Functional characterization of the three mitogen‐activated protein kinase kinases (MAP2Ks) present in the C ryphonectria parasitica genome reveals the necessity of Cpkk1 and Cpkk2, but not Cpkk3, for pathogenesis on chestnut (C astanea spp.)

The biological function(s) of the cpkk1 , cpkk2 and cpkk3 genes, encoding the three mitogen‐activated protein kinase kinases ( MAP2Ks ) of C ryphonectria parasitica , the causal agent of chestnut blight, were examined through knockout strains. C pkk1, the M kk1 orthologue, acts in a phosphorylation cascade essential for cell integrity; C pkk2 is the S te7 orthologue involved in the pheromone response pathway; C pkk3 is the P bs2 orthologue, the MAP2K activated during the high‐osmolarity response. Our analysis confirmed the role of each MAP2K in its respective signalling cascade with some peculiarities: abnormal hyphae with a reduced number of septa and thinner cell walls were observed in Δcpkk1 mutants, and a strong growth defect on solid media was evident in Δcpkk2 mutants, when compared with the controls. Virulence on chestnut was affected in both the Δcpkk1 and Δcpkk2 strains, which were also unable to complete the developmental steps essential for mating. No alterations were reported in Δcpkk3 , except under hyperosmotic conditions and in the presence of fludioxonil. Δcpkk2 mutants, however, showed higher sensitivity during growth in medium containing the antibiotic G418 ( G eneticin).