Cloning, characterization and expression analysis of resistant gene analogues for wilt resistant in chickpea

Management of chickpea wilt incited by Fusarium oxysporum f. sp. ciceris has been done primarily through development and use of resistant cultivars as part of an integrated management approach as the resistance genes are a major factor behind protecting plants from various pathogens. Thus, isolation and characterization of the RGAs of chickpea provide a critical foundation for deciphering host-pathogen interactions and development of novel methods to manage pathogens of crop plant. Eleven RGAs were identified in resistant variety (WR 315) of chickpea by degenerate PCR amplification followed by sequencing, cloning and database search. The restriction pattern generated using enzyme Hinf1 as well as analysis of phylogenetic tree of derived amino acid sequences classified chickpea RGA sequences into four classes of RGAs. Amino acid sequence alignment of these clones clearly showed the presence of consensus motifs namely, P-loop and Non-TIR linked sequences. The finding further supports that these consensus motifs are extensively present in dicot plant species. Three conserved motifs within the aligned sequences were also identified using MEME analysis. These conserve motifs exhibited the high level of similarity with NB-ARC domain as shown by pfam protein motif analyse. Quantitative real time-PCR analysis clearly revealed that both SA and JA induce the expression of RGA genes, however, the expression was higher in the plants treated with SA as compared to the control or JA treated plants. The differentially expressed signaling molecules suggesting that SA and JS as stimuli mainly involved in responding defence and activating signaling pathways.