PGPBS, a mitogen-activated protein kinase kinase, is required for vegetative differentiation, cell wall integrity, and pathogenicity of the barley leaf stripe fungus Pyrenophora graminea

Abstract

The high-osmolarity glycerol (HOG) signaling pathway regulates the adaptation of fungi to environmental stressors. The mitogen-activated protein kinase kinase (MAPKK) PBS2 of Saccharomyces cerevisiae serves as a scaffold protein in the HOG pathway. We characterized the pgpbs gene of Pyrenophora graminea, which encodes a MAPKK that is 56% orthologous to PBS2 of S. cerevisiae. A cloning technique based on homology was applied to amplify the pgpbs gene. Specific silent mutations then were generated in pgpbs. We evaluated the potential roles of PGPBS in the osmotic response, vegetative differentiation, cell wall integrity, drug resistance, and pathogenicity. Our findings indicated that the pgpbs coding region comprises 2075 base pairs and encodes a protein of 676 amino acids. Mutants deficient in pgpbs expression had significant reductions in vegetative growth and were sensitive to calcofluor white (CFW), an inhibitor of cell wall synthesis. Mutants also lost pathogenicity and were sensitive to an osmotic stress–inducing medium containing NaCl and sorbitol. Moreover, mutants had increased resistance to the dicarboximide fungicide iprodione and the triazole fungicide tebuconazole. These findings suggest that pgpbs is involved in the osmotic and ionic stress responses, vegetative differentiation, cell wall integrity, virulence, and tolerance to iprodione and tebuconazole. We expect that our findings will help elucidate the pathogenesis of barley leaf stripe and will inform strategies for breeding resistance to this disease.