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.

Fig 1. Defects of the cpkA cpk2 mutant in growth, appressorium formation, and plant infection.

A. Seven-day-old cultures of the wild-type Guy11 and cpkA, cpk2, and cpkA cpk2 mutants. Scale bar = 1 cm. B. Conidia of Guy11 and mutant strains were incubated on the hydrophobic (upper panel) and hydrophilic (lower panel) surface of GelBond membranes for 24 h. Scale bar = 10 μm. C. Leaves of two-week-old rice seedlings were inoculated by spray or injection with conidium suspensions of labelled strains. Inoculation with 0.25% gelatin was used as the negative control. Typical leaves were photographed 7 dpi.

Fig 2. Assays for the intracellular cAMP level and activation of Pmk1 or Mps1 MAP kinase.

A. The intracellular cAMP level was assayed with vegetative hyphae of Guy11 and the cpkA, cpk2, and cpkA cpk2 mutants. Mean and standard deviation were calculated with results from three independent biological replicates. Different letters mark statistically significant differences (P = 0.05). B. Western blots of proteins isolated from Guy11 and the pmk1, mps1, cpkA, cpk2, and cpkA cpk2 mutant were detected with an anti-TpEY specific (upper panel) or anti-MAPK (lower panel) antibody to assay the phosphorylation of Pmk1 (42-kD) and Mps1 (46-kD).

Fig 3. Phenotypes of the spontaneous suppressor strain CCS1 of cpkA cpk2.

A. Seven-day-old OTA cultures of the wild type strain Guy11, cpkA cpk2 mutant, and suppressor strain CCS1. The fast-growing suppressor of cpkA cpk2 was marked with an arrow. B. Hyphal tips of Guy11 and CCS1 from 7-day-old OTA cultures. Arrows point to melanized hyphal tips. Scale bar = 10 μm. C. Rice leaves inoculated by injection with conidia of Guy11 and CCS1 were examined 7 days-post-inoculation. D. Appressoria formed by Guy11 and CCS1 on the hydrophobic and hydrophilic surfaces after incubation for 24 h. Scale bar = 10 μm. E. Abnormal appressoria formed by CCS1 on the hydrophobic surface after incubation for 24 h. Scale bar = 10 μm.

Fig 4. Deletion of MoSFL1 and MoSFL^CT in the cpkA cpk2 mutant.

A. Seven-day-old oatmeal agar plates of Guy11, cpkA cpk2 mutant, cpkA cpk2 Mosfl1 (TKO4), and cpkA cpk2 MoSFL1^ΔCT (CTD2) triple mutants. B. Appressorium formation assays with Guy11, TKO4, and CTD2 strains on the hydrophobic surface of GelBond membranes for 24 h. Scale bar = 10 μm. C. Leaves of two-week-old rice seedlings were injected with conidium suspensions of Guy11, TKO4, and CTD2 strains. Typical leaves were photographed 7 dpi.

Fig 5. Co-IP assays for the interaction of MoCyc8 with MoSfl1 and MoSfl1^ΔCT.

Western blots of total proteins (In for input) isolated from transformants expressing the MoCYC8-S and 3×Flag-MoSFL1 (CYS15) or 3×Flag-MoSfl1^ΔCT (CNC19) constructs and proteins immuno-precipitated (IP) with anti-S-Tag agarose beads were detected with the anti-S and anti-Flag antibodies. Detection with an anti-actin antibody was included as the negative co-IP control. The expected sizes of MoCyc8-S, 3×Flag-MoSfl1, and 3×Flag-MoSfl1^ΔCT were labelled on the right.

Fig 6. Assays for the effects of cAMP or PKA inhibitor H-89 on the MoSfl1-MoCyc8 interaction.

A. Western blots of total proteins (In for input) and proteins immuno-precipitated (IP) with anti-S-Tag agarose beads were detected with an anti-S, anti-Flag, or anti-actin antibody. Proteins were isolated from transformant GCS1 expressing MoCYC8-S and 3×Flag-MoSFL1 constructs cultured under labelled treatments (+) or not (−). B. Western blots of total proteins and proteins immuno-precipitated with anti-S-Tag agarose beads of transformant GTS9 expressing MoTUP1-S and 3×Flag-MoSFL1 constructs were detected with an anti-S, anti-Flag, or anti-actin antibody. Total proteins isolated from the wild-type strain Guy11 (WT) were included as the control. C. Seven-day-old CM cultures of the Motup1 mutant had limited growth and apical or subapical swollen bodies in hyphae.

Fig 7. Site-directed mutagenesis of putative PKA phosphorylation sites in MoSfl1.

A. Schematic drawing of the MoSfl1 protein and alignment of the marked region with its orthologs from F. graminearum (Fg) and S. cerevisiae (Sc). The consensus PKA phosphorylation sites were boxed with red lines. The putative PKA phosphorylation residues were marked with stars. B. Five-day-old OTA cultures of the wild-type strain Guy11, cpkA cpk2 mutant, and transformants of cpkA cpk2 expressing the MoSFL1^S211D (ASD5), MoSFL1^S211A (ASA9), MoSFL1^T441D (HTD17), MoSFL1^T441A (HTA17), MoSFL1^S554D (GSD22), or MoSFL1^S554A (GSA22) allele. C. Conidia of Guy11 and MoSFL1^S211D transformant ASD5 were assayed for appressorium formation on the hydrophobic side of GelBond membranes.

Fig 8. Spontaneous suppressors of the cpk1 cpk2 mutant in F. graminearum.

A. Four-day-old V8 cultures of the wild-type strain PH-1, cpk1 cpk2 mutant (DM1), and suppressor strains HS8 and HS20. B. Corn silks inoculated with culture blocks of suppressor strains HS13, HS25, and HS20. C. Suppressor mutations identified in FgSFL1.

Fig 9. A model of the repressive role of MoSfl1 and its phosphorylation by PKA in transcriptional regulation.

A. In the wild-type strain, phosphorylation of MoSfl1 by PKA disrupts its interaction with the Cyc8-Tup1 co-repressor. Without its association with MoSfl1, the Cyc8-Tup1 complex fails to block the transcription of MoSfl1 target genes that are important for hyphal growth and other developmental or infection processes. B. In the cpkA cpk2 mutant, the Cyc8-Tup1 co-repressor interacts with un-phosphorylated MoSfl1 to repress subsets of its target genes important for growth. C. Suppressor mutations in MoSFL1 block or reduce the association of MoSfl1 with the Cyc8-Tup1 co-repressor to bypass the requirement for its phosphorylation by PKA. P, phosphorylation; MBS, MoSfl1-binding site.