Rice blast resistant gene, Pi54 cloned from rice line, Tetep, is effective against diverse isolates of Magnaporthe oryzae. In this study, we prospected the allelic variants of the dominant blast resistance gene from a set of 92 rice lines to determine the nucleotide diversity, pattern of its molecular evolution, phylogenetic relationships and evolutionary dynamics, and to develop allele specific markers. High quality sequences were generated for homologs of Pi54 gene. Using comparative sequence analysis, InDels of variable sizes in all the alleles were observed. Profiling of the selected sites of SNP (Single Nucleotide Polymorphism) and amino acids (N sites ≥ 10) exhibited constant frequency distribution of mutational and substitutional sites between the resistance and susceptible rice lines, respectively. A total of 50 new haplotypes based on the nucleotide polymorphism was also identified. A unique haplotype (H_3) was found to be linked to all the resistant alleles isolated from indica rice lines. Unique leucine zipper and tyrosine sulfation sites were identified in the predicted Pi54 proteins. Selection signals were observed in entire coding sequence of resistance alleles, as compared to LRR domains for susceptible alleles. This is a maiden report of extensive variability of Pi54 alleles in different landraces and cultivated varieties, possibly, attributing broad-spectrum resistance to Magnaporthe oryzae. The sequence variation in two consensus region: 163 and 144 bp were used for the development of allele specific DNA markers. Validated markers can be used for the selection and identification of better allele(s) and their introgression in commercial rice cultivars employing marker assisted selection.

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Magnaporthe oryzae infection causes rice blast, a destructive disease that is responsible for considerable decrease in rice yield. Development of resistant varieties via introgressing resistance genes with marker-assisted breeding can eliminate pesticide use and minimize crop losses. Here, resistant near-isogenic line (NIL) of Pusa Basmati-1(PB1) carrying broad spectrum rice blast resistance gene Pi9 was used to investigate Pi9-mediated resistance response. Infected and uninfected resistant NIL and susceptible control line were subjected to RNA-Seq. With the exception of one gene (Pi9), transcriptional signatures between the two lines were alike, reflecting basal similarities in their profiles. Resistant and susceptible lines possessed 1043 (727 up-regulated and 316 down-regulated) and 568 (341 up-regulated and 227 down-regulated) unique and significant differentially expressed loci (SDEL), respectively. Pathway analysis revealed higher transcriptional activation of kinases, WRKY, MYB, and ERF transcription factors, JA-ET hormones, chitinases, glycosyl hydrolases, lipid biosynthesis, pathogenesis and secondary metabolism related genes in resistant NIL than susceptible line. Singular enrichment analysis demonstrated that blast resistant NIL is significantly enriched with genes for primary and secondary metabolism, response to biotic stimulus and transcriptional regulation. The co-expression network showed proteins of genes in response to biotic stimulus interacted in a manner unique to resistant NIL upon M. oryzae infection. These data suggest that Pi9 modulates genome-wide transcriptional regulation in resistant NIL but not in susceptible PB1. We successfully used transcriptome profiling to understand the molecular basis of Pi9-mediated resistance mechanisms, identified potential candidate genes involved in early pathogen response and revealed the sophisticated transcriptional reprogramming during rice-M. oryzae interactions.

Rice blast caused by Magnaporthe oryzae is one of the most important diseases of rice. Pi54, a rice gene that imparts resistance to M. oryzae isolates prevalent in India, was already cloned but its avirulent counterpart in the pathogen was not known. After decoding the whole genome of an avirulent isolate of M. oryzae, we predicted 11440 protein coding genes and then identified four candidate effector proteins which are exclusively expressed in the infectious structure, appresoria. In silico protein modeling followed by interaction analysis between Pi54 protein model and selected four candidate effector proteins models revealed that Mo-01947_9 protein model encoded by a gene located at chromosome 4 of M. oryzae, interacted best at the Leucine Rich Repeat domain of Pi54 protein model. Yeast-two-hybrid analysis showed that Mo-01947_9 protein physically interacts with Pi54 protein. Nicotiana benthamiana leaf infiltration assay confirmed induction of hypersensitive response in the presence of Pi54 gene in a heterologous system. Genetic complementation test also proved that Mo-01947_9 protein induces avirulence response in the pathogen in presence of Pi54 gene. Here, we report identification and cloning of a new fungal effector gene which interacts with blast resistance gene Pi54 in rice.