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A protein kinase coordinates cycles of autophagy and glutaminolysis in invasive hyphae of the fungus Magnaporthe oryzae within rice cells

ABSTRACT: The devastating blast fungus Magnaporthe oryzae elaborates invasive hyphae (IH) in living rice cells during early infection, separated from host cytoplasm by plant-derived interfacial membranes, but the metabolic strategies underpinning this fundamental intracellular biotrophic growth phase are poorly understood. Eukaryotic cell growth depends on activated target-of-rapamycin (TOR) kinase signaling, which inhibits autophagy. Here, using live-cell imaging coupled with multiomic approaches, we show how the M. oryzae serine/threonine protein kinase Rim15 coordinates cycles of autophagy and glutaminolysis in IH – the latter through phosphorylation of NAD-dependent glutamate dehydrogenase – to reactivate TOR and promote biotrophic growth. Deleting RIM15 attenuated IH growth and triggered plant immunity; these defects were fully remediated by exogenous α-ketoglutarate treatment, but glucose treatment only suppressed host defenses. Our results together suggest 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.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Magnaporthe Sp. Mg08

TISSUE(S): Mycelial Felt, Liver

SUBMITTER: Gang Li

LAB HEAD: Richard A. Wilson

PROVIDER: PXD043076 | Pride | 2023-07-10

REPOSITORIES: Pride

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			Action	DRS
	20210522_GL_Guy11-R1_1ug.raw	Raw
	20210522_GL_Guy11-R2_1ug.raw	Raw
	20210522_GL_Guy11-R3_1ug.raw	Raw
	20210522_GL_Mascot-1.msf	Msf
	20210522_GL_rim15-R1_1ug.raw	Raw

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Project description:Rice blast is the most threatening disease to cultivated rice worldwide. Magnaporthe oryzae, its causal agent is likely to encounter environmental challenges during invasive growth in the host plants that require shifts in gene expression to establish a compatible interaction. Here, we tested the hypothesis that M. oryzae have similar gene expression patterns in planta, versus during in vitro stresses. Gene expression data was collected from in vitro experiments of heat shock, oxidative burst, and three different types of nutrient starvation. These data were compared to fungal gene expression during the invasive growth phase in compatible interactions on two grass hosts, rice and barley. We have identified 4,973 differentially expressed genes, from which 1,909 genes showed similar regulation patterns between at least one of the in vitro stresses and rice and/or barley. Hierarchical clustering of these 1,909 genes showed three major clusters in which growth in planta conditions closely grouped with the starving nutrient conditions. Out of these 1,909 genes, 55 and 129 genes were induced and repressed in all seven treatments, respectively. The functional categorization of those 55 induced genes revealed that most of them are either related to carbon metabolism, membrane proteins, or are involved in oxidoreduction reactions. The 129 repressed genes are putatively involved in vesicle trafficking, signal transduction, nitrogen metabolism, or molecular transport. These findings indicated that M. oryzae is likely coping with nutrient deprived environments in its hosts during the invasive growth stage about 72 hours post-inoculation, and not as much with host defense responses, or a temperature stress. Eight M. oryzae treatments: mycelia grown in liquid complete medium (set as the control); 72 hours post-incoulation (hpi) in rice leaves; 72 hours post-incoulation in barley leaves; mycelia under heat shock; mycelia under oxidative stress; mycelia under minimal medium; mycelia under nitrogen starvation; mycelia under carbon starvation. Three biological replicates had their total RNA pulled after RNA extraction. Dye-swaps used as technical replicates.

Project description:The potential for sexual reproduction in Aspergillus oryzae was assessed by investigating the presence and functionality of MAT genes. Previous genome studies had identified a MAT1-1 gene in the RIB40 reference strain. We now report the existence of a complimentary MAT1-2 gene and sequencing of an idiomorph region from A. oryzae strain AO6. This allowed the development of a PCR diagnostic assay, which detected isolates of MAT1-1 and MAT1-2 genotype among 180 strains assayed including industrial tane-koji isolates. Strains used for sake and miso production showed a near 1:1 ratio of MAT1-1 and MAT1-2 mating-types, whereas strains used for soy sauce production showed a significant bias towards the MAT1-2 mating type. MAT1-1 and MAT1-2 isogenic strains were then created by genetic manipulation of the resident idiomorph, and comparisons were made of gene expression by DNA microarray and RT-PCR methodologies under conditions when MAT genes were expressed. 33 genes were found to be up-regulated greater than 10-fold in either the MAT1-1 host or MAT1-2 gene replacement strains relative to each other, showing that both MAT1-1 and MAT1-2 genes functionally regulate gene expression in A. oryzae in a mating-type dependent manner, the first such report from a supposedly asexual fungus. MAT1-1 expression specifically up-regulated an a-pheromone precursor gene, but most genes affected were of unknown function. Results are consistent with a heterothallic breeding system in A. oryzae, and prospects for the discovery of a sexual cycle are discussed. On a condition that induces mating-type genes descrived in growth protocol section, each cogenic MAT1-1 and MAT1-2 mating-type strains were processed into RNA extraction and hybridization on Affymetrix microarrays. In a speculation that genes involved in putative mating process were reguated in mating-type dependent manner, we designed the cogenic strains that differs only in mating-type gene locus to analyze differential gene expression of MAT1-1 vs MAT1-2 strains.

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A protein kinase coordinates cycles of autophagy and glutaminolysis in invasive hyphae of the fungus Magnaporthe oryzae within rice cells

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