Understanding the genetic pathways that regulate how pathogenic fungi respond to their environment is paramount to developing effective mitigation strategies against disease. Carbon catabolite repression (CCR) is a global regulatory mechanism found in a wide range of microbial organisms that ensures the preferential utilization of glucose over less favourable carbon sources, but little is known about the components of CCR in filamentous fungi. Here we report three new mediators of CCR in the devastating rice blast fungus Magnaporthe oryzae: the sugar sensor Tps1, the Nmr1-3 inhibitor proteins, and the multidrug and toxin extrusion (MATE)–family pump, Mdt1. Using simple plate tests coupled with transcriptional analysis, we show that Tps1, in response to glucose-6-phosphate sensing, triggers CCR via the inactivation of Nmr1-3. In addition, by dissecting the CCR pathway using Agrobacterium tumefaciens-mediated mutagenesis, we also show that Mdt1 is an additional and previously unknown regulator of glucose metabolism. Mdt1 regulates glucose assimilation downstream of Tps1 and is necessary for nutrient utilization, sporulation, and pathogenicity. This is the first functional characterization of a MATE–family protein in filamentous fungi and the first description of a MATE protein in genetic regulation or plant pathogenicity. Perturbing CCR in Δtps1 and MDT1 disruption strains thus results in physiological defects that impact pathogenesis, possibly through the early expression of cell wall–degrading enzymes. Taken together, the importance of discovering three new regulators of carbon metabolism lies in understanding how M. oryzae and other pathogenic fungi respond to nutrient availability and control development during infection. To succeed as pathogens, fungi such as the rice blast fungus M. oryzae must adapt their metabolism to nutrient availability within the host, but little is known about the genetic regulatory mechanisms involved. M. oryzae destroys enough rice to feed 60 million people annually, and understanding how the infection process is controlled would afford new targets for anti-rice blast strategies and shed light on regulatory pathways common to other pathogenic fungi. Here we use M. oryzae to identify and describe three new regulators of global carbon metabolism in filamentous fungi: the sugar-sensor Tps1; the transcription factor inhibitor proteins Nmr1-3; and a transmembrane efflux pump Mdt1 (the first pump of its type to be described in pathogenic filamentous fungi), which is essential for sporulation and pathogenicity. Tps1, Nmr1-3, and Mdt1 are shown to control the fungal response to glucose availability, and perturbation of this regulatory pathway abolishes disease. This work gives fresh insights into nutrient adaptation and the control of fungal development during infection and is thus applicable to a wide range of fungal pathogens.

The blast fungus Magnaporthe oryzae threatens global food security through the widespread destruction of cultivated rice. Foliar infection requires a specialized cell called an appressorium that generates turgor to force a thin penetration hypha through the rice cuticle and into the underlying epidermal cells, where the fungus grows for the first days of infection as a symptomless biotroph. Understanding what controls biotrophic growth could open new avenues for developing sustainable blast intervention programs. Here, using molecular genetics and live-cell imaging, we dismantled M. oryzae glucose-metabolizing pathways to reveal that the transketolase enzyme, encoded by TKL1, plays an essential role in facilitating host colonization during rice blast disease. In the absence of transketolase, Δtkl1 mutant strains formed functional appressoria that penetrated rice cuticles successfully and developed invasive hyphae (IH) in rice cells from primary hyphae. However, Δtkl1 could not undertake sustained biotrophic growth or cell-to-cell movement. Transcript data and observations using fluorescently labeled histone H1:RFP fusion proteins indicated Δtkl1 mutant strains were alive in host cells but were delayed in mitosis. Mitotic delay could be reversed and IH growth restored by the addition of exogenous ATP, a metabolite depleted in Δtkl1 mutant strains. We show that ATP might act via the TOR signaling pathway, and TOR is likely a downstream target of activation for TKL1. TKL1 is also involved in controlling the migration of appressorial nuclei into primary hyphae in host cells. When taken together, our results indicate transketolase has a novel role in mediating - via ATP and TOR signaling - an in planta-specific metabolic checkpoint that controls nuclear migration from appressoria into primary hyphae, prevents mitotic delay in early IH and promotes biotrophic growth. This work thus provides new information about the metabolic strategies employed by M. oryzae to enable rice cell colonization. The blast fungus Magnaporthe oryzae destroys rice and wheat harvests and could compromise global food security. Following penetration into the rice cell, M. oryzae elaborates bulbous invasive hyphae that grow in living rice cells for most of the infection cycle without causing disease symptoms. Little is known about the physiological processes governing this important biotrophic stage of fungal growth. Here, we used gene functional analysis to show how the primary metabolic enzyme transketolase is essential for hyphal growth in rice cells. Loss of transketolase did not affect the ability of the fungus to gain entry into rice cells, but invasive hyphal growth was curtailed in transketolase null mutants. Biotrophic growth was restored in transketolase mutants by the addition of exogenous ATP. We conclude that M. oryzae metabolism is dedicated to metabolizing glucose through transketolase in planta in order to provide ATP as a trigger for biotrophic growth and infection. This work is significant because it reveals important—but previously unknown—metabolic strategies employed by M. oryzae to facilitate rice infection. These strategies might be open to abrogation by chemical or biological means and are likely relevant to other rapidly proliferating intracellular pathogens.

Foliar fungal pathogens challenge global food security, but how they optimize growth and development during infection is understudied. Despite adopting several lifestyles to facilitate nutrient acquisition from colonized cells, little is known about the genetic underpinnings governing pathogen adaption to host-derived nutrients. Homologs of common global and pathway-specific gene regulatory elements are likely to be involved, but their contribution to pathogenicity, and how they are connected to broader genetic networks, is largely unspecified. Here, we focus on carbon and nitrogen metabolism in foliar pathogens and consider what is known, and what is not known, about fungal exploitation of host nutrient and ask how common metabolic regulators have been co-opted to the plant-pathogenic lifestyle as well as how nutrients are utilized to drive infection.

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.