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

Abstract

Understanding how microorganisms manipulate plant innate immunity and colonize host cells is a major goal of plant pathology. Here, we report that the fungal nitrooxidative stress response suppresses host defenses to facilitate the growth and development of the important rice pathogen Magnaporthe oryzae in leaf cells. Nitronate monooxygenases encoded by NMO genes catalyze the oxidative denitrification of nitroalkanes. We show that the M. oryzae NMO2 gene is required for mitigating damaging lipid nitration under nitrooxidative stress conditions and, consequently, for using nitrate and nitrite as nitrogen sources. On plants, the Δnmo2 mutant strain penetrated host cuticles like wild type, but invasive hyphal growth in rice cells was restricted and elicited plant immune responses that included the formation of cellular deposits and a host reactive oxygen species burst. Development of the M. oryzae effector-secreting biotrophic interfacial complex (BIC) was misregulated in the Δnmo2 mutant. Inhibiting or quenching host reactive oxygen species suppressed rice innate immune responses and allowed the Δnmo2 mutant to grow and develop normally in infected cells. NMO2 is thus essential for mitigating nitrooxidative cellular damage and, in rice cells, maintaining redox balance to avoid triggering plant defenses that impact M. oryzae growth and BIC development.Global rice yields are significantly and negatively impacted each year by blast disease caused by the hemibiotrophic fungus Magnaporthe oryzae 1-3 (synonym of Pyricularia oryzae). Defining the full spectrum of molecular pro- Marroquin-Guzman et al. in Nature Microbiology 2 (2017) 2 cesses used by M. oryzae to manipulate rice innate immunity and allow fungal colonization of host cells might reveal additional sources of pathogen resistance and improve crop health. M. oryzae infects hosts by first forming specialized infection structures, appressoria, at the tips of germ tubes emerging from spores adhered to the leaf surface. 4,5 A thin penetration peg emerging from an unmelanized patch on the base of the appressorium 6 is forced through the rice leaf cuticle under hydrostatic turgor pressure 1 . In the first penetrated cell, the peg differentiates into primary hyphae then bulbous invasive hyphae (IH) that are surrounded by the plant-derived extra-invasive hyphal membrane (EIHM). Branching IH fill the first invaded cell before spreading into neighboring living rice cells at around 44 h post-inoculation. 7,8 This biotrophic growth phase progresses for 4-5 days before M. oryzae enters its necrotrophic phase.To colonize rice cells, M. oryzae must first suppress or avoid triggering two types of plant innate immunity that protect against microbial attack [9][10][11] : pathogen-associated molecular pattern (PAMP) triggered immunity (PTI), which can be suppressed by microbial effectors, and effector-triggered immunity (ETI), if effectors are detected. The biotrophic interfacial complex (BIC), a host membrane-derived structure, is formed behind M. oryzae IH in each in...

Reactive oxygen species metabolism and plant-fungal interactions

“…Suppressing the host oxidative burst restored single BIC development and Δnmo2 growth in rice cells. This work consequently demonstrated how a mutation in the fungus resulted in a response from the plant that in turn affected the development of the fungus (Marroquin-Guzman et al, 2017). Thus, investigations into basic fungal metabolism have uncovered molecular and metabolic decisions underlying fungal growth and plant defense suppression in rice cells.…”

“…This co-evolution between plant and pathogens serves as the basis of the zig-zag model of plant pathology (Hillmer et al, 2017;Jones and Dangl, 2006). However, oxidative bursts are not effective in preventing proliferation of the necrotrophic fungi B. cinerea and Sclerotinia sclerotiorum, and can be tolerated by the hemibiotrophs, Septoria tritici and M. oryzae (Govrin and Levine, 2000;Shetty et al, 2007;Samalova et al, 2014;Marroquin-Guzman et al, 2017). How do fungi deal with host ROS and maintain redox balance during host infection?…”

“…Tps1 controls the expression of NADPH-dependent enzymes-including those of the thioredoxin and glutathione antioxidation system-in response to NADPH availability while balancing the production of NADPH with glucose-6-phosphate availability, thus providing molecular links between primary metabolism and antioxidation during host infection (Fernandez et al, 2012;Fernandez and Wilson, 2014;Wilson et al, 2010). Building on this concept, a more recent study in M. oryzae showed that the NMO2 gene, encoding a nitronate monooxygenase enzyme catalyzing oxidative denitrification of nitroalkanes, is carbon and nitrogen responsive, regulated by Tps1, and required for axenic growth on nitrate media (Marroquin-Guzman et al, 2017). Nmo2 was subsequently discovered to protect fungal cells from reactive nitrogen species (RNS) as a component of a previously unknown stress pathway in eukaryotes that mediates nitrooxidative stress.…”

“…These ROS react readily with lipids, DNA, proteins and other macromolecules, leading to death and aging of the cell (Beckman and Ames, 1998). ROS can also react with oxides of nitrogen such as NO to generate damaging reactive nitrogen species (RNS) such as peroxynitrite (Marroquin-Guzman et al, 2017). To eliminate ROS (and RNS), fungal pathogens have developed robust antioxidation systems involving superoxide dismutases (SODs), catalases, peroxidases, glutathione and thioredoxin.…”

“…To eliminate ROS (and RNS), fungal pathogens have developed robust antioxidation systems involving superoxide dismutases (SODs), catalases, peroxidases, glutathione and thioredoxin. Interestingly, although ROS was thought to act as a toxifying component in hosts against plant pathogens, it is not produced in amounts sufficient to overcome these pathogens (Marroquin-Guzman et al, 2017;Samalova et al, 2014) and instead is emerging as a signaling component of the plant defense response.…”