Glucose-\u3ci\u3eABL1\u3c/i\u3e-TOR Signaling Modulates Cell Cycle Tuning to Control Terminal Appressorial Cell Differentiation

The conserved target of rapamycin (TOR) pathway integrates growth and development with available nutrients, but how cellular glucose controls TOR function and signaling is poorly understood. Here, we provide functional evidence from the devastating rice blast fungus Magnaporthe oryzae that glucose can mediate TOR activity via the product of a novel carbon- responsive gene, ABL1, in order to tune cell cycle progression during infection-related development. Under nutrient-free conditions, wild type (WT) M. oryzae strains form terminal plant-infecting cells (appressoria) at the tips of germ tubes emerging from three-celled spores (conidia). WT appressorial development is accompanied by one round of mitosis followed by autophagic cell death of the conidium. In contrast, Δabl1 mutant strains undergo multiple rounds of accelerated mitosis in elongated germ tubes, produce few appressoria, and are abolished for autophagy. Treating WT spores with glucose or 2-deoxyglucose phenocopied Δabl1. Inactivating TOR in Δabl1 mutants or glucose-treated WT strains restored appressorium formation by promoting mitotic arrest at G1/G0 via an appressorium- and autophagy-inducing cell cycle delay at G2/M. Collectively, this work uncovers a novel glucose- ABL1-TOR signaling axis and shows it engages two metabolic checkpoints in order to modulate cell cycle tuning and mediate terminal appressorial cell differentiation. We thus provide new molecular insights into TOR regulation and cell development in response to glucose

Chromatin Immunoprecipitation (ChIP) Assay for Detecting Direct and Indirect Protein -DNA Interactions in \u3ci\u3eMagnaporthe oryzae\u3c/i\u3e

Chromatin immunoprecipitation (ChIP) is a powerful technology for analyzing protein-DNA interactions in cells. Robust ChIP procedures have been established for investigating direct interactions between protein and DNA. However, detecting indirect protein-DNA interactions in vivo is challenging. Recently, we used ChIP to analyze an indirect protein-DNA interaction between a putative histone demethylase, MoJmjC, and the promoter of the superoxide dismutase 1-encoding gene MoSOD1 in the rice blast fungus Magnaporthe oryzae (M. oryzae) (Fernandez et al., 2014). We tagged MoJmjC with the 3x FLAG epitope (Fernandez et al., 2014), instead of the larger and more commonly used GFP epitope, to mitigate against steric hindrance. We also employed a two-step cross-linking strategy using DSG and formaldehyde-rather than the one-step formaldehyde cross-linking procedure more frequently employed for analyzing direct protein-DNA interactions - in order to better capture the indirect MoJmjC-MoSOD1 DNA interactions in vivo. In addition, we have shown that two-step cross-linking is suitable for ChIP analysis of direct protein-DNA interactions between a GATA transcription factor, Asd4, and its cognate binding site (Marroquin-Guzman and Wilson, 2015). Here, we provide a detailed protocol for chromatin immunoprecipitation, with versatile two-step cross-linking, in M. oryzae

Chromatin Immunoprecipitation (ChIP) Assay for Detecting Direct and Indirect Protein -DNA Interactions in \u3ci\u3eMagnaporthe oryzae\u3c/i\u3e

Chromatin immunoprecipitation (ChIP) is a powerful technology for analyzing protein-DNA interactions in cells. Robust ChIP procedures have been established for investigating direct interactions between protein and DNA. However, detecting indirect protein-DNA interactions in vivo is challenging. Recently, we used ChIP to analyze an indirect protein-DNA interaction between a putative histone demethylase, MoJmjC, and the promoter of the superoxide dismutase 1-encoding gene MoSOD1 in the rice blast fungus Magnaporthe oryzae (M. oryzae) (Fernandez et al., 2014). We tagged MoJmjC with the 3x FLAG epitope (Fernandez et al., 2014), instead of the larger and more commonly used GFP epitope, to mitigate against steric hindrance. We also employed a two-step cross-linking strategy using DSG and formaldehyde-rather than the one-step formaldehyde cross-linking procedure more frequently employed for analyzing direct protein-DNA interactions - in order to better capture the indirect MoJmjC-MoSOD1 DNA interactions in vivo. In addition, we have shown that two-step cross-linking is suitable for ChIP analysis of direct protein-DNA interactions between a GATA transcription factor, Asd4, and its cognate binding site (Marroquin-Guzman and Wilson, 2015). Here, we provide a detailed protocol for chromatin immunoprecipitation, with versatile two-step cross-linking, in M. oryzae

A protein kinase coordinates cycles of autophagy and glutaminolysis in invasive hyphae of the fungus \u3ci\u3eMagnaporthe oryzae\u3c/i\u3e within rice cells

The blast fungus Magnaporthe oryzae produces invasive hyphae in living rice cells during early infection, separated from the host cytoplasm by plantderived interfacial membranes. However, the mechanisms underpinning this intracellular biotrophic growth phase are poorly understood. Here, we show that the M. oryzae serine/threonine protein kinase Rim15 promotes biotrophic growth by coordinating cycles of autophagy and glutaminolysis in invasive hyphae. Alongside inducing autophagy, Rim15 phosphorylates NADdependent glutamate dehydrogenase, resulting in increased levels of α- ketoglutarate that reactivate target-of-rapamycin (TOR) kinase signaling, which inhibits autophagy. Deleting RIM15 attenuates invasive hyphal growth and triggers plant immunity; exogenous addition of α-ketoglutarate prevents these effects, while glucose addition only suppresses host defenses. Our results indicate that Rim15-dependent cycles of autophagic flux liberate α-ketoglutarate – via glutaminolysis – to reactivate TOR signaling and fuel biotrophic growth while conserving glucose for antioxidation-mediated host innate immunity suppression

Plant defense suppression is mediated by a fungal sirtuin during rice infection by \u3ci\u3eMagnaporthe oryzae\u3c/i\u3e

Crop destruction by the hemibiotrophic rice pathogen Magnaporthe oryzae requires plant defense suppression to facilitate extensive biotrophic growth in host cells before the onset of necrosis. How this is achieved at the genetic level is not well understood. Here, we report that a M. oryzae sirtuin, MoSir2, plays an essential role in rice defense suppression and colonization by controlling superoxide dismutase (SOD) gene expression. Loss of MoSir2 function in Δsir2 strains did not affect appressorial function, but biotrophic growth in rice cells was attenuated. Compared to wild type, Δsir2 strains failed to neutralize plant-derived reactive oxygen species (ROS) and elicited robust defense responses in rice epidermal cells that included elevated pathogenesis-related gene expression and granular depositions. Deletion of a SOD-encoding gene under MoSir2 control generated Δsod1 deletion strains that mimicked Δsir2 for impaired rice defense suppression, confirming SOD activity as a downstream output of MoSir2. In addition, comparative protein acetylation studies and forward genetic analyses identified a JmjC domain-containing protein as a likely target of MoSir2, and a Δsir2 Δjmjc double mutant was restored for MoSOD1 expression and defense suppression in rice epidermal cells. Together, this work reveals MoSir2 and MoJmjC as novel regulators of early rice cell infection

A protein kinase coordinates cycles of autophagy and glutaminolysis in invasive hyphae of the fungus \u3ci\u3eMagnaporthe oryzae\u3c/i\u3e within rice cells

The blast fungus Magnaporthe oryzae produces invasive hyphae in living rice cells during early infection, separated from the host cytoplasm by plantderived interfacial membranes. However, the mechanisms underpinning this intracellular biotrophic growth phase are poorly understood. Here, we show that the M. oryzae serine/threonine protein kinase Rim15 promotes biotrophic growth by coordinating cycles of autophagy and glutaminolysis in invasive hyphae. Alongside inducing autophagy, Rim15 phosphorylates NADdependent glutamate dehydrogenase, resulting in increased levels of α- ketoglutarate that reactivate target-of-rapamycin (TOR) kinase signaling, which inhibits autophagy. Deleting RIM15 attenuates invasive hyphal growth and triggers plant immunity; exogenous addition of α-ketoglutarate prevents these effects, while glucose addition only suppresses host defenses. Our results indicate that Rim15-dependent cycles of autophagic flux liberate α-ketoglutarate – via glutaminolysis – to reactivate TOR signaling and fuel biotrophic growth while conserving glucose for antioxidation-mediated host innate immunity suppression

A protein kinase coordinates cycles of autophagy and glutaminolysis in invasive hyphae of the fungus \u3ci\u3eMagnaporthe oryzae\u3c/i\u3e within rice cells

The blast fungus Magnaporthe oryzae produces invasive hyphae in living rice cells during early infection, separated from the host cytoplasm by plantderived interfacial membranes. However, the mechanisms underpinning this intracellular biotrophic growth phase are poorly understood. Here, we show that the M. oryzae serine/threonine protein kinase Rim15 promotes biotrophic growth by coordinating cycles of autophagy and glutaminolysis in invasive hyphae. Alongside inducing autophagy, Rim15 phosphorylates NADdependent glutamate dehydrogenase, resulting in increased levels of α- ketoglutarate that reactivate target-of-rapamycin (TOR) kinase signaling, which inhibits autophagy. Deleting RIM15 attenuates invasive hyphal growth and triggers plant immunity; exogenous addition of α-ketoglutarate prevents these effects, while glucose addition only suppresses host defenses. Our results indicate that Rim15-dependent cycles of autophagic flux liberate α-ketoglutarate – via glutaminolysis – to reactivate TOR signaling and fuel biotrophic growth while conserving glucose for antioxidation-mediated host innate immunity suppression

The \u3ci\u3eMagnaporthe oryzae\u3c/i\u3e nitrooxidative stress response suppresses rice innate immunity during blast disease

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

Nutrient Acquisition Strategies Employed by the Rice Blast Fungus Magnaporthe Oryzae During Infection

Magnaporthe oryzae is the causal agent of rice blast, one of the most devastating rice diseases worldwide. Understanding the metabolic strategies by which plant pathogenic fungi adapt to the changing nutrient landscapes within their hosts, would be a major step towards identifying potential targets to generate durable resistant rice cultivars. Using genetic, physiological, and biochemical approaches, here we describe novel insights into the metabolic mechanisms and strategies that underpin appressorium development and biotrophic growth in M. oryzae. On the leaf surface, Asd4-dependent glutaminolysis drives appressorium formation by suppressing the conserved target of rapamycin (TOR) inhibition of cAMP/PKA signaling pathways. This is the first report indicating that TOR is a negative-acting regulator of appressoria formation in M. oryzae, thus adding to our knowledge about how these specialized infection cells develop. Moreover, we demonstrate that appressorial differentiation is controlled by a novel glucose-ABL1-TOR signalling axis, which modulates cell cycle progression in response to glucose. Inside the rice cell, M. oryzae must manipulate plant innate immunity in order to grow undetected by the plant during its biotrophic phase. We show NMO2 (nitronate monooxygense) is essential for mitigating nitrooxidative cellular damage and, in rice cells, maintain redox balance to avoid triggering rice innate immunity. This provide insight on a previously unknown aspect of molecular plant-microbe interactions and, more broadly, identify a new requisite for nitrate and nitrite metabolism in fungi. Finally, we show what host-derived nutrients might facilitate rice cell invasion. Transcript and genetic analysis demonstrate ammonium is acquired from the plant under nitrogen starvation conditions via the ammonium transporters MEP1 and MEP2 during early biotrophy, while at least some amino acids are synthesized de novo through TCA cycle intermediates. Amino acid uptake via the general amino acid permease GAP1 occurs later in biotrophy in a Nut1-dependent manner

GATA-Dependent Glutaminolysis Drives Appressorium Formation in \u3ci\u3eMagnaporthe oryzae\u3c/i\u3e by Suppressing TOR Inhibition of cAMP/PKA Signaling

Fungal plant pathogens are persistent and global food security threats. To invade their hosts they often form highly specialized infection structures, known as appressoria. The cAMP/ PKA- and MAP kinase-signaling cascades have been functionally delineated as positive-acting pathways required for appressorium development. Negative-acting regulatory pathways that block appressorial development are not known. Here, we present the first detailed evidence that the conserved Target of Rapamycin (TOR) signaling pathway is a powerful inhibitor of appressorium formation by the rice blast fungus Magnaporthe oryzae. We determined TOR signaling was activated in an M. oryzae mutant strain lacking a functional copy of the GATA transcription factor-encoding gene ASD4. Δasd4 mutant strains could not form appressoria and expressed GLN1, a glutamine synthetase-encoding orthologue silenced in wild type. Inappropriate expression of GLN1 increased the intracellular steady-state levels of glutamine in Δasd4 mutant strains during axenic growth when compared to wild type. Deleting GLN1 lowered glutamine levels and promoted appressorium formation by Δasd4 strains. Furthermore, glutamine is an agonist of TOR. Treating Δasd4 mutant strains with the specific TOR kinase inhibitor rapamycin restored appressorium development. Rapamycin was also shown to induce appressorium formation by wild type and Δcpka mutant strains on non-inductive hydrophilic surfaces but had no effect on the MAP kinase mutant Δpmk1. When taken together, we implicate Asd4 in regulating intracellular glutamine levels in order to modulate TOR inhibition of appressorium formation downstream of cPKA. This study thus provides novel insight into the metabolic mechanisms that underpin the highly regulated process of appressorium development