An NADPH-dependent genetic switch regulates plant infection by the rice blast fungus

Abstract

To cause rice blast disease, the fungus Magnaporthe oryzae breaches the tough outer cuticle of the rice leaf by using specialized infection structures called appressoria. These cells allow the fungus to invade the host plant and proliferate rapidly within leaf tissue. Here, we show that a unique NADPH-dependent genetic switch regulates plant infection in response to the changing nutritional and redox conditions encountered by the pathogen. The biosynthetic enzyme trehalose-6-phosphate synthase (Tps1) integrates control of glucose-6-phosphate metabolism and nitrogen source utilization by regulating the oxidative pentose phosphate pathway, the generation of NADPH, and the activity of nitrate reductase. We report that Tps1 directly binds to NADPH and, thereby, regulates a set of related transcriptional corepressors, comprising three proteins, Nmr1, Nmr2, and Nmr3, which can each bind NADP. Targeted deletion of any of the Nmr-encoding genes partially suppresses the nonpathogenic phenotype of a Δtps1 mutant. Tps1-dependent Nmr corepressors control the expression of a set of virulence-associated genes that are derepressed during appressorium-mediated plant infection. When considered together, these results suggest that initiation of rice blast disease by M. oryzae requires a regulatory mechanism involving an NADPH sensor protein, Tps1, a set of NADP-dependent transcriptional corepressors, and the nonconsuming interconversion of NADPH and NADP acting as signal transducer.fungal pathogenicity | ascomycete | cofactor R ice blast disease represents a significant constraint on worldwide rice production, resulting in severe epidemics and overall global yield losses of 10-30% each year (1). Rice constitutes 23% of the calories consumed annually by the global human population, so understanding and controlling rice blast disease could play an important role in ensuring global food security in the future (2). To infect rice plants, the blast fungus Magnaporthe oryzae produces specialized infection cells called appressoria, which rupture the leaf cuticle and allow fungal hyphae to invade and colonize the host. The fungus is able to proliferate rapidly within rice cells, deriving nutrition from living tissue while evading or suppressing plant defenses (1). Understanding the regulatory mechanisms that allow the fungus to undergo these developmental transitions and to grow so effectively within its host may provide new means to control rice blast disease.In this study, we set out to investigate the gene regulatory mechanisms that condition the ability of M. oryzae to respond to the nutrient status of its environment during plant infection-moving from the nutrient-free conditions of the rice leaf surface to the relatively nutrient-rich interior of the leaf. Previously, we observed a pivotal role for the biosynthetic enzyme trehalose-6-phosphate synthase (Tps1) in the regulation of carbon and nitrogen metabolism in M. oryzae (3, 4). Tps1 is required for production of the nonreducing disaccharide trehalose from glucose-6-phosphate (G6...

The Magnaporthe oryzae nitrooxidative stress response suppresses rice innate immunity during blast disease

“…21 We could find no phenotype for the resulting Δnmo4 mutant strain, which was fully pathogenic on rice and, unlike Δnmo2, showed no growth defects on nitrate media ( Supplementary Fig. 1a,b).…”

“…Targeted gene deletion was confirmed for two transformants by PCR (ref. 21), which were subsequently tested for pathogenicity on rice seedlings. To ensure that the Δnmo2 phenotype resulted solely from targeted replacement of the NMO2 gene, the full-length NMO2 gene (plus 1 kb each of 5′ and 3′ flanking sequences) was amplified by PCR using the primers listed in Supplementary Table 2 and transformed into protoplasts of one of the Δnmo2 mutant strains.…”

“…38 Strains were growth-tested on defined minimal medium containing 1% (wt/vol) glucose (GMM), with indicated nitrogen sources, as previously described. 21 Strains were tested for susceptibility to nitrooxidative stress on CM with H 2 O 2 (30% in water, Fisher) and sodium nitroprusside dehydrate (NPS, MP Biomedicals) at the indicated concentrations. The peroxynitrite scavenger MnTBAP chloride (AdipoGen Life Sciences) was added to GMM with 10 mM NO 3 − as the sole nitrogen source.…”

“…Targeted gene replacement of the M. oryzae NMO genes was achieved using the split marker strategy described by Wilson and associates. 21 Briefly, the sequence of the NMO2,4 genes was obtained from the M. oryzae genome database at the Broad Institute and was used to design gene-specific primers (Supplementary Table 2). A total of 1 kb of the gene of interest was replaced with the sulfonylurea resistance-conferring gene, ILV1 (2.8 kb).…”

Effector-Mediated Suppression of Chitin-Triggered Immunity by Magnaporthe oryzae Is Necessary for Rice Blast Disease

“…Sequencing of both constructs was performed using T7 primer to ensure the constructs were in frame. Yeast two-hybrid analysis was then performed using the Matchmaker GAL4 Two-Hybrid System 3 (Clontech) according to the manufacturer's instructions, as described previously (Wilson et al, 2010).…”