Glutamate dehydrogenase 2 is required for virulence by facilitating fungal growth in the host hemocoel

<i>Metarhizium robertsii</i>, a fungal pathogen employed in pest biocontrol, can alkalize the surrounding environment, although the biological implications remain unclear. Here, we found that <i>M. robertsii</i> glutamate dehydrogenase 2 (<i>MrGDH2</i>) was responsible for alkalization during fungal growth on insect wings or media containing cuticle powder. Loss of <i>MrGDH2</i> function resulted in significantly reduced virulence during both cuticle-passing and cuticle-bypassing infections but did not affect appressorium formation or cuticle penetration. Deletion of <i>MrGDH2</i> failed to alkalize amino acid-containing media under carbon deprivation, leading to impaired mycelia growth and conidiation. Hemolymph carbohydrates were decreased during <i>M. robertsii</i> infection, and the Δ<i>Mrgdh2</i> mutant exhibited delayed fungal growth and impaired alkalization in hemolymph cultures. Notably, expression of <i>PacC</i>, a pH-responsive transcription factor critical for virulence, was downregulated in hyphal bodies of the Δ<i>Mrgdh2</i> mutant. Contrary to the established models in plant and human fungal pathogens, we demonstrate that Gdh2 activity is dispensable for appressorium formation but essential for fungal colonization in insect hemocoel during <i>M. robertsii</i> infection.