosequencing and propose a method of rapid and efficient transcriptome characterization in a non-model species with little prior molecular information. We also report on the development and validation of a strategy to distinguish plant and fungal ESTs using the tBLASTx program and "proxy-reference" genomes in the absence of true reference genomes.

Results
=======

Contiguous EST assembly
-----------------------

10,158 contigs were obtained by *de novo* assembly of 128,720 high-quality reads produced on a Roche 454 GS FLX sequencer (see Additional file [1](#S1){ref-type="supplementary-material"}). Minimum contig length was 50 bp, maximum length was 1,015 bp and average length was 200 bp. Average read coverage of contigs was 4.5X, and the maximum read coverage was 2,303X (Figure [1](#F1){ref-type="fig"}).

![**Depth of 454 pyrosequence read coverage (X) for the 10,158 pea-*S.**sclerotiorum*transcriptome EST contigs** .](1471-2164-13-668-1){#F1}

Filter for virus or viroid contamination
----------------------------------------

The tBLASTx program identified 51 contigs with a BLAST hit (alignment identity) to virus or viroid DNA with an e-value cutoff threshold of less than 1e^-3^. Further evaluation of these 51 EST contigs with tBLASTx against 3 legume and 7 fungal genome databases (which acted as proxy-reference genomes) revealed that 46 contigs showed significant alignment with the proxy plant genome database, 43 showed significant alignment with the fungal genome database and 40 showed significant alignment with both databases, 2 contigs showed significant alignment only with virus genomes. By comparing the e-value ratio (virus/fungi or virus/plant) of all proxy-reference genome alignments, 10 contigs were assigned to pea and 9 contigs were assigned to *S*. *sclerotiorum* based on an e-value ratio \>1e^20^, 30 contigs were difficult to distinguish, with e-value ratios between 1e^-20^ and 1e^20^. BLASTn analysis of the 32 unassigned contigs against pea and *S*. *sclerotiorum* ESTs from known sources revealed that 20 contigs, including the 2 that only aligned with virus genomes, had high identity matches to pea with 95% accuracy and 95% query coverage, and 1 to *S*. *sclerotiorum*. The 11 contigs which were difficult to assign to a genome database by either tBLASTx or BLASTn methods were far more similar to plant or fungi than to virus genomes.

Development and testing a method to distinguish pea and *S*. *sclerotiorum* ESTs using an artificially mixed pool
-----------------------------------------------------------------------------------------------------------------

Pea and *S*. *sclerotiorum* ESTs were downloaded from GenBank to test the tBLASTx sorting method. Three hundred twenty-one ESTs with vector contamination and 71 ESTs highly similar to virus or viroids were removed from the total pool of 36,080 known pea and *S*. *sclerotiorum* ESTs. Using an e-value threshold of 1e^-3^, 35,688 mixed ESTs from pea and *S*. *sclerotiorum* were compared to legume and fungal proxy-reference genome databases and parsed using the tBLASTx program (Figure [2](#F2){ref-type="fig"}). 11,191 ESTs only aligned with the legume proxy-reference genomes, 11,259 ESTs only aligned with the fungal proxy-reference genomes, 11,266 ESTs similar to both plant and fungal proxy-reference genomes and 1,972 ESTs did not match either proxy-reference genome database. The ESTs with tBLASTx results to both plant and fungal genomes were analyzed further by comparing the e-value ratio (fungi/plant) of fungal and plant proxy-reference genome alignments. 4,098 ESTs were assigned to pea based on an e-value ratio \>1e^20^, 5,649 ESTs were assigned to *S*. *sclerotiorum* based on an e-value ratio \<1e^-20^, while 1,519 ESTs were difficult to distinguish due to high e-value alignments to both proxy-reference genome databases, with e-value ratios between 1e^-20^ and 1e^20^. This method successfully separated 90.1% of the known ESTs into pea or *S*. *sclerotiorum* categories, with only a 0.1% misallocation rate. Only 5.5% of ESTs had zero similarity to either of the proxy-reference genomes, and 4.3% of ESTs had high similarity to both the plant and fungal proxy-reference genome databases (Table [1](#T1){ref-type="table"}).

![**Flow diagram of host and pathogen EST parsing method for mixed transcriptomes from plant and fungi based on a modified tBLASTx method** .](1471-2164-13-668-2){#F2}

###### 

**Preliminary testing of the tBLASTx method with pooled known pea and*S*.*sclerotiorum*ESTs**

  **Category of EST**   **Numbers of ESTs**   **ESTs assigned successfully**
  --------------------- --------------------- --------------------------------
  Plant                 15,289                99.9% (14 wrong)
  Fungi                 16,908                99.8% (23 wrong)
  Ambiguous             1,519                 N/A
  Unassigned            1,972                 N/A
  **Total**             35,688\*              90.1%

\*To assess the potential of the tBLASTx method for the parsing of pea and *S*. *sclerotiorum* EST contigs, 17,533 and 18,547 known *S*. *sclerotiorum* and pea ESTs from online databases were pooled, the ESTs were then parsed using the tBLASTx method. Only 9.8% of ESTs could not be assigned and only 37 (0.1%) ESTs were mis-assigned to the wrong class (plant or fungi).

Parsing 454 pyrosequence pea and *S*. *sclerotiorum* ESTs with tBLASTx and BLASTn
---------------------------------------------------------------------------------

Initial tBLASTx parsing of 10,158 contigs with an e-value threshold of 1e^-3^ resulted in identification of 4,523 pea ESTs, 2,304 *S*. *sclerotiorum* ESTs, 1,974 ESTs that matched both pea and *S*. *sclerotiorum*, and 1,357 ESTs that did not matched either proxy-reference genome database. The 1,974 ESTs that matched both proxy-reference genomes at the e-value threshold of 1e^-3^ were further subdivided using the e-value ratio method into 544 pea ESTs (fungi/plant e-value ratio \>1e^20^), 355 *S*. *sclerotiorum* ESTs (fungi/plant e-value ratio \<1e^-20^) and 1,075 that were ambiguous (fungi/plant e-value ratio \<1e^20^ and \>1e^-20^). This brought the number of classified ESTs for each category to 5,067 for pea, 2,659 for *S*. *sclerotiorum*, 1,075 as ambiguous with high matches to both proxy-reference genomes, and 1,357 with no significant alignment. The remaining 2,432 EST contigs that were ambiguous or showed no significant alignment were further parsed with BLASTn analysis against known pea and *S*. *sclerotiorum* ESTs if identity and query coverage were both equal to or greater than 95%. 1,232 ESTs of this pool were assigned with BLASTn to pea and 121 ESTs were assigned to *S*. *sclerotiorum*, leaving 310 ambiguous and 769 EST contigs with no significant alignment. In total with tBLASTx and BLASTn, 10,158 contigs were separated into 6,299 pea ESTs, 2,780 *S*. *sclerotiorum* ESTs, 310 ambiguous ESTs and 769 unassigned ESTs (Figure [3](#F3){ref-type="fig"}).

![**Distribution and assignment of 10**,**158 EST contigs derived from pyrosequencing of the pea-*Sclerotinia sclerotiorum*transcriptome using the tBLASTx method.** EST contigs with an e-value score \<1e^-3^ to only plant or fungal EST reference databases were automatically assigned. Those EST contigs with hits (\<1e^-3^) to both databases were assigned based on a cutoff ratio of e-values (fungi/plant), those ESTs with a ratio \> =1e^20^ were classified as pea, those \< =1e^-20^ as Sclerotinia and those between \>1e^-20^ and \<1e^20^ as ambiguous. ESTs without any hits (\>1e^-3^) were classified as unassigned ESTs.](1471-2164-13-668-3){#F3}

Validation of tBLASTx and BLASTn EST parsing results by PCR
-----------------------------------------------------------

Validation of the tBLASTx and BLASTn assignment was performed for 50 *S*. *sclerotiorum* and 50 pea EST contigs randomly sampled from the two assigned categories. All 50 primer sets designed to the pea EST contigs amplified the expected amplicon size in both the pea-*S*. *sclerotiorum* and non-inoculated pea cDNA indicating correct parsing assignment of the pea ESTs (Figure [4](#F4){ref-type="fig"}). Of the 50 PCR primers designed to the *S*. *sclerotiorum* ESTs, 47 amplified a PCR product from both the pea-*S*. *sclerotiorum* and *S*. *sclerotiorum* only cDNA samples and most of them amplified the same size amplicon in both cDNA samples. Two of the 50 *S*. *sclerotiorum* PCR primer pairs amplified the expected PCR products from the pea-*S*. *sclerotiorum* cDNA sample but not the *S*. *sclerotiorum* only cDNA, perhaps indicating that this transcript is only expressed during the interaction with pea. One *S*. *sclerotiorum* primer set failed to amplify any PCR product from either template.

![**PCR results for validation of 50 pea and 50** ***S.**sclerotiorum*pyrosequence-classified EST contigs against a mix of host and pathogen interaction cDNA, host cDNA only and pathogen cDNA only.** All 50 PCR pea primer sets amplified the expected amplicons from the pea-*S*. *sclerotiorum* and pea cDNA samples. Forty-seven of the 50 PCR *S*. *sclerotiorum* primer sets amplified expected amplicons from the pea-*S*. *sclerotiorum* and *S*. *sclerotiorum* cDNA samples, one primer pair failed to amplify from either cDNA and the remaining two only amplified product from the *pea*-*S*. *sclerotiorum* cDNA possibly indicating interaction induced transcripts.](1471-2164-13-668-4){#F4}

Unique ESTs expressed in the pea-*S*. *sclerotiorum* interaction
----------------------------------------------------------------

To detect unique genes expressed in our pea-*S*. *sclerotiorum* interaction, the 6,299 classified pea ESTs in our data set were compared with BLASTn against 81,449 recently published pea ESTs from flowers, leaves, cotyledons, epi- and hypocotyl, and etiolated and light treated etiolated seedlings \[[@B7]\]. Of these 6,299 ESTs, 3,459 ESTs had significant alignments with an e-value cutoff of 1e^-10^, in which 1,668 contigs had a percentage identity threshold of 95% for 95% or more of the query sequence, leaving 2,840 potentially unique pea ESTs to the pea-*S*. *sclerotiorum* interaction. It was possible to annotate 1,631 of these ESTs of which 67 contigs encode transcription factors (Table [2](#T2){ref-type="table"}), 69 were involved in signaling pathways (Table [3](#T3){ref-type="table"}) and 82 contigs were involved in encoding defense-associated proteins (Table [4](#T4){ref-type="table"}).

###### 

Description of genes encoded by 67 unique pea ESTs encoding transcription factors

  **Seq.Name**                       **Seq**. **Description**                                                    **Seq.Length**   **min.e-value**   **mean Similarity**
  ---------------------------------- --------------------------------------------------------------------------- ---------------- ----------------- ---------------------
  MYB transcription factors                                                                                                                         
  746                                myb-like protein                                                            102              7.8E-12           92.1%
  914                                myb-cc transcription factor                                                 143              7.0E-18           94.8%
  1535                               myb transcription factor myb50                                              120              1.5E-07           81.3%
  3483                               myb-cc transcription factor                                                 264              1.00E-32          84.80%
  5012                               myb family transcription factor                                             226              5.90E-20          68.90%
  5326                               myb family transcription factor                                             209              3.50E-17          80.70%
  5825                               myb-related transcription factor                                            368              8.50E-32          65.60%
  AP2/EREBPs transcription factors                                                                                                                  
  1311                               ethylene insensitive transcription factor                                   220              1.11E-34          79.50%
  2887                               dehydration responsive element-binding protein 3 (DREB3)                    149              6.8E-08           73.5%
  4803                               ap2 domain-containing transcription factor                                  150              1.5E-07           66.1%
  7472                               ap2 erf domain-containing transcription factor                              376              7.3E-23           81.30%
  7671                               ein3-like protein                                                           150              1.1E-13           88.2%
  8261                               ethylene insensitive transcription factor                                   163              1.43E-18          90.00%
  8894                               ap2-like ethylene-responsive transcription factor                           228              2.2E-27           78.60%
  9486                               ap2 erf domain-containing transcription factor                              282              5.4E-13           57.80%
  WRKY transcription factors                                                                                                                        
  413                                wrky transcription                                                          166              2.4E-18           83.90%
  6039                               wrky transcription factor 2                                                 277              1.2E-04           47.00%
  9142                               wrky 10                                                                     116              2.2E-14           82.10%
  9626                               wrky transcription factor 40                                                124              3.4E-12           85.90%
  bZIP transcription factors                                                                                                                        
  1563                               bZIP transcription factor bZIP122                                           236              2E-28             64.2%
  5518                               bZip transcription factor                                                   120              1.9E-07           77.7%
  Others                                                                                                                                            
  177                                transcription factor                                                        196              6.7E-08           98.5%
  200                                zinc finger family protein                                                  264              3.3E-23           67.4%
  257                                transcription factor iiia                                                   255              4.8E-30           76.6%
  446                                histone acetyltransferase                                                   123              1.3E-11           76.6%
  642                                kruppel-like zinc finger protein                                            276              3.0E-21           80.1%
  677                                integral membrane family protein                                            186              2.4E-21           86.9%
  918                                ring finger and chy zinc finger domain-containing protein 1                 305              1.7E-43           78.3%
  1245                               zinc finger                                                                 178              5.0E-24           72.9%
  1407                               zinc finger                                                                 546              5.7E-60           72.5%
  1541                               phosphoprotein phosphatase                                                  271              2.3E-45           99.2%
  1818                               nuclear transcription x-box                                                 246              6.7E-24           77.3%
  2070                               25.7 kda protein                                                            185              3.5E-12           67.8%
  2739                               DNA binding protein                                                         254              9.7E-23           80.9%
  2864                               zinc finger protein 622                                                     156              5.3E-13           88.0%
  2982                               homeodomain transcription factor                                            113              2.0E-07           79.7%
  3354                               dna binding                                                                 179              5.5E-15           75.4%
  3477                               prefoldin subunit 2                                                         218              3.1E-18           87.8%
  3616                               constans-like b-box zinc finger protein                                     140              1.7E-11           78.2%
  3637                               global transcription factor group                                           108              3.1E-05           80.0%
  3724                               zinc finger protein                                                         566              1.6E-47           65.2%
  3916                               knotted I class homeodomain protein                                         173              7.3E-15           89.7%
  4012                               ring zinc finger ankyrin protein                                            141              5.5E-15           87.6%
  4123                               zinc finger family protein                                                  189              2.0E-28           87.2%
  4349                               zinc finger ccch domain-containing protein 32                               133              2.3E-16           75.2%
  4390                               C2H2 type zinc finger family protein                                        232              2.4E-26           81.8%
  4632                               transcription factor                                                        205              2.7E-17           68.3%
  4694                               pinoresinol-lariciresinol reductase                                         347              2.4E-53           92.6%
  5015                               rna recognition motif and cchc-type zinc finger domain-containing protein   185              1.8E-21           95.4%
  5038                               transcription factor                                                        198              7.6E-12           82.8%
  5161                               stress responsive gene 6 srg6                                               169              5.4E-10           89.8%
  5445                               ring zinc finger protein                                                    105              2.5E-10           78.4%
  6289                               transcription factor                                                        247              1.7E-16           88.0%
  6595                               transcription initiation factor tfiid subunit d5                            172              1.4E-13           81.6%
  6596                               zinc finger ccch domain-containing protein 53                               267              4.2E-18           70.1%
  6877                               transcription factor iiia                                                   217              5.7E-20           73.4%
  7318                               zinc finger family protein                                                  179              4.7E-14           67.2%
  7691                               dna binding                                                                 167              9.3E-10           69.6%
  7815                               ring zinc finger protein                                                    154              3.2E-10           71.0%
  8103                               homeobox protein knotted-1-like 6                                           183              1.8E-24           86.6%
  8133                               ribulose bisphosphate carboxylase activase                                  215              9.1E-34           96.8%
  8849                               zinc finger (b-box type) family protein                                     119              4.1E-13           89.4%
  9097                               zinc finger ccch domain-containing protein 67                               225              8.8E-08           62.4%
  9224                               transcription factor                                                        205              6.8E-05           58.0%
  9432                               zinc finger                                                                 241              3.3E-31           61.4%
  9754                               Peptide transporter                                                         124              4.2E-10           81.2%
  10046                              mads-box protein                                                            247              1.8E-26           81.9%

###### 

Description of genes encoded by 69 unique pea ESTs involved in the signaling pathways

  **Seq.Name**                                                 **Seq.Description**                                                                   **Seq.Length**   **min.e-value**   **mean Similarity**
  ------------------------------------------------------------ ------------------------------------------------------------------------------------- ---------------- ----------------- ---------------------
  Abscisic acid mediated signaling pathway                                                                                                                                              
  2884                                                         abscisic acid receptor pyl8                                                           137              2.3E-16           91.5%
  3332                                                         serine threonine-protein kinase                                                       116              9.0E-13           89.3%
  3469                                                         calcium-dependent protein                                                             190              1.2E-28           96.2%
  Auxin mediated signaling pathway                                                                                                                                                      
  109                                                          saur family protein                                                                   287              3.8E-27           69.3%
  1547                                                         auxin-binding protein                                                                 247              9.4E-39           87.3%
  1737                                                         auxin signaling f-box 3                                                               109              2.2E-11           99.1%
  2013                                                         auxin-induced protein                                                                 229              2.9E-11           61.8%
  7004                                                         germin-like protein                                                                   245              5.9E-33           88.4%
  7084                                                         auxin down-regulated-like protein                                                     343              4.4E-15           86.9%
  8570                                                         auxin-induced protein                                                                 177              5.7E-20           80.6%
  9301                                                         auxin-induced protein                                                                 279              7.4E-44           93.9%
  Brassinosteroid mediated signaling pathway                                                                                                                                            
  104                                                          glutamate binding protein                                                             190              9.7E-15           89.7%
  516                                                          kinase family protein                                                                 250              5.0E-40           97.2%
  908                                                          vf14-3-3c protein                                                                     290              1.7E-43           92.1%
  3415                                                         14-3-3-like protein gf14 lambda                                                       105              3.2E-10           91.5%
  8120                                                         kinase family protein                                                                 103              1.5E-10           96.5%
  9939                                                         BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 precursor                  451              5.5E-63           79.2%
  Calcium-mediated signaling                                                                                                                                                            
  2711                                                         calcium ion binding protein                                                           359              8.9E-21           93.3%
  5090                                                         calcium-dependent protein kinase                                                      236              1.2E-36           94.9%
  5443                                                         calcium-dependent protein kinase 19                                                   111              2.6E-12           98.2%
  5757                                                         calcium ion binding protein                                                           309              3.9E-45           86.2%
  8209                                                         calcium-dependent protein kinase 15                                                   123              2.5E-15           85.00%
  8909                                                         plasma membrane-type calcium atpase                                                   247              1.5E-36           91.3%
  9449                                                         calcium calmodulin-regulated receptor-like kinase                                     158              7.4E-15           81.4%
  10142                                                        calmodulin                                                                            365              3.1E-50           100%
  Ethylene mediated signaling pathway                                                                                                                                                   
  1250                                                         ethylene receptor                                                                     232              4.0E-37           73.2%
  8377                                                         zinc finger protein, putative                                                         185              1.2E-17           78.6%
  Gibberellic acid mediated signaling pathway                                                                                                                                           
  3093                                                         gibberellin oxidase-like protein                                                      205              1.4E-13           78.6%
  7394                                                         TPA: putative GID1-like gibberellin receptor                                          141              4.4E-20           90.05%
  7551                                                         gibberellin 2-oxidase                                                                 183              4.5E-28           83.0%
  10003                                                        gibberellin receptor                                                                  259              1.6E-33           75.0%
  small GTPase mediated signal transduction                                                                                                                                             
  895                                                          ras-related protein                                                                   421              3.9E-48           88.2%
  2498                                                         gtp binding protein                                                                   159              1.4E-10           96.7%
  3413                                                         adp-ribosylation factor                                                               372              7.3E-55           100%
  3434                                                         ras-related protein rab7                                                              130              8.6E-16           99.9%
  3696                                                         adp-ribosylation factor                                                               140              1.4E-18           99.9%
  4176                                                         RAS-related GTP-binding protein                                                       224              9.0E-37           99.1%
  4656                                                         small gtp-binding protein                                                             178              3.2E-15           87.4%
  5075                                                         small gtp-binding protein                                                             193              3.8E-27           99.0%
  5134                                                         gtp-binding protein                                                                   282              4.1E-37           95.4%
  5318                                                         adp-ribosylation factor                                                               231              1.1E-18           99.9%
  7015                                                         adp-ribosylation factor 3                                                             141              1.2E-17           100%
  7249                                                         ras-like protein                                                                      199              2.1E-30           99.7%
  7488                                                         rab2 -family small gtpase                                                             142              9.7E-20           99.7%
  8652                                                         gtp binding protein                                                                   134              3.9E-16           95.0%
  Jasmonic acid or Salicylic acid mediated signaling pathway                                                                                                                            
  2086                                                         wus-interacting protein 1                                                             236              1.3E-32           91.9%
  9051                                                         syntaxin 121                                                                          268              2.4E-21           90.3%
  9545                                                         bifunctional inhibitor lipid-transfer protein seed storage 2 s albumin-like protein   303              7.0E-34           64.4%
  Others                                                                                                                                                                                
  376                                                          senescence-associated-like protein                                                    194              2.8E-30           93.9%
  1216                                                         diacylglycerol kinase 5                                                               156              3.0E-16           82.3%
  1266                                                         26 s proteasome non-atpase regulatory subunit 14                                      158              1.3E-24           99.3%
  1756                                                         typical p-type r2r3 myb protein                                                       140              1.8E-05           66.5%
  1815                                                         pas lov protein 1                                                                     102              1.7E-06           88.0%
  2847                                                         peptidyl-prolyl cis-trans isomerase cyp20-3                                           155              1.3E-22           93.9%
  2964                                                         flavin-containing monooxygenase 1                                                     227              2.7E-25           79.6%
  4308                                                         small nuclear                                                                         231              1.6E-25           94.9%
  4474                                                         b regulatory subunit of pp2a ( gamma)                                                 247              7.2E-39           97.8%
  5301                                                         actin depolymerizing factor 1                                                         117              5.8E-12           99.6%
  5774                                                         atp binding                                                                           212              3.5E-25           86.8%
  5809                                                         autoinhibited calcium atpase                                                          246              1.2E-04           65.0%
  6377                                                         integrin-linked protein kinase family protein                                         106              3.2E-10           93.6%
  6424                                                         protein phosphatase                                                                   243              3.1E-34           91.4%
  7633                                                         leucine-rich repeat-containing protein                                                149              2.9E-19           96.4%
  8149                                                         dormancy auxin associated protein                                                     193              2.6E-28           80.3%
  8514                                                         big map kinase                                                                        235              4.4E-28           88.9%
  8781                                                         Mitogen-activated protein kinase homolog MMK2                                         246              1.6E-30           91.3%
  9004                                                         protein ralf-like 33                                                                  270              3.7E-43           86.4%
  9508                                                         ring finger                                                                           248              3.2E-31           86.2%
  9933                                                         histidine-containing phosphotransfer                                                  221              4.8E-06           75.4%

###### 

Description of 82 unique pea ESTs encoding defense-associated proteins

  **Seq.Name**                                                  **Seq.Description**                                                                        **Seq.Length**   **min.e-value**   **mean Similarity**
  ------------------------------------------------------------- ------------------------------------------------------------------------------------------ ---------------- ----------------- ---------------------
  Pathogenesis-related protein                                                                                                                                                                
  5422                                                          pathogenesis-related protein                                                               224              5.3E-21           83.9%
  6766                                                          pathogenesis-related protein 1                                                             201              8.1E-14           73.1%
  7235                                                          pathogenesis-related protein 4a                                                            139              3.2E-07           97.0%
  9781                                                          pathogenesis-related protein 1                                                             647              6.3E-58           82.3%
  Genes involved in disassembly of fungal cell wall                                                                                                                                           
  196                                                           endo-beta-1,3-glucanase                                                                    319              8.7E-48           82.8%
  491                                                           chitinase                                                                                  117              3.4E-12           79.4%
  589                                                           chitinase                                                                                  646              1.5E-96           85.8%
  1243                                                          beta-1,3-glucanase                                                                         638              1.3E-84           88.0%
  1317                                                          transferring glycosyl                                                                      257              5.5E-34           77.4%
  1622                                                          basic chitinase class 3                                                                    451              4.8E-75           84.2%
  1687                                                          glycosyl transferase family 8                                                              135              4.7E-14           87.2%
  2311                                                          chitinase                                                                                  184              6.7E-16           79.9%
  2469                                                          glycosyltransferase-like protein                                                           225              3.0E-32           85.3%
  2880                                                          glycosyl hydrolases family 17 domain-containing protein                                    206              7.8E-25           84.8%
  3896                                                          Glycoside hydrolase, family 17                                                             231              6.4E-19           86.7%
  3940                                                          glycosyltransferase family protein                                                         160              8.7E-16           80.7%
  4388                                                          beta-1,3-glucanase                                                                         243              1.5E-28           85.8%
  4424                                                          acidic glucanase                                                                           316              4.2E-50           91.3%
  5082                                                          aspartyl protease family protein                                                           163              3.2E-10           87.0%
  5129                                                          udp-glycosyltransferase-like protein                                                       122              3.1E-13           92.1%
  5742                                                          chitinase                                                                                  123              2.6E-12           86.0%
  5908                                                          aspartyl protease family protein                                                           303              2.2E-25           81.0%
  6193                                                          acidic glucanase                                                                           102              2.7E-12           100.0%
  6919                                                          glycoside hydrolase family 47 protein                                                      254              4.5E-12           93.0%
  7308                                                          transferase, transferring glycosyl groups                                                  148              2.7E-17           80.9%
  8804                                                          glycosyl hydrolase family 81 protein                                                       201              3.7E-19           76.6%
  9211                                                          aspartyl protease-like protein                                                             337              8.6E-08           68.7%
  Genes involved in biosynthesis of plant cell wall structure                                                                                                                                 
  1677                                                          neutral alpha-glucosidase ab precursor                                                     361              1.6E-38           76.9%
  2757                                                          protein cobra                                                                              228              4.6E-26           88.2%
  4004                                                          cellulose synthase                                                                         301              1.7E-11           66.5%
  8822                                                          dolichyl-diphosphooligosaccharide\--protein glycosyltransferase 48 kda subunit precursor   144              1.8E-16           92.5%
  Lignin related genes                                                                                                                                                                        
  1432                                                          o-methyltransferase 1                                                                      525              4.4E-72           79.0%
  4536                                                          laccase 8                                                                                  447              2.4E-82           74.5%
  5158                                                          lignin biosynthetic peroxidase                                                             106              1.8E-08           81.3%
  5257                                                          caffeic acid 5-hydroxyferulic acid 35-o-methyltransferase ferulic acid complex chain a     171              2.0E-12           83.3%
  6534                                                          putative copper ion-binding laccase                                                        208              7.7E-33           77.6%
  8248                                                          caffeic acid 5-hydroxyferulic acid 35-o-methyltransferase ferulic acid complex chain a     256              1.9E-26           84.5%
  8686                                                          lignin biosynthetic peroxidase                                                             245              7.5E-28           74.8%
  Pectin related genes                                                                                                                                                                        
  150                                                           pectin methylesterase                                                                      242              4.1E-26           75.5%
  531                                                           21 kda protein                                                                             158              1.2E-20           75.9%
  892                                                           pectin methylesterase                                                                      152              9.1E-21           93.6%
  2699                                                          multicopper oxidase                                                                        259              1.8E-32           83.6%
  7076                                                          pectin methyltransferase qua2                                                              234              1.3E-24           87.9%
  8878                                                          pectin lyase-like protein                                                                  126              9.2E-10           86.2%
  9386                                                          pectin methylesterase                                                                      220              2.6E-36           86.3%
  Others                                                                                                                                                                                      
  79                                                            threonyl-trna synthetase                                                                   184              3.7E-22           89.9%
  643                                                           xyloglucan endotransglycosylase hydrolase                                                  215              4.3E-36           97.0%
  1215                                                          prolyl 4-hydroxylase alpha                                                                 176              7.0E-26           94.5%
  1353                                                          (iso)flavonoid glycosyltransferase                                                         240              1.5E-36           85.5%
  1357                                                          peroxidase 52                                                                              109              2.2E-11           90.8%
  1527                                                          60S ribosomal protein L10                                                                  530              7.7E-93           97.0%
  1813                                                          vacuolar atp synthase subunit                                                              552              1.8E-80           87.2%
  2018                                                          gamma-glutamylcysteine synthetase                                                          286              2.4E-37           95.9%
  2083                                                          polygalacturonase-inhibiting protein precursor                                             523              3.0E-89           84.7%
  2201                                                          xyloglucan endotransglucosylase hydrolase-like protein                                     178              1.4E-26           94.1%
  2261                                                          3,5-epimerase/4-reductase                                                                  199              2.4E-26           92.5%
  2418                                                          udp-glucosyl transferase 74b1                                                              224              1.7E-19           79.8%
  2760                                                          peptide deformylase 1a                                                                     199              6.4E-27           88.6%
  2779                                                          alpha-galactosidase 1                                                                      296              1.6E-30           77.3%
  3004                                                          protein disulfide                                                                          245              8.0E-30           92.5%
  3304                                                          dolichyl-diphosphooligosaccharide\--protein glycosyltransferase subunit dad1               109              1.4E-13           99.6%
  3349                                                          pentose-5-phosphate 3-epimerase                                                            171              5.0E-24           95.8%
  3877                                                          AKIN gamma                                                                                 392              1.5E-57           88.0%
  4111                                                          polygalacturonase                                                                          117              3.4E-12           95.0%
  4189                                                          family 8 glycosyl transferase                                                              233              2.1E-17           86.5%
  4244                                                          beta-galactosidase                                                                         145              1.3E-19           90.9%
  4473                                                          nad-dependent epimerase dehydratase                                                        164              9.9E-20           88.6%
  4711                                                          udp-d-glucose udp-d-galactose 4-epimerase 2                                                209              2.6E-28           90.9%
  4836                                                          alpha-expansin 4                                                                           227              1.4E-37           90.4%
  4942                                                          flavonoid glycosyltransferase                                                              368              1.7E-40           73.1%
  5625                                                          prolyl 4-hydroxylase alpha                                                                 117              4.9E-11           75.7%
  5777                                                          nad-dependent epimerase dehydratase                                                        131              1.1E-05           94.0%
  6800                                                          putative beta-D-xylosidase                                                                 205              1.4E-18           66.9%
  6916                                                          UDP-glucose 4-epimerase, putative                                                          322              2.0E-20           88.4%
  7235                                                          af137351_1pathogenesis-related protein 4a                                                  139              3.2E-07           97.0%
  7414                                                          glycine-rich protein                                                                       109              4.5E-12           96.0%
  7526                                                          NAD dependent epimerase/dehydratase, putative                                              273              1.3E-43           97.1%
  7528                                                          polygalacturonase precursor                                                                229              2.5E-10           62.0%
  8194                                                          protein disulfide isomerase                                                                179              5.0E-24           92.9%
  8631                                                          beta-galactosidase like protein                                                            198              1.7E-16           92.3%
  8646                                                          nad-dependent epimerase dehydratase                                                        230              2.4E-29           74.1%
  8682                                                          polygalacturonase inhibitor protein                                                        582              5.0E-71           80.3%
  8736                                                          alanine-2-oxoglutarate aminotransferase 2                                                  221              6.9E-29           87.9%
  9036                                                          alcohol dehydrogenase, putative                                                            330              1.3E-35           84.6%
  9052                                                          nad-dependent epimerase dehydratase                                                        202              5.6E-23           81.5%

The 2,780 *S*. *sclerotiorum* EST contigs were also assessed with BLASTn against 57,751 *S*. *sclerotiorum* ESTs (from mycelia at neutral pH, developing apothecia and developing sclerotia). Of these, 1,784 ESTs matched with an e-value cutoff of 1e^-10^, in which 294 ESTs matched with 95% identity for 95% of more of the query length to the *S*. *sclerotiorum* EST growth libraries. Of the remaining 996 unique ESTs, it was possible to annotate 438 ESTs of which 95 ESTs were described as being related to pathogen virulence or pathogenicity (Table [5](#T5){ref-type="table"}).

###### 

**Description of 95 unique*S*.*sclerotiorum*EST contigs with putative involvement in virulence or pathogenicity**

  **Seq.Name**                                                               **Seq.Description**                                                               **Seq.Length**   **min.e-value**   **mean Similarity**
  -------------------------------------------------------------------------- --------------------------------------------------------------------------------- ---------------- ----------------- ---------------------
  4 genes involved in the recognition of the host and in signaling pathway                                                                                                                        
  1457                                                                       c2h2 type zinc finger containing protein                                          222              2.2E-35           75.5%
  1796                                                                       guanine nucleotide-binding protein alpha subunit                                  171              2.7E-17           95.0%
  3623                                                                       importin beta-2                                                                   109              1.2E-09           85.7%
  6785                                                                       c2h2 transcription factor                                                         135              2.8E-19           79.9%
  18 genes affecting biosynthesis and the integrin of fungal cell walls                                                                                                                           
  330                                                                        alpha-1,2-mannosyltransferase kre5                                                196              8.4E-19           70.9%
  2296                                                                       cell wall biogenesis protein glutathione transferase                              122              2.8E-14           90.5%
  2424                                                                       endoglucanase-4 precursor                                                         326              6.4E-27           76.0%
  2930                                                                       alpha-1,2-mannosyltransferase alg11                                               154              1.7E-22           95.3%
  3362                                                                       alpha-glucan-branching enzyme                                                     234              6.8E-29           82.6%
  3825                                                                       alpha-mannosyltransferase                                                         248              3.4E-41           71.1%
  4843                                                                       dolichol-phosphate mannosyltransferase                                            253              1.9E-34           87.1%
  5737                                                                       endoglucanase ii                                                                  122              8.7E-16           86.0%
  5773                                                                       dolichol-phosphate mannosyltransferase                                            255              1.9E-26           89.7%
  6654                                                                       endochitinase 42                                                                  189              1.5E-28           87.0%
  6797                                                                       chitinase                                                                         105              2.3E-11           79.9%
  6956                                                                       endoglucanase ii                                                                  267              6.8E-45           86.7%
  7397                                                                       gpi-anchored cell wall beta-1,3-endoglucanase                                     159              3.0E-24           69.3%
  8474                                                                       af215732_1class iv chitin synthase                                                113              2.0E-07           86.0%
  8681                                                                       chitin synthase                                                                   138              2.0E-20           95.6%
  8785                                                                       mannan polymerase ii complex anp1 subunit \[[@B61]\]                              173              8.6E-24           92.8%
  9176                                                                       gpi-anchored cell wall beta-1,3-endoglucanase                                     295              9.2E-34           66.5%
  9570                                                                       mannosyl transferase                                                              122              3.3E-15           82.6%
  12 genes involved in the production of infection structures                                                                                                                                     
  97                                                                         siderophore biosynthesis                                                          106              5.9E-12           81.5%
  239                                                                        mannosyl transferase                                                              221              1.3E-22           94.3%
  253                                                                        adenylate kinase                                                                  140              6.3E-19           88.3%
  427                                                                        serine threonine-protein phosphatase pp2a catalytic subunit                       289              4.9E-51           99.4%
  1554                                                                       adenylate cyclase                                                                 135              1.4E-18           83.2%
  4181                                                                       mfs toxin efflux pump                                                             194              1.1E-21           69.4%
  5419                                                                       alcohol dehydrogenase                                                             190              3.4E-28           75.7%
  5981                                                                       adp-ribosylation factor 1                                                         455              7.3E-23           93.7%
  6192                                                                       adp-ribosylation factor 1                                                         103              3.5E-12           100.0%
  6325                                                                       nadp-dependent alcohol dehydrogenase                                              347              4.0E-53           82.8%
  7407                                                                       1-phosphatidylinositol phosphodiesterase                                          102              9.2E-05           76.0%
  9579                                                                       alcohol oxidase                                                                   112              9.7E-15           90.7%
  39 genes involved in the penetration of the cuticle and cell wall                                                                                                                               
  47                                                                         cellulose 1,4-beta-cellobiosidase                                                 165              7.3E-23           77.1%
  473                                                                        xyloglucan-specific endo-beta-1,4-glucanase precursor                             228              7.6E-36           84.0%
  485                                                                        cutinase                                                                          375              2.5E-63           68.5%
  555                                                                        endo-1,4-beta-xylanase                                                            265              3.8E-19           79.3%
  717                                                                        endo-1,4-beta-xylanase                                                            348              2.8E-62           78.4%
  970                                                                        acetyl xylan                                                                      118              2.3E-16           73.5%
  1533                                                                       endopolygalacturonase 2                                                           181              1.3E-27           89.5%
  1562                                                                       carbohydrate esterase family 1 and carbohydrate-binding module family 1 protein   241              4.4E-28           76.1%
  2427                                                                       4-coumarate:CoA ligase                                                            185              1.8E-29           79.8%
  2450                                                                       acetolactate synthase                                                             236              1.9E-39           93.3%
  3006                                                                       aspartic-type endopeptidase                                                       171              1.5E-23           71.2%
  3115                                                                       fungal alpha-l-arabinofuranosidase                                                110              3.1E-13           91.6%
  3147                                                                       extracellular exo-polygalacturonase                                               269              1.5E-12           83.5%
  3513                                                                       beta-xylosidase                                                                   219              9.1E-37           84.6%
  3712                                                                       feruloyl esterase b                                                               187              1.7E-27           78.6%
  3861                                                                       pectin methylesterase                                                             170              2.4E-26           80.0%
  3907                                                                       extracellular endo-1,5-alpha-l-arabinosidase A                                    172              1.6E-25           80.0%
  4037                                                                       aspartic endopeptidase                                                            129              3.2E-18           82.4%
  4295                                                                       cutinase                                                                          278              2.6E-36           66.3%
  4446                                                                       xyloglucan-specific endo-beta- -glucanase precurso                                626              8.3E-89           77.3%
  5554                                                                       beta-glucosidase 1b                                                               267              8.0E-46           83.7%
  5562                                                                       alpha-l-arabinofuranosidase                                                       130              1.1E-15           74.2%
  5679                                                                       polygalacturonase 1                                                               427              9.8E-60           96.6%
  5918                                                                       acetyl xylan esterase                                                             345              5.1E-16           84.0%
  6412                                                                       exoglucanase 2 precursor                                                          141              4.8E-19           90.3%
  6383                                                                       alpha-L-arabinofuranosidase A                                                     130              1.1E-13           78.3%
  6431                                                                       exopolygalacturonase                                                              135              1.3E-16           89.0%
  6759                                                                       endo-1,4-beta-xylanase                                                            359              8.8E-45           81.1%
  6824                                                                       pectin methylesterase                                                             107              3.1E-13           79.7%
  7085                                                                       pectinesterase family protein                                                     109              7.7E-12           82.5%
  7661                                                                       polygalacturonase 1                                                               601              3.9E-45           93.2%
  7834                                                                       extracellular exo-polygalacturonase                                               386              3.0E-61           68.0%
  8501                                                                       pectin methylesterase                                                             252              1.6E-41           62.5%
  9093                                                                       carbohydrate esterase family 8 protein                                            244              9.4E-31           71.6%
  9509                                                                       extracellular endo-1,5-alpha-l-arabinase                                          322              2.0E-36           74.7%
  9525                                                                       cellulase family protein                                                          239              1.8E-37           72.8%
  9583                                                                       acetolactate synthase                                                             123              1.3E-16           90.6%
  9655                                                                       acetyl xylan esterase                                                             129              2.3E-16           82.8%
  10069                                                                      endopolygalacturonase 2                                                           460              1.2E-73           91.7%
  9 genes involved in responding to host immune system                                                                                                                                            
  1220                                                                       efflux transporter                                                                120              1.9E-15           80.3%
  4783                                                                       abc bile acid transporter                                                         142              1.6E-17           72.5%
  5659                                                                       mfs transporter                                                                   236              8.0E-38           80.8%
  6180                                                                       ATP-binding cassette transporter                                                  229              1.7E-40           81.1%
  6538                                                                       glutathione transferase                                                           486              3.7E-91           70.2%
  7926                                                                       glutathione s-transferase ure2-like protein                                       123              1.3E-16           79.8%
  8029                                                                       ornithine decarboxylase                                                           234              9.5E-39           90.3%
  8188                                                                       glutathione s-transferase                                                         527              6.5E-100          79.7%
  9716                                                                       salicylate hydroxylase                                                            202              5.4E-34           85.8%
  3 genes involved in fungal nutrition (virulence-associated)                                                                                                                                     
  1446                                                                       methionine aminopeptidase 2                                                       337              7.2E-47           84.8%
  1889                                                                       vacuolar atpase proteolipid subunit c                                             314              2.6E-23           92.5%
  5131                                                                       vacuolar atpase proteolipid subunit c                                             157              3.1E-21           90.4%
  Others 10 genes related with pathogen virulence or pathogenicity                                                                                                                                
  145                                                                        acid proteinase                                                                   583              1.8E-89           63.5%
  1387                                                                       methylcitrate synthase precursor                                                  182              1.7E-27           96.2%
  2840                                                                       centromere kinetochore protein                                                    217              4.1E-34           73.0%
  3499                                                                       vacuolar protein 8                                                                255              7.7E-28           97.4%
  3747                                                                       acid proteinase                                                                   246              6.5E-40           66.9%
  5648                                                                       dipeptidyl peptidase                                                              226              1.2E-12           83.1%
  7192                                                                       alpha-amylase precursor                                                           207              2.9E-32           62.7%
  7273                                                                       vanillyl-alcohol oxidase                                                          146              5.3E-21           80.3%
  10090                                                                      phospholipase d active site motif protein                                         235              3.3E-39           69.9%
  10153                                                                      alpha-ketoglutarate dependent xanthine dioxygenase                                249              1.2E-41           87.6%

Prediction of secretory/signal peptides for the *S*. *sclerotiorum* contigs
---------------------------------------------------------------------------

A total of 2,754 coding regions were predicted with OrfPredictor from the set of 2,780 *S*. *sclerotiorum* ESTs. The peptide sequences were then used as a query for SignalP 3.0, which predicts the presence and location of signal peptide cleavage sites in amino acid sequences and identifies them as secretory proteins. The neural network (NN) method predicted 244 secretory signals, and the Hidden Markov Model (HMM) predicted 216. A total of 142 ESTs were identified by both NN and HMM and can be considered putative secretory peptides with high confidence (see Additional file [2](#S2){ref-type="supplementary-material"}). Of these 142 predicted secretory proteins, 21 were reported to be involved in pathogen virulence or pathogenicity (Table [6](#T6){ref-type="table"}).

###### 

***S*.*sclerotiorum*EST contigs encoding potential secretory/signal peptides involved in virulence or pathogenicity**

  **Seq**. **Name**   **Seq**. **Description**                                     **Seq.Length**   **min.e-value**   **mean Similarity**
  ------------------- ------------------------------------------------------------ ---------------- ----------------- ---------------------
  355                 Enolase                                                      120              3.7E-14           97.8%
  395                 bzip transcription factor                                    108              2.5E-10           86.1%
  1352                fkbp-type peptidyl-prolyl                                    479              2.4E-58           81.5%
  1434                chitin synthase 1                                            220              2.6E-36           86.1%
  2605                cysteine desulfurase                                         157              3.7E-14           93.8%
  3499                vacuolar protein 8                                           255              7.7E-28           97.4%
  3632                autophagy protein                                            253              2.8E-38           60.3%
  4181                mfs toxin efflux pump                                        194              1.1E-21           69.4%
  4467                nadh:ubiquinone oxidoreductase subunit                       117              1.3E-16           90.8%
  5493                formate nitrite transporter                                  145              9.4E-18           77.1%
  6251                v-atpase proteolipid subunit                                 398              3.8E-43           89.4%
  6759                endo-1,4-beta-xylanase                                       359              8.8E-45           81.1%
  7392                rhamnogalacturonan acetylesterase                            330              3.6E-38           73.3%
  7736                phosphoethanolamine transferase pigf                         186              7.7E-20           76.1%
  8184                tetraspanin tsp3                                             225              2.5E-31           96.7%
  8501                pectin methylesterase                                        252              1.6E-41           62.5%
  9219                adenylate kinase                                             225              4.5E-12           100.0%
  9240                glycosyl hydrolase family 61                                 320              1.5E-55           65.9%
  9375                chd5 domain-containing protein                               327              1.2E-41           82.2%
  9461                phosphatidylglycerol phosphatidylinositol transfer protein   551              9.8E-92           70.5%
  9847                Glutaredoxin                                                 158              6.9E-21           83.9%

Discussion
==========

Significance of study and summary of the main findings
------------------------------------------------------

Despite *Pisum sativum* being used by Gregor Mendel to propose a model of particulate inheritance and being a highly nutritious food source for populations worldwide, few genomic resources exist for pea. One of the pathogens of pea, *S*. *sclerotiorum* is not only capable of causing devastating disease of pea but is able to infect over 400 plant species \[[@B1]\]. By sequencing a normalized cDNA pool of the pea-*S*. *sclerotiorum* interaction with next generation sequencing we have catalogued a number of novel genes putatively involved in pathogenicity and resistance. To our knowledge this is the first study to examine the pea-*S*. *sclerotiorum* "interactome". Sequencing the transcriptome (RNA-seq) is the method of choice in non-model systems for transcript discovery and genome annotation \[[@B8]\]. However, it has rarely been used to study plant-fungal interactions; one reason for this is the difficulty in distinguishing plant and fungal ESTs, particularly when reference genomes are not available. Using genomes of closely related species and tBLASTx to parse pea and *S*. *sclerotiorum* ESTs we demonstrated that Roche 454-pyrosequencing is a useful technique to characterize the host-pathogen interactome when genome resources are limited.

tBLASTx parsing method
----------------------

Two different strategies have been utilized previously to identify transcript origins in mixed plant and fungal EST datasets. One is a predictive method based on triplet nucleotide usage frequencies \[[@B9]\] and the other is a homology method using the BLASTp algorithm \[[@B10]\]. One shortcoming of the BLASTp method is that it could not be applied to novel genes or sequences from the non-coding regions of genes. Although the triplet nucleotide frequency method extends the application of the algorithm to both coding and non-coding sequences, the classification accuracy is approximately 90%, and required the use of a training set of ESTs to develop the nucleotide frequency for separation. A combined method was also used by Fernandez et al. \[[@B11]\], although this method distinguished 91% of the ESTs from the *Coffea arabicaHemileia vastatrix* interaction no validation of the method was presented \[[@B11]\].

Classification of genes from a pool of mixed cDNA by traditional sequence similarity analysis (BLAST) is of interest to many investigations into plant-pathogen interactions. DNA sequencing is becoming more affordable and whole genome sequences of many organisms are becoming available and will aid in plant-pathogen interaction studies. However, in pea these resources are not available, therefore, we used a standalone BLAST approach against proxy-reference genome databases with high genetic similarity to pea or *S*. *sclerotiorum* to distinguish mixed transcripts. Using an artificial mixture of known pea and *Sclerotinia* ESTs, we found the error rate using the BLAST method was significantly lower than the triplet nucleotide frequencies method (Table [7](#T7){ref-type="table"}). We also demonstrated that the tBLASTx algorithm provided improved sorting of contigs relative to the BLASTn algorithm, and results in fewer ambiguous reads (see Additional file [3](#S3){ref-type="supplementary-material"}). In addition, although one individual genome of *S*. *sclerotiorum* (strain 1980) has been sequenced \[[@B12]\], there are still 1.6 Mb of predicted gaps in the 39.6 Mb assembly. To avoid ignoring unique genes between two different strains of the same species, a multi-fungal genome approach was adopted in this study. It was demonstrated that the assignment error rate based on 7 closely related fungal genomes was slightly decreased relative to assignment based on the single *S*. *sclerotiorum* genome (see Additional file [4](#S4){ref-type="supplementary-material"}). The e-value and e-value ratio utilized in our study to differentiate pea and *S*. *sclerotiorum* reads chosen selected after comparing several e-values, to maximize discrimination while reducing the error rate (see Additional file [5](#S5){ref-type="supplementary-material"}). Additionally, we determined error rates for this method using the artificial EST mix and validated the technique using our EST data set. We found that the percentage of unassigned ESTs (23.9%) in the 454 data set was higher than in the test EST data set (9%). One hypothesis to explain this difference is the average sequence length in the 454 data (200 bp) was shorter than known pea (496 bp) or *Sclerotinia* ESTs (674 bp) used in test data, which may result in no significant alignment against the proxy-reference plant or fungal genome databases, particularly when non-coding mRNA is considered. The remaining unassigned EST contigs (21%) were parsed using BLASTn searches against known pea and *S*. *sclerotiorum* ESTs, which assigned 44.4% of the unassigned contigs. Using this combined tBLASTx and BLASTn approach 89.4% of the 10,158 contigs were identified as pea (6,299) or *S*. *sclerotiorum* EST (2,780). Additionally, the EST parsing method was validated by PCR demonstrating that the parsing method was able to correctly assign ESTs from the 454 data set with a low error rate.

###### 

**Comparison of triplet nucleotide frequencies method and tBlastx method to assign artificial EST mixture of pea-*S*.*sclerotiorum*(including 18,490 pea ESTs and 17,198** ***S*.*sclerotiorum*ESTs)**

  **Category of EST**    **Number of ESTs by triplet nucleotide frequencies method**   **Number of ESTs by tBLASTx method**
  --------------------- ------------------------------------------------------------- --------------------------------------
  Plant                                   17,660 (*2*,*180 wrong*)                             15,289 (*14 wrong*)
  Fungi                                   18,028 (*3*,*010 wrong*)                             16,908 (*23 wrong*)
  Ambiguous                                           0                                               1,519
  Unassigned                                          0                                               1,972
  Total                                            35,688                                             35,688

Pea ESTs unique to the pea-*S*. *sclerotiorum* interaction
----------------------------------------------------------

In response to pathogen attack, plants have evolved complex signaling and defense pathways. Putatively unique ESTs in our pea-*S*. *sclerotiorum* interactome were defined and identified by comparing EST contigs in our library against those of non-interaction EST libraries of pea and *S*. *sclerotiorum*. Although we identified a total of 2,840 (45.1%) putatively unique pea ESTs it was only possible to annotate 1,631 of these and only 451 had annotations suggesting roles in defense or response to biotic and abiotic stress. Most of the annotated genes are consistent with previous expression profiling analyses in *Brassica napus* infected with *Sclerotinia sclerotiorum*\[[@B13]\]. Following infection, many genes, including those encoding defense-associated proteins, enzymes involved in signaling pathways, and genes encoding transcription factors were induced.

Transcriptional control of the expression of stress-responsive genes is a crucial part of plant response to a range of abiotic and biotic stresses \[[@B14]\]. We demonstrated that 67 putative transcription factors were detected. These genes were classified into the MYB family, the Apetala2/Ethylene responsive element binding protein (AP2/EREBP) family, WRKY family and others (Table [2](#T2){ref-type="table"}). Seven MYB family transcription factors were detected in our data and they play a key role in hormone signal transduction and disease resistance \[[@B15]\]. Eight AP2/EREBP transcription factors, including 3 ethylene insensitive transcription factors (contig 1311, 7671 and 8261) and 3 AP2/ERF genes (7472, 8894 and 9486), are key regulatory elements for ethylene signaling and response for biotic or abiotic stresses \[[@B16],[@B17]\].WRKY40 act as negative regulators of defense signaling and have been associated with negatively regulating resistance to *P*. *syringae* in Arabidopsis \[[@B18]\].

Plant defenses are regulated through a complex network of transduction pathways \[[@B19]\]. Sixty-nine unique pea ESTs involved in signaling pathways were detected in this study. The signaling pathways were mediated by different signaling molecules, like abscisic acid (ABA), auxin, brassinosteroid, calcium ion, ethylene (ET), gibberellic acid (GA), jasmonic acid (JA), salicylic acid (SA) and small GTPase (Table [3](#T3){ref-type="table"}). Those results were consistent with previous studies of signaling pathways involved in plant resistance to *Sclerotinia sclerotiorum*\[[@B20],[@B21]\].

Expression of downstream proteins, including defense-associated proteins, was induced through signal transduction and transcription factor regulation after pathogen infection. In this study, 82 unique pea ESTs encoding defense related proteins were detected (Table [4](#T4){ref-type="table"}). Four contigs (5422, 6766, 7235 and 9781), encoding putative pathogenesis-related (PR) proteins involved in the response to pathogen attack were prominent. Numbers of cell-wall-related genes were also detected; those contigs involved in the biosynthesis of plant cell wall structures and the disassembly of fungal cell walls. Chitinase (encoding by contig 491, 589, 1622, 2311 and 5742), beta-1, 3-glucanase (encoding by 196, 1243 and 4388) and other glycoside hydrolases are known to possess anti-fungal activity by degrading fungal cell walls \[[@B22]\].

*S*. *sclerotiorum* ESTs unique to the pea-*S*. *sclerotiorum* interaction
--------------------------------------------------------------------------

Pathogens have evolved a number of strategies to gain entry into the host cell and to overcome the plant defense system. In this study, we identified 996 *S*. *sclerotiorum* contigs as specifically expressed during pea-*S*. *sclerotiorum* interaction through comparison of EST contigs against *S*. *sclerotiorum* ESTs from growth libraries. Ninety-five of 438 annotated contigs were described as being involved in pathogen virulence or pathogenicity (Table [5](#T5){ref-type="table"}).

Fungi produce enzymes that degrade the cell wall and wall-associated polymers to penetrate plant cells. There were 39 specifically expressed contigs involved in the penetration of the plant cuticle and cell wall. Contig 6412 encodes an exoglucanase 2 precursor, which has cellulolytic activity \[[@B23]\] and is involved in cellulose degradation; enzymes encoded by 11 contigs (473, 555, 717, 970, 3115, 3513, 5562, 5918, 6759, 6383 and 9655) are involved in hemicellulose degradation; enzymes encoded by 11 contigs (1533, 3147, 3861, 5679, 6431, 6824, 7085, 7661, 7834, 8501 and 10069) are involved with pectin degradation. In addition, carbohydrate esterase encoded by contig 1562 was also involved in plant polysaccharide degradation. Integrity of the fungal cell wall is also very important for pathogenesis and some reports showed the deletion of biosynthetic cell wall enzymes resulted in dramatically reduced virulence \[[@B24]\]. In our data, 18 contigs were identified as affecting biosynthesis and integrity of fungal cell walls. Enzymes encoded by contigs 6654, 6797, 8474 and 8681 were involved in chitin synthesis; contigs 2424, 3362, 5737, 6956, 7397 and 9176 were involved in glucan synthesis; and 8 contigs (239, 330, 2930, 3825, 4843, 5773, 8785 and 9570) were involved in mannan synthesis.

*Sclerotinia sclerotiorum* differentiates appressoria into infection cushions prior to invasion and we found 12 genes involved in the formation of infection structures. Eight contigs were involved in response to the host immune system, of which 3 efflux transporters encoded by contigs 1220, 4783 and 6180 are responsible not only for export of compounds involved in pathogenesis such as secondary metabolites, but also export of host-derived antimicrobial compounds \[[@B25]-[@B27]\]. Contig 1769 had similarity to the guanine nucleotide-binding protein (G protein) alpha subunit which is an important signal transducing molecule in cells, essential for growth, asexual and sexual development, and virulence in both animal and plant pathogenic filamentous fungal species \[[@B28]\]. Importin beta-2 encoded by contig 3623 belongs to the importin β family which mediates transport between the nucleus and cytoplasm of macromolecules that contain nuclear import or export signals. All importin β members have the ability to recognize and bind specific cargo involved in the recognition of the host and signaling \[[@B29],[@B30]\].

Secreted/signaling proteins
---------------------------

Proteins secreted by fungi play a key role in the development of plant disease and the evolution of pathogenicity \[[@B31]\]. Some secreted proteins can degrade polymers encountered, such as cellulose, lipid, protein, and lignin, and transport the resulting simple sugars, amino acids, and fatty acids into the growing cell for use \[[@B32]\]. Using the SignalP3.0 program with stringent criteria, 142 contigs encoding putative secreted proteins were identified in the 2,780 *S*. *sclerotiorum* contigs. Twenty-one of the 66 annotated contigs were described as involved in pathogen virulence/pathogenicity in previous research (Table [6](#T6){ref-type="table"}). Contig 355 encodes an enolase which is usually present on the cell surface or even secreted and is a potential virulence factor. In bacterial systems enolase has been demonstrated to contribute to pathogenicity by binding plasminogen in the infected host, potentially allowing the bacteria to acquire surface-associated proteolytic activity \[[@B33]-[@B35]\]. The basic leucine zipper transcription factor, encoded by contig 395, is a member of the bZIP family, one bZIP family member (Moatf1) from the rice fungus *Magnaporthe oryzae* mediates oxidative stress responses and is necessary for full virulence \[[@B36]\]. Contig 1352 encoding fkbp-type peptidylprolyl isomerase, with high homology to the Mip (macro-phage infectivity potentiator) protein, has been shown to be an essential virulence factor in *Legionella pneumophila*\[[@B37]-[@B39]\]. Chitin synthase 1 (contig 1434) plays a major role in cell wall biogenesis. Disruption of *Botrytis cinerea* class I chitin synthase gene *Bcchs1* results in cell wall weakening and reduced virulence \[[@B40],[@B41]\]. Autophagy is necessary for turnover of organic matter during the formation of conidia and appressoria and for normal development and pathogenicity in *Magnaporthe grisea*. Autophagy is required for the virulence of some eukaryotic pathogens \[[@B42]-[@B44]\]. Contig 6759 encodes endo-β-1,4 xylanase which plays a significant role in the virulence of *Magnaporthe oryzae*, affects both penetration and expansion of *M*. *oryzae* in infected plants \[[@B45]\]. Pectin methylesterase (PME) produced by phytopathogenic bacteria and fungi catalyses the demethoxylation of pectin, a major plant cell wall polysaccharide \[[@B46]\]. The possible role of secreted adenylate kinase (AK), encoded by contig 9219, as a virulence factor is in producing and keeping an intact pool of toxic mixtures of AMP, ADP, and ATP, which allows *Pseudomonas aeruginosa* to exert its full virulence \[[@B47]\]. Glutathione reductase is important to nitric oxide and macrophage resistance and is essential for virulence \[[@B48]\] and in *Candida albicans* GRX2, a putative glutaredoxin, is required for virulence in a murine model \[[@B49]\].

Conclusions
===========

Here we present an EST resource that is specific for the pea-*S*. *sclerotiorum* interaction. We demonstrate and validate a method to reliably parse host and pathogen ESTs without the need for reference genomes. The ESTs were compared to non-interaction EST libraries to identify candidate resistance and pathogenicity genes. We also catalogued 145 proteins putatively secreted by *S*. *sclerotiorum*. The EST dataset will be a useful reference for further plant-fungus interaction studies, particularly for the *Sclerotinia* and legume research communities. Additionally, the *S*. *sclerotiorum* ESTs will be a valuable resource for the annotation of the *S*. *sclerotiorum* genome. Although the depth of our sequencing was not sufficient to obtain a global view of transcripts expressed during the pea-*S*. *sclerotiorum* interaction, the results are still very useful for the identification of plant resistance, fungal pathogenicity and virulence genes. This study sets the ground work and will be a resource for our current pea-*S*. *sclerotiorum* RNAseq expression profiling studies.

Methods
=======

Plant, fungal growth and inoculation
------------------------------------

Three plants of pea cultivar 'Lifter' (PI628276) were established per 1 gallon plastic pot in Sunshine LA 4 potting mix (Sun Gro Horticulture, Bellevue, WA). The plants were maintained in a greenhouse for 4 weeks with supplemental lighting extending the day length to approximately 14 h (October). Day and night temperatures were 22 ± 2°C and 16 ± 2°C, respectively. *S*. *sclerotiorum* isolate WMA-1 was isolated from a diseased pea plant in 2003 from a pea field (Washington, USA) with white mold disease symptoms and stored as air dry sclerotia at room temperature. Isolate WMA-1 (=ATCC MYA-4521) was demonstrated to be genetically representative of eight *S*. *sclerotiorum* strains sampled from legume hosts from various geographic locations using randomly amplified polymorphic DNA (RAPD) analysis (Kawabe and Peever, *unpublished*). Plants were inoculated with a 5 mm plug collected from the leading edge of an actively growing colony on a potato dextrose agar (PDA). The plug was placed fungal side down on the stem between the 4^th^ and 5^th^ detectable nodes and held in place by wrapping with Parafilm. Plants were transferred to a growth chamber with a 12 h photoperiod, an approximate 60% relative humidity, temperature of 20 ± 1°C and a 12 h photoperiod, for 72 hours to allow disease lesion development prior to RNA extraction.

Total RNA extraction and purification of mRNA from total RNA
------------------------------------------------------------

A 1 cm stem section was collected from each of 18 infected plants by cutting above and below the lesion front advancing toward the base of the plant. The stem section included both necrotic and green tissue with the advancing lesion front located in the center of the section. Stem sections were snap-frozen in liquid nitrogen and ground to a fine powder with a mortar and pestle. A total of 3 ml of TRIzol (Invitrogen, Carlsbad, CA, USA) was added to the ground tissue and the sample was split in half for column purification with the TRIzol Plus RNA purification kit (Invitrogen, Carlsbad, CA, USA). The additional step of on-column DNA digestion was performed with DNase I (Invitrogen, Carlsbad, CA, USA) to remove contaminating DNA. RNA was eluted in 250 μl of water per spin column. Poly-A RNA was isolated from total RNA with the Oligotex kit using the mRNA spin-column protocol (Qiagen, Valencia, CA, USA). Purified mRNA was eluted in a total of 100 μl of 5 mM Tris (pH 7.5). RNA and mRNA quantity was determined with a spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA). Total RNA and mRNA quality was assessed with an RNA Nano LabChip on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).

cDNA synthesis, normalization and 454 pyrosequencing
----------------------------------------------------

Purified mRNA was used to construct a full length normalized cDNA pool through the services of Evrogen \[[@B50]\]. Briefly, the service utilized the SMART cDNA cloning methodology to generate a full length cDNA pool \[[@B51]\], which was normalized using a duplex-specific nuclease \[[@B52]\]. The double stranded normalized cDNA pool was sheared by nebulization and prepared for and sequenced as per manufacturer's instructions on a Roche 454 GS FLX sequencer using an entire plate at Washington State University.

Data filtering and *de novo* assembly
-------------------------------------

35 Mb of sequence data representing 162,729 reads were generated by 454 sequencing. Quality trimming, adaptor sequence removal and size selection of reads was performed with Galaxy software (<http://main.g2.bx.psu.edu/>) \[[@B53]\]. After trimming adaptors, 128,720 reads with quality scores over 20 and sequence length longer than 50 bp were assembled with Abyss \[[@B54]\]. Parameters were adjusted for optimal assembly as measured by N50 statistic (a weighted median statistic such that 50% of the entire assembly is contained in contigs or scaffolds equal to or larger than this value).

Virus or viroid contamination detection
---------------------------------------

To determine whether any viruses or viroids were present in the fungi-infected plant cDNA sample, viroid and virus databases \[[@B55]\], including 41 complete viroid genomes and 2628 virus genomes, were downloaded from NCBI (released in April 2011). All EST contigs were analyzed with tBLASTx against viroid and virus databases. The e-value cutoff threshold was set at 1e^-3^. Contigs with a BLAST hit to viroid and virus databases were further analyzed by tBLASTx program against 3 legume genomes database and 7 fungi genomes database individually using the same cutoff threshold (see next section "development of a *S*. *sclerotiorum* and *P*. *sativum* parsing method").

Development of a *S*. *sclerotiorum* and *P*. *sativum* parsing method
----------------------------------------------------------------------

To separate *S*. *sclerotiorum* and pea ESTs from the mixed pool, a procedure based on that proposed by Hsiang et al. in 2003 \[[@B56]\] was employed with modifications (Figure [2](#F2){ref-type="fig"}). Briefly, the mixed ESTs were compared with tBLASTx (NCBI-BLAST-2.2.24+) to fungal and plant "proxy-reference" genome databases (Table [8](#T8){ref-type="table"}). These proxy reference databases were established as the pea genome is not available and the inclusion of additional ascomycetes genomes to *S*. *sclerotiorum* (strain 1980) improved the assignment rate. The proxy-fungal genome database was a mixture of *Sclerotinia sclerotiorum* (strain 1980) and 6 closely related Ascomycete fungi (*Botrytis cinerea*, *Chaetomium globosum*, *Fusarium graminearum*, *Magnaporthe grisea*, *Neurospora crassa and Verticillium dahlia*) and a plant genome database including 3 sequenced legume genomes (*Glycine max*, *Lotus japonicus and Medicago truncatula*). ESTs that only matched to fungal or plant genome database with an e-value of 1e^-03^ or better were automatically classified into *S*. *sclerotiorum* or pea ESTs, respectively. ESTs, which matched (e-value \<1e^-3^) to both fungi and plant databases, were further analyzed by comparing the e-value of best-hit from fungi and plant genome results. An e-value ratio was determined by dividing the best-hit e-value to fungi and plant genomes from the tBLASTx searches. A cutoff ratio were set at \> =1e^20^ for pea ESTs, \<=1e^-20^ for *S*. *sclerotiorum* ESTs and those that fell between 1e^-20^ and \<1e^20^ were considered to be ambiguous. To acquire a final sort of results, those ESTs without a BLAST hit or those found to be ambiguous were assigned with BLASTn against known *S*. *sclerotiorum* or pea ESTs if their identity was above 95% in similarity across 95% of the sequence length. 81,449 pea ESTs (from flowers, leaves, cotyledons, epi- and hypocotyl, and etiolated and light treated etiolated seedlings) \[[@B7]\] and 57,751 *S*. *sclerotiorum* ESTs (from mycelia growing at neutral pH, developing apothecia and developing sclerotia\-\--downloaded from BROAD database) were used to assist in the classification and annotation of contigs.

###### 

Source of fungal plant genome databases used for tBLASTx EST assignment

  **7 ascomycete fungal genome databases**    **Size**   **Database**   **Version**
  ------------------------------------------ ---------- -------------- -------------
  *Botrytis cinerea*                          42.66 Mb      BROAD         2005-10
  *Chaetomium globosum*                       34.89 Mb      BROAD         2005-06
  *Fusarium graminearum*                      36.45 Mb      BROAD         2007-03
  *Magnaporthe grisea*                        38.76 Mb      BROAD         2011-04
  *Neurospora crassa*                         41.04 Mb      BROAD         2010-06
  *Sclerotinia sclerotiorum*                  38.33 Mb      BROAD         2011-06
  *Verticillium dahlia*                       33.83 Mb      BROAD         2008-07
  **3 legume genome databases**               **Size**   **Database**   **Version**
  *Glycine max*                               \~975 Mb     GenBank        2010-01
  *Lotus japonicus*                           \~500 Mb     GenBank        2008-06
  *Medicago truncatula*                       \~305 Mb     GenBank        2009-03

To verify the feasibility of the EST parsing method, 17,533 *S*. *sclerotiorum* ESTs derived from developing *S*. *sclerotiorum* libraries were downloaded from BROAD institute and 18,547 *P*. *sativum* ESTs were obtained from the GenBank EST database by search keyword '*Pisum sativum*'. Vector contamination was removed from the downloaded ESTs by BLAST search with UniVec database (GenBank) in *P*. *sativum* and *S*. *sclerotiorum* ESTs were trimmed. After vector trimming, tBLASTx analysis of the downloaded ESTs was performed separately against the proxy-reference fungal and plant databases (Table [8](#T8){ref-type="table"}). The following relevant data from tBLASTx output were extracted to an Excel file: query sequence name, query sequence length, fungi database target name, fungi database e-value for top match, total query sequence length for all match to fungi database, plant database target name, plant database top match e-value, total query sequence length for all match to plant database.

PCR to confirm validity of classified contigs
---------------------------------------------

Fifty contigs from *S*. *sclerotiorum* and 50 contigs from pea were randomly sampled to check the validity of EST contig classification. Primers were designed for each contig using the program Primer3 \[[@B57]\]. cDNA from pea inoculated with *S*. *sclerotiorum*, cDNA from non-inoculated pea, cDNA from *S*. *sclerotiorum* growing on PDA medium, and genomic DNA extracted from pea and *S*. *sclerotiorum* using DNeasy plant mini kit (Qiagen, Valencia, CA, USA ) were used as template in PCR with primer pairs for each contig. PCR contained 4 μl of 5 × GoTaq PCR Buffer (Promega, Madison, WI, USA), 200 μM each dNTP, 2.5 μM each primer, 0.4 U of GoTaq polymerase, and approximately 50 ng of DNA template in a final volume of 20 μl. PCR were held at 94°C for 2 min; followed by 40 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min; with a final extension at 72°C for 10 min. PCR products from each contig were separated on a 1% agarose gel and visualized with ethidium bromide.

Gene annotation and analysis
----------------------------

The biological function of EST contigs was predicted with gene ontology (GO) terms based on BLASTx analysis using the program BLAST2GO \[[@B58],[@B59]\]. Default BLASTx parameters with an e-value threshold of 1e^-3^ and a high-scoring segment pairs (hsp) filter of 33 were retained so as to assign function to as many contigs as possible while ensuring short matching sequences less than 100 nucleotides were excluded. An annotation configuration with e-value-hit-filter 1.0E^-6^, annotation cut off "55" and GO weight "10" was selected.

Prediction of secretory/signal peptides for the *S*. *sclerotiorum* ESTs
------------------------------------------------------------------------

The secretory/signal peptides for each *S*. *sclerotiorum* EST were analyzed using prediction algorithms. Firstly, OrfPredictor \[[@B60]\] was used to predict protein coding regions for the assembled ESTs. The output for OrfPredictor was a file of predicted coding regions from the ESTs in FASTA format, where the definition line contains the query identifier, the frame, the beginning and the end position of the predicted coding region, and the predicted protein peptide sequences. The peptide sequences were then used as a query for SignalP 3.0 (<http://www.cbs.dtu.dk/services/SignalP/>) with default settings, which predicts the presence and location of signal peptide cleavage sites \[[@B13]\]. Both a neural network (NN) and Hidden Markov Model (HMM) approach were used. EST contigs identified by both NN and HMM were considered to be secretory/signal peptides with high confidence.

Competing interests
===================

The authors declare that they have no competing interest.

Authors' contributions
======================

XF performed bioinformatics analysis, validated the EST parsing and drafted the manuscript. KM provided pea lines for analysis and contributed to direction of the study. TC participated in design of the study and conducted signal peptide analysis. TP initiated the project and directed the study. MC directed the project, performed inoculations and initial experiments to develop the normalized cDNA and drafted the manuscript. All authors read, edited and approved the final manuscript.

Authors' information
====================

XF is a Research Associate at Michigan State University with a background and interest in expression profiling. KM is an Associate Professor and legume breeder in the Department of Plant Sciences at North Dakota State University. TC is Trait Production Manager at Dow AgroSciences LLC. TP is an Associate Professor of Plant Pathology at Washington State University, with particular interests in the mechanisms of fungal speciation and the genetics of host specificity. MC is an Assistant Professor of Plant Pathology at Michigan State University.

Supplementary Material
======================

###### Additional file 1

**Fasta files for 10158 contigs (pea and Sclerotinia ESTs), parsed into ambiguous contigs (1a), no blast-hit contigs (1b), pea contigs (1c) and*S. sclerotiorum*contigs (1d).**

###### 

Click here for file

###### Additional file 2

***S. sclerotiorum*EST contigs encoding potential secretory/signal peptides.**

###### 

Click here for file

###### Additional file 3

**Comparison of BLASTn and tBLASTx method to assign artificial EST mixture of pea-*S. sclerotiorum*(including 18,490 pea ESTs and 17,198 *S. sclerotiorum*ESTs).**

###### 

Click here for file

###### Additional file 4

**The assignment results of an artificial EST mixture using the tBlastx method against 7 fungal genome databases and the*S. sclerotiorum*genome only.**

###### 

Click here for file

###### Additional file 5

Comparison of different e-value ratios (fungi/plant) to distinguish species ESTs from the artificial EST mixture.

###### 

Click here for file

Acknowledgements
================

This project was supported by USDA-ARS Specific Cooperative Agreement \#58-5442-9-239 (National Sclerotinia Initiative). We thank Ahmit Dhingra, Department of Horticulture, Washington State University for 454 pyrosequencing.
