Surface recognition and penetration are critical steps in the infection cycle of many plant pathogenic fungi. In Magnaporthe oryzae, cAMP signaling is involved in surface recognition and pathogenesis. Deletion of the MAC1 adenylate cyclase gene affected appressorium formation and plant infection. In this study, we used the affinity purification approach to identify proteins that are associated with Mac1 in vivo. One of the Mac1-interacting proteins is the adenylate cyclase-associated protein named Cap1. CAP genes are well-conserved in phytopathogenic fungi but none of them have been functionally characterized. Deletion of CAP1 blocked the effects of a dominant RAS2 allele and resulted in defects in invasive growth and a reduced intracellular cAMP level. The Δcap1 mutant was defective in germ tube growth, appressorium formation, and formation of typical blast lesions. Cap1-GFP had an actin-like localization pattern, localizing to the apical regions in vegetative hyphae, at the periphery of developing appressoria, and in circular structures at the base of mature appressoria. Interestingly, Cap1, similar to LifeAct, did not localize to the apical regions in invasive hyphae, suggesting that the apical actin cytoskeleton differs between vegetative and invasive hyphae. Domain deletion analysis indicated that the proline-rich region P2 but not the actin-binding domain (AB) of Cap1 was responsible for its subcellular localization. Nevertheless, the AB domain of Cap1 must be important for its function because CAP1ΔAB only partially rescued the Δcap1 mutant. Furthermore, exogenous cAMP induced the formation of appressorium-like structures in non-germinated conidia in CAP1ΔAB transformants. This novel observation suggested that AB domain deletion may result in overstimulation of appressorium formation by cAMP treatment. Overall, our results indicated that CAP1 is important for the activation of adenylate cyclase, appressorium morphogenesis, and plant infection in M. oryzae. CAP1 may also play a role in feedback inhibition of Ras2 signaling when Pmk1 is activated.
In Magnaporthe oryzae, cAMP signaling is known to play an important role in surface recognition and plant penetration. The Mac1 adenylate cyclase is essential for plant infection. To better understand Mac1 activation mechanisms, in this study we used the affinity purification approach to identify proteins that are associated with Mac1 in vivo. One of the Mac1-interacting protein is the adenylate cyclase associated protein (CAP) encoded by the CAP1 gene. Results from our study indicated that Cap1 is important for Mac1 activation and plant infection in M. oryzae. The Δcap1 mutant was defective in germ tube growth and appressorium formation and failed to cause typical blast lesions. Like LifeAct, Cap1 localized to apical patches in vegetative hyphae but not in invasive hyphae. The P2 proline-rich region was important for Cap1 localization but the actin-binding domain played a role in feedback inhibition of Ras signaling. To our knowledge, functional characterization of CAP genes has not been reported in filamentous fungi. Our results indicate that CAP1 is important for regulating adenylate cyclase activities, appressorium morphogenesis, and plant infection. Further characterization of CAP1 will be important to better understand the interaction between cAMP signaling and the PMK1 pathway in M. oryzae.