Analysis of genome and secretome of Ascochyta rabiei causal agent of Ascochyta blight in chickpea

Ascochyta blight disease caused by Ascochyta rabiei is a major constraint to chickpea (Cicer arietinum) production worldwide and remains an unresolved problem. The interaction of this pathogen with its host involves diverse strategies due to the necrotrophic nature of the A. rabiei pathogen. A. rabiei is a member of the order Pleosporales which contains most fungal species known to produce crop diseases involving necrotrophic fungal effectors; we therefore hypothesised that this A. rabiei pathogen would use a similar mechanism to those employed by other Pleosporales in its own interaction with the host. The analysis of the genome assembly of A. rabiei isolate ME-14 assembled from Illumina Genome Analyzer 75 bp paired-end reads was carried out with approximately 20x coverage. The genome is approximately 34 Mb in length with 11,391 proteins (≥ 50 amino acids) predicted by the self-training GeneMark-ES software. Of these, 1,057 were predicted by SignalP and WoLF PSORT to be secreted by A. rabiei. Validation of gene annotations was carried out with the support of expressed sequence tag (EST) transcripts, proteomics and protein alignment analysis. Despite the small scale and limited data of this study, several hundreds of gene models were validated. This finding suggests the A. rabiei genome assembly offers a reasonably accurate representation of its gene-coding regions. Assembly and annotation of the fungal genome were performed with the main aims of providing insight into biological aspects of pathogenesis and facilitating effector candidate identification. Mining the in silico predicted genes through comparative genome analyses, using several published databases, provided information on which genes potentially were involved in pathogenesis. A total of 7 PKS, 2 NRPS and 1 PKS/NRPS hybrid were predicted in the A. rabiei genome. Of these, one striking PKS gene, ARA11537, was highly homologous to SOL1, an Alternaria solani solanapyrone polyketide synthase gene. Comparative genomic and phylogenetic analyses with other Pleosporales genome data showed that the solanapyrone biosynthesis gene cluster (SBGC) was only present in A. solani and A. rabiei but not in other species, suggesting possible horizontal gene transfer (HGT) events between these two species. Infiltration of A. rabiei culture filtrate (CF) into chickpea leaves produced necrotic and chlorotic symptoms. Different chickpea cultivars also showed different responses upon infiltration. Partial purification of the CF using ion exchange (IEX) and hydrophobic interaction chromatography (HIC) identified the active fractions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis suggested the effector protein(s) responsible are within a size range of 10–50 kDa. In silico analysis of effector candidates predicted 266 small secreted proteins (SSPs) (< 30 kDa). The assembly provided an invaluable resource and in conjunction with mass spectrometry peptide analysis, several potential effector proteins were identified. In summary, this study has advanced further understanding of the mechanism of pathogenicity in A. rabiei, bringing us one step closer to achieving a solution for control of this pathogen.