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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. 

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.