Characterizing Roles for the Glutathione Reductase, Thioredoxin Reductase and Thioredoxin Peroxidase-Encoding Genes of Magnaporthe oryzae during Rice Blast Disease

Abstract

Understanding how pathogenic fungi adapt to host plant cells is of major concern to securing global food production. The hemibiotrophic rice blast fungus Magnaporthe oryzae, cause of the most serious disease of cultivated rice, colonizes leaf cells asymptomatically as a biotroph for 4–5 days in susceptible rice cultivars before entering its destructive necrotrophic phase. During the biotrophic growth stage, M. oryzae remains undetected in the plant while acquiring nutrients and growing cell-to-cell. Which fungal processes facilitate in planta growth and development are still being elucidated. Here, we used gene functional analysis to show how components of the NADPH-requiring glutathione and thioredoxin antioxidation systems of M. oryzae contribute to disease. Loss of glutathione reductase, thioredoxin reductase and thioredoxin peroxidase-encoding genes resulted in strains severely attenuated in their ability to grow in rice cells and that failed to produce spreading necrotic lesions on the leaf surface. Glutathione reductase, but not thioredoxin reductase or thioredoxin peroxidase, was shown to be required for neutralizing plant generated reactive oxygen species (ROS). The thioredoxin proteins, but not glutathione reductase, were shown to contribute to cell-wall integrity. Furthermore, glutathione and thioredoxin gene expression, under axenic growth conditions, was dependent on both the presence of glucose and the M. oryzae sugar/ NADPH sensor Tps1, thereby suggesting how glucose availability, NADPH production and antioxidation might be connected. Taken together, this work identifies components of the fungal glutathione and thioredoxin antioxidation systems as determinants of rice blast disease that act to facilitate biotrophic colonization of host cells by M. oryzae.

Reactive oxygen species metabolism and plant-fungal interactions

“…The thioredoxin system contains thioredoxins (Trx), and the NADPH-dependent enzyme thioredoxin reductase (TrxR) (Lu and Holmgren, 2014;Fernandez and Wilson, 2014). Thioredoxins are ubiquitous redox proteins containing cysteine residues that are reversibly redox active, cycling between oxidized disulfide (Trx-S 2 ) and reduced dithiol [Trx-(SH) 2 ] in a TrxR dependent manner (Holmgren, 1989;Zhang et al, 2016).…”

“…The cell-intrinsic glucose-6-phosphate/ NADPH-sensing Tps1 enzyme mediates activation of the thioredoxin and glutathione systems in M. oryzae. 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).…”

“…However, H 2 O 2 has a longer halflife than most, thus leading to the development of a number of tools for H 2 O 2 detection (Cruz de Carvalho, 2008). 3,3′-diaminibenzidine (DAB) staining in conjunction with the NADPH oxidase inhibitor Diphenylene iodium (DPI) have frequently been used to understand the dynamics of H 2 O 2 during plant-pathogen interactions (Moore et al, 2002;Marschall and Tudzynski, 2014;Fernandez et al, 2014;Marroquin-Guzman et al, 2017). However, DAB is a slow-acting stain (8-12 h) (Thordal-Christensen et al, 1997).…”

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

“…[13][14][15][16] The M. oryzae glucose-6-phosphate (G6P) sensor, Tps1, regulates carbon metabolism and connects glucose availability 17 to glutathione-dependent antioxidation and the establishment of biotrophy. 18 Other examples linking fungal metabolic regulation and plant defense suppression are not known. We hypothesized that nitrogen-regulated processes might also contribute to the colonization of rice cells by M. oryzae.…”

Distinct roles of two cytoplasmic thioredoxin reductases (Trr1/2) in the redox system involving cysteine synthesis and host infection of Beauveria bassiana

“…Function loss of a Trr homologue in Magnaporthe oryzae resulted in attenuated ability to grow in rice cells and a failure to produce spreading necrotic lesions on the leaf surface (Fernandez and Wilson 2014). Deletion of trr1 from Botrytis cinerea impaired the fungal virulence and antioxidant capability due to enhanced H 2 O 2 production and retarded growth (Viefhues et al 2014).…”