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Abstract The fungusMagnaporthe oryzaecauses blast, the most devastating disease of cultivated rice. After penetrating the leaf cuticle,M. oryzaegrows as a biotroph in intimate contact with living rice epidermal cells before necrotic lesions develop. Biotrophic growth requires maintaining metabolic homeostasis while suppressing plant defenses, but the metabolic connections and requirements involved are largely unknown. Here, we characterized theM. oryzaenucleoside diphosphate kinase‐encoding geneNDK1and discovered it was essential for facilitating biotrophic growth by suppressing the host oxidative burst—the first line of plant defense. NDK enzymes reversibly transfer phosphate groups from tri‐ to diphosphate nucleosides. Correspondingly, intracellular nucleotide pools were perturbed inM. oryzaestrains lackingNDK1through targeted gene deletion, compared to WT. This affected metabolic homeostasis: TCA, purine and pyrimidine intermediates, and oxidized NADP⁺, accumulated in Δndk1. cAMP and glutathione were depleted. ROS accumulated in Δndk1hyphae. Functional appressoria developed on rice leaf sheath surfaces, but Δndk1invasive hyphal growth was restricted and redox homeostasis was perturbed, resulting in unsuppressed host oxidative bursts that triggered immunity. We conclude Ndk1 modulates intracellular nucleotide pools to maintain redox balance via metabolic homeostasis, thus quenching the host oxidative burst and suppressing rice innate immunity during biotrophy. 

Summary Fungal phytopathogens can suppress plant immune mechanisms in order to colonize living host cells. Identifying all the molecular components involved is critical for elaborating a detailed systems‐level model of plant infection probing pathogen weaknesses; yet, the hierarchy of molecular events controlling fungal responses to the plant cell is not clear.Here we show how, in the blast fungusMagnaporthe oryzae, terminating rice innate immunity requires a dynamic network of redox‐responsive E3 ubiquitin ligases targeting fungal sirtuin 2 (Sir2), an antioxidation regulator required for suppressing the host oxidative burst.Immunoblotting, immunopurification, mass spectrometry and gene functional analyses showed that Sir2 levels responded to oxidative stress via a mechanism involving ubiquitination and three antagonistic E3 ubiquitin ligases: Grr1 and Ptr1 maintained basal Sir2 levels in the absence of oxidative stress; Upl3 facilitated Sir2 accumulation in response to oxidative stress. Grr1 and Upl3 interacted directly with Sir2 in a manner that decreased and scaled with oxidative stress, respectively.DeletingUPL3depleted Sir2 during growth in rice cells, triggering host immunity and preventing infection. OverexpressingSIR2in the Δupl3mutant remediated pathogenicity. Our work reveals how redox‐responsive E3 ubiquitin ligases inM. oryzaemediate Sir2 accumulation‐dependent antioxidation to modulate plant innate immunity and host susceptibility. 

Animal and plant microbial pathogens deploy effector proteins and virulence factors to manipulate host cell innate immunity, often using unconventional secretion routes that are poorly understood. Transfer RNA (tRNA) anticodon modifications occur across taxa, but few biological functions are known. Here, in the devastating blast fungus Magnaporthe oryzae, we find that unconventional protein secretion in living host rice cells depends on tRNA modification and codon usage. Using gene deletions, mass spectrometry and live-cell imaging, we characterized the M. oryzae Uba4-Urm1 sulfur relay system mediating tRNA anticodon wobble uridine 2-thiolation (s2U34), a conserved modification required for efficient decoding of AA-ending cognate codons. In M. oryzae, cytoplasmic effectors like Pwl2 and AVR-Pita are translocated into host cells via an unconventional secretion route; apoplastic effectors like Bas4 are secreted by the conventional ER-Golgi pathway. Loss of U34 thiolation abolished PWL2 and AVR-PITA (but not BAS4) mRNA translation in host cells. Paromomycin treatment, which increases near-cognate tRNA acceptance, restored Pwl2 and AVR-Pita production in U34 thiolation-deficient mutant strains. Synonymous AA- to ¬¬AG-ending codon changes remediated PWL2 mRNA translation in uba4; in UBA4+, expressing recoded PWL2 resulted in Pwl2 super-secretion that destabilized the microbe-host cell interface. Thus, wobble U34 tRNA thiolation and codon usage tune pathogen unconventional protein secretion in host cells. 

Eukaryotic filamentous plant pathogens with biotrophic growth stages like the devastating hemibiotrophic rice blast fungus Magnaporthe oryzae grow for extended periods in living host plant cells without eliciting defense responses. M. oryzae elaborates invasive hyphae (IH) that grow in and between living rice cells while separated from host cytoplasm by plant-derived membrane interfaces. However, although critical to the plant infection process, the molecular mechanisms and metabolic strategies underpinning this intracellular 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 plate growth tests and RNAseq, proteomic, quantitative phosphoproteomics and metabolic approaches, we show how cycles of autophagy in IH modulate TOR reactivation via α-ketoglutarate to sustain biotrophic growth and maintain biotrophic interfacial membrane integrity in host rice cells. Deleting the M. oryzae serine-threonine protein kinase Rim15-encoding gene attenuated biotrophic growth, disrupted interfacial membrane integrity and abolished the in planta autophagic cycling we observe here for the first time in wild type. Δrim15 was also impaired for glutaminolysis and depleted for α-ketoglutarate. α-ketoglutarate treatment of Δrim15-infected leaf sheaths remediated Δrim15 biotrophic growth. In WT, α-ketoglutarate treatment suppressed autophagy. α-ketoglutarate signaling is amino acid prototrophy- and GS-GOGAT cycle-dependent. We conclude that, following initial IH elaboration, cycles of Rim15- dependent autophagic flux liberate α-ketoglutarate – via the GS-GOGAT cycle – as an amino acid-sufficiency signal to trigger TOR reactivation and promote fungal biotrophic growth in nutrient-restricted host rice cells. 

Protein–protein interactions underlie cellular structure and function. In recent years, a number of methods have been developed for the identification of protein complexes and component proteins involved in the control of various biological pathways. Tandem affinity purification (TAP) coupled with mass spectrometry (MS) is a powerful method enabling the isolation of high-purity native protein complexes under mild conditions by performing two sequential purification steps using two different epitope tags. In this protocol, we describe a TAP-MS methodology for identifying protein-protein interactions present at very low levels in the fungal cell. Using the 6xHis-3xFLAG double tag, we start the affinity purification process for our protein of interest using high-capacity Ni²⁺ columns. This allows for greatly increased sample input compared to antibody-based first-step purification in conventional TAP protocols and provides a large amount of highly concentrated and preliminarily purified protein complexes to be used in a second purification step involving FLAG immunoprecipitation. The second step greatly facilitates the capture of low-level interacting partners under in vivo conditions. Our TAP-MS method has been proven to secure the characterization of low-abundance protein complexes under physiological conditions with high efficiency, specificity, and economy in the filamentous fungus Magnaporthe oryzae and might benefit gene function and proteomics studies in plants and other research fields. 

Electron microscopy (EM) allows characterization of the morphology and ultrastructure of a cell. However, challenges concerning cryo sample fixation are still one of the main roadblocks to its widespread adoption. In this protocol, we describe two alternative EM preparation methods employed to study Magnaporthe oryzae appressoria on artificial hydrophobic surfaces.