Identification and Characterization of a Nonhost Resistance Gene in Arabidopsis against Rice Blast Pathogen Magnaporthe oryzae

Abstract

Nonhost resistance (NHR) is a naturally occurring phenomena that confers broad spectrum resistance to non-adapted pathogens in plants. Arabidopsis is a well-established nonhost model to study disease resistance against Magnaporthe oryzae that causes the devastating disease of rice blast. Although many NHR genes have been identified in Arabidopsis, the exact mechanism of such resistance is not known and the mode of activation of systemic defences is still not deciphered. Arabidopsis PEN2 is indispensable for both pre- and post-penetration resistance to M. oryzae. Forward genetic screen of the M3 population derived from EMS mutagenized pen2-3 seeds for susceptibility to M. oryzae identified mosA as the mutant of interest. Further, bulked segregant analysis by map-based cloning and next generation sequencing delineated two candidate genes with C→T substitution mutations in the lower arm of chromosome 2. Screening of the two candidate genes using T-DNA insertion mutants identified AT2G44160 as a novel NHR candidate responsible for mosA susceptibility. Based on the observation of the extent of penetration in single mutants, pen2-3 and at2g44160 and comparison with mosA, we assume that mosA phenotype is the additive effect of two mutations- pen2-3 and at2g44160. Further characterization of mosA susceptibility revealed that mosA has severely compromised pre-penetration resistance, while the post-penetration resistance remained intact and active. Homology modelling of wild type and mutant AT2G44160-encoded MTHFR2 protein confirmed conformational changes in the active site of the mutated protein due to alanine to valine substitution. This substitution caused steric hindrance in ligand binding of mthfr2 as observed by molecular docking experiment. Also, mosA extracts comparatively allowed M. oryzae conidial germination and hyphal growth that substantiated the putative role of MTHFR2 in Arabidopsis secondary metabolism. Furthermore, in silico data mining for the expression profile of Arabidopsis and rice orthologs of MTHFR2 showed significant downregulation of MTHFR2 expression, which stated the importance of this developmentally required gene in pre-penetration resistance. Hence, we present MTHFR2 as a novel NHR gene that, like PEN2, is required for pre-penetration resistance to M. oryzae.