PKA activity is essential for relieving the suppression of hyphal growth and appressorium formation by MoSfl1 in Magnaporthe oryzae

In the rice blast fungus Magnaporthe oryzae, the cAMP-PKA pathway regulates surface recognition, appressorium turgor generation, and invasive growth. However, deletion of CPKA failed to block appressorium formation and responses to exogenous cAMP. In this study, we generated and characterized the cpk2 and cpkA cpk2 mutants and spontaneous suppressors of cpkA cpk2 in M. oryzae. Our results demonstrate that CPKA and CPK2 have specific and overlapping functions, and PKA activity is essential for appressorium formation and plant infection. Unlike the single mutants, the cpkA cpk2 mutant was significantly reduced in growth and rarely produced conidia. It failed to form appressoria although the intracellular cAMP level and phosphorylation of Pmk1 MAP kinase were increased. The double mutant also was defective in plant penetration and Mps1 activation. Interestingly, it often produced fast-growing spontaneous suppressors that formed appressoria but were still non-pathogenic. Two suppressor strains of cpkA cpk2 had deletion and insertion mutations in the MoSFL1 transcription factor gene. Deletion of MoSFL1 or its C-terminal 93-aa (MoSFL1ΔCT) was confirmed to suppress the defects of cpkA cpk2 in hyphal growth but not appressorium formation or pathogenesis. We also isolated 30 spontaneous suppressors of the cpkA cpk2 mutant in Fusarium graminearum and identified mutations in 29 of them in FgSFL1. Affinity purification and co-IP assays showed that this C-terminal region of MoSfl1 was essential for its interaction with the conserved Cyc8-Tup1 transcriptional co-repressor, which was reduced by cAMP treatment. Furthermore, the S211D mutation at the conserved PKA-phosphorylation site in MoSFL1 partially suppressed the defects of cpkA cpk2. Overall, our results indicate that PKA activity is essential for appressorium formation and proper activation of Pmk1 or Mps1 in M. oryzae, and phosphorylation of MoSfl1 by PKA relieves its interaction with the Cyc8-Tup1 co-repressor and suppression of genes important for hyphal growth.

Author summary The cAMP-PKA signaling pathway plays a critical role in regulating various cellular processes in eukaryotic cells in response to extracellular cues. In the rice blast fungus, this important pathway is involved in surface recognition, appressorium morphogenesis, and infection. However, the exact role of PKA is not clear due to the functional redundancy of two PKA catalytic subunits CPKA and CPK2. To further characterize their functions in growth and pathogenesis, in this study we generated and characterized the cpkA cpk2 double mutant and its suppressor strains. Unlike the single mutants, cpkA cpk2 mutant had severe defects in growth and conidiation and was defective in appressorium formation and plant infection. Interestingly, the double mutant was unstable and produced fast-growing suppressors. In two suppressor strains, mutations were identified in a transcription factor gene orthologous to SFL1, a downstream target of PKA in yeast. Deletion of the entire or C-terminal 93 residues of MoSFL1 could suppress the growth defect of cpkA cpk2. Furthermore, the terminal region of MoSfl1 was found to be essential for its interaction with the MoCyc8 co-repressor, which may be negatively regulated by PKA. Therefore, loss-of-function mutations in MoSFL1 can bypass PKA activity to suppress the growth defect of cpkA cpk2.