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

Abstract

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.

MoPpe1 partners with MoSap1 to mediate TOR and cell wall integrity signalling in growth and pathogenicity of the rice blast fungus Magnaporthe oryzae

“…Magnaporthe oryzae causes devastating rice blast worldwide and is also a widely adopted model organism for studying the plant-pathogen interaction (Wilson and Talbot, 2009;Zhang et al, 2016). In Magnaporthe oryzae signalling pathways, including the cAMP-PKA pathway and MoPmk1 and MoMps1 MAP kinase signalling pathways, sense and transduce extracellular signals to regulate growth, differentiation and pathogenicity (Adachi and Hamer, 1998;Marroquin-Guzman and Wilson, 2015;Li et al, 2017c). The cell wall integrity (CWI) MAP kinase pathway, consisting of MoMck1, MoMkk1 and MoMps1, is also important for appressorium function and virulence (Jeon et al, 2008;Yin et al, 2016).…”

Regulatory network of genes associated with stimuli sensing, signal transduction and physiological transformation of appressorium inMagnaporthe oryzae

“…The cAMP/PKA signal cascade is further mediated via its binding with the PKA regulatory subunit SUM 1, allowing the release of the PKA catalytic subunit for phosphorylating the downstream targets in the nucleus like transcriptional factors including CDTF 1, SOM 1, MSTU 1 in M. oryzae (Filippi 2004) and SFL 1 in budding yeast (Song and Carlson 1998; Galeote et al 2007). The two catalytic subunits CpkA and Cpk2 play specific and overlapping functions in regulating infection-related morphogenesis and pathogenicity of M. oryzae (Li et al 2017b). Recent findings show that PKA activity is essential for appressorium formation and phosphorylation of SFL1 in M. oryzae (Li et al 2017b).…”

“…The two catalytic subunits CpkA and Cpk2 play specific and overlapping functions in regulating infection-related morphogenesis and pathogenicity of M. oryzae (Li et al 2017b). Recent findings show that PKA activity is essential for appressorium formation and phosphorylation of SFL1 in M. oryzae (Li et al 2017b). The activation of Sfl1 relieves Cy8-Tup1 which is a co-repressor that suppresses genes required for hyphal growth and appressorium development in M. oryzae (Li et al 2017b).…”

“…Recent findings show that PKA activity is essential for appressorium formation and phosphorylation of SFL1 in M. oryzae (Li et al 2017b). The activation of Sfl1 relieves Cy8-Tup1 which is a co-repressor that suppresses genes required for hyphal growth and appressorium development in M. oryzae (Li et al 2017b). …”

Arginine methylation is required for remodelling pre‐mRNA splicing and induction of autophagy in rice blast fungus

“…The split‐marker strategy was used in this study to generate the targeted gene knockout (ko) mutant strains, while protoplast isolation and the fungal transformation were performed as previously reported (Park & Xu, ; Saitoh et al ., ; Y. Li et al ., ). Briefly, the first round PCR used the LBCK/LB‐R and RBCK/RB‐F primers to amplify 2000 bp upstream and downstream of the target gene sequence.…”