<scp>CWI</scp> ‐ <scp>MAPKs</scp> Regulate the Formation of Hyphopodia Required for Virulence in <scp> <i>Ceratocystis fimbriata</i> </scp>

Ceratocystis fimbriata is a destructive fungal pathogen that infects various economic crops. Nevertheless, the infection mechanism of this fungus is still unclear. Our previous studies have shown that the transcription factor CfSwi6 downstream of the cell wall integrity pathway is involved in regulating the pathogenicity of C. fimbriata. To further clarify the pathogenic mechanism of this pathway, upstream MAPKs (CfBck1-CfMkk1-CfSlt2) were characterised in this study. Deletion of CWI-MAPK genes resulted in an almost complete loss of pathogenicity of C. fimbriata. Importantly, CWI-MAPKs are associated with the formation of hyphopodia, which are infection structures required for C. fimbriata, and are reported for the first time in this work. Mutants lacking CWI-MAPK genes had defects in forming hyphopodia. The ability of mutants to penetrate cellophane membranes and host cells was reduced. CWI-MAPKs or CfSwi6 deletion affected CfSep4 assembly at penetration pegs, while CfSep4 was important for septin-ring and penetration peg formation. These results indicate that CWI-MAPKs regulate infection structure formation by modulating septin-ring organisation. RNA-seq analysis revealed that some downstream genes co-regulated by CfSlt2 and CfSwi6 are cellophane surface-induced genes. Knockout of PHH50197 and CfHSP30_1, two CfSlt2-CfSwi6-dependent genes, affected hyphopodium formation and pathogenicity. Additionally, other downstream genes, including PHH51274, CfHSP30_0, CfSTE11 and PHH55780, are not necessary for hyphopodium morphogenesis but are important for pathogenicity. Our study reveals a molecular mechanism by which CWI-MAPKs regulate pathogenicity through downstream genes mediated by CfSwi6 in C. fimbriata.