Screening of binding proteins that interact with two components of the HOG MAPK pathway by the yeast two-hybrid method in Colletotrichum siamense

In phytopathogenic fungi, the Hog1 MAPK pathway plays roles in osmoregulation, oxidation responses, fungicide sensitivity, among others. However, how fungi depend on the HOG MAPK pathway to regulate their functions is poorly understood. To further elucidate the molecular mechanisms of the HOG MAPK pathway, a yeast two-hybrid (Y2H) system was adopted to screen interacting proteins of two key components (CsPbs2 and CsHog1) of the HOG MAPK pathway in a cDNA library of Colletotrichum siamense . The results showed that 19 proteins may interact with CsPbs2, including cytochrome P450 4F5, ceramide very-long-chain fatty acid hydroxylase SCS7, the oxidative stress response two-component system protein SSK1, and other proteins. Twenty-one proteins may interact with CsHog1, including the cell wall protein PhiA, acriflavine sensitivity control protein acr-2, arachidonate 15-lipoxygenase, respiratory supercomplex factor 2-like protein, and others. Five proteins interact with both CsHog1 and CsPbs2: antigenic thaumatin-like protein, aspirochlorine biosynthesis protein N, secreted beta-glucosidase sun 1, alcohol dehydrogenase 1, and hypothetical protein GQ607_005106. Furthermore, the interaction of CsSCS7 and CsCytP450 4F5 with CsPbs2 was verified by His pull-down. qPCR revealed significantly upregulated CsPbs2 , CsHog1 , CsSCS7 and CsCytP450 4F5 expression in Colletotrichum treated with 50 μg/mL fludioxonil for 2 h. This research identified candidate CsHog1- and CsPbs2-interacting proteins via a Y2H system and confirmed the interactions of CsSCS7 and CsCytP450 4F5 with CsPbs2. The findings lay the foundation for further studies of HOG MAPK pathway function and regulatory mechanisms, especially regarding fungicide sensitivity.