Three Mitogen-Activated Protein Kinases Required for Cell Wall Integrity Contribute Greatly to Biocontrol Potential of a Fungal Entomopathogen

<div><p>Bck1, Mkk1 and Slt2 are three mitogen-activated protein (MAP) kinases constituting cell wall integrity (CWI) pathway that may control multi-stress responses via crosstalk with high-osmolarity glycerol (HOG) pathway in budding yeast. In this study, Bck1, Mkk1 and Slt2 orthologues in <i>Beauveria bassiana</i> were confirmed as the three-module cascade essential for CWI because cell wall impairment occurred in the hyphae and conidia of Δ<i>bck1</i>, Δ<i>mkk1</i> and Δ<i>slt2</i> examined in multiple experiments. Strikingly, all the deletion mutants became more sensitive to hyperosmotic NaCl and sorbitol with the Western blot of Hog1 phosphorylation being weakened in Δ<i>bck1</i> and absent in Δ<i>mkk1</i> and Δ<i>slt2</i>. Apart from crossing responses to cell wall perturbation and high osmolarity, three deletion mutants exhibited faster growth and conidiation on nutrition-rich medium, much less virulence to <i>Galleria mellonella</i> larvae, and higher sensitivity to nutritional, fungicidal, thermal and UV-B irradiative stresses, accompanied with less accumulation of intracellular mannitol and trehalose. Moreover, Δ<i>mkk1</i> and Δ<i>slt2</i> were equally more sensitive to all the stresses of different types except wet-heat stress than wild type and more or less different from Δ<i>bck1</i> in sensitivity to most of the stresses despite their null responses to two oxidants. All the changes in three deletion mutants were restored by each targeted gene complementation. Taken together, the CWI-required Bck1, Mkk1 and Slt2 are all positive, but differential, regulators of multi-stress tolerance and virulence perhaps due to interplay with the HOG pathway essential for osmoregulation, thereby contributing greatly to the biocontrol potential of the fungal entomopathogen.</p>