F-box proteins MoFwd1, MoCdc4 and MoFbx15 regulate development and pathogenicity in the rice blast fungus Magnaporthe oryzae

The Skp1-Cul1-F-box-protein (SCF) ubiquitin ligases are important parts of the ubiquitin system controlling many cellular biological processes in eukaryotes. However, the roles of SCF ubiquitin ligases remain unclear in phytopathogenic Magnaporthe oryzae. Here, we cloned 24 F-box proteins and confirmed that 17 proteins could interact with MoSkp1, showing their potential to participate in SCF complexes. To determine their functions, null mutants of 21 F-box-containing genes were created. Among them, the F-box proteins MoFwd1, MoCdc4 and MoFbx15 were found to be required for growth, development and full virulence. Fluorescent-microscopy observations demonstrated that both MoFbx15 and MoCdc4 were localized to the nucleus, compared with MoFwd1, which was distributed in the cytosol. MoCdc4 and MoFwd1 bound to MoSkp1 via the F-box domain, the deletion of which abrogated their function. Race tube and qRT-PCR assays confirmed that MoFwd1 was involved in circadian rhythm by regulating transcription and protein stability of the core circadian clock regulator MoFRQ. Moreover, MoFWD1 also orchestrates conidial germination by influencing conidial amino acids pools and oxidative stress release. Overall, our results indicate that SCF ubiquitin ligases play indispensable roles in development and pathogenicity in M. oryzae.