PKR Protects the Major Catalytic Subunit of PKA Cpk1 from FgBlm10-Mediated Proteasome Degradation in <i>Fusarium graminearum</i>

For optimal proteolytic function, the proteasome core (CP or 20S) must associate with activators. The cAMP-PKA pathway is reported to affect the activity of the proteasome in humans. However, the relationship between the proteasome and PKA is not well characterized. Our results showed that the major catalytic subunit Cpk1 was degraded without the protection of Pkr. Eleven (out of 67) pkr suppressors had FgBlm10 C-terminal truncation, one suppressor had an amino acid change mutation in the PRE6 ortholog (FGRRES_07282), and one in the PRE5 ortholog (FGRRES_05222). These mutations rescued the defects in growth and conidial morphology, Cpk1 stability, and PKA activities in the pkr mutant. The interaction of FgBlm10 with FgPre5 and FgPre6 were detected by co-immunoprecipitation, and the essential elements for their interaction were characterized, including the FgBlm10 C-terminus, amino acid D82 of FgPre6 and K62 of FgPre5. Additional FgBlm10-interacting proteins were identified in the wild type and pkr mutant, suggesting that PKA regulates the preference of FgBlm10-mediated proteasome assembly. In addition, PKA indirectly affected the phosphorylation of FgBlm10, and its localization in the nucleus. The truncation of the FgBlm10 C terminus also enhanced nuclear import and bleomycin resistance, suggesting its role in proteasome assembly at DNA damage sites. Collectively, our data demonstrated that regulation between PKA and proteasome degradation is critical for the vegetative growth of F. graminearum

FgSnt1 of the Set3 HDAC complex plays a key role in mediating the regulation of histone acetylation by the cAMP-PKA pathway in Fusarium graminearum

The cAMP-PKA pathway is critical for regulating growth, differentiation, and pathogenesis in fungal pathogens. In Fusarium graminearum, mutants deleted of PKR regulatory-subunit of PKA had severe defects but often produced spontaneous suppressors. In this study eleven pkr suppressors were found to have mutations in FgSNT1, a component of the Set3C histone deacetylase (HDAC) complex, that result in the truncation of its C-terminal region. Targeted deletion of the C-terminal 98 aa (CT98) in FgSNT1 suppressed the defects of pkr in growth and H4 acetylation. CT98 truncation also increased the interaction of FgSnt1 with Hdf1, a major HDAC in the Set3 complex. The pkr mutant had no detectable expression of the Cpk1 catalytic subunit and PKA activities, which was not suppressed by mutations in FgSNT1. Cpk1 directly interacted with the N-terminal region of FgSnt1 and phosphorylated it at S443, a conserved PKA-phosphorylation site. CT98 of FgSnt1 carrying the S443D mutation interacted with its own N-terminal region. Expression of FgSNT1S443D rescued the defects of pkr in growth and H4 acetylation. Therefore, phosphorylation at S443 and suppressor mutations may relieve self-inhibitory binding of FgSnt1 and increase its interaction with Hdf1 and H4 acetylation, indicating a key role of FgSnt1 in crosstalk between cAMP signaling and Set3 complex. Author summary The cAMP-protein kinase A (PKA) pathway is one of the well-conserved signal transduction pathways in F. graminearum that regulates various infection and developmental processes. In this study we showed that deletion of the C-terminal region of FgSnt1 suppressed the defects of the pkr mutants deleted of the regulator subunit of PKA in growth and H4 histone acetylation. Snt1 is a component of the well-conserved Set3 histone deacetylase (HDAC) complex that is important for regulating histone acetylation and gene expression. Truncation of FgSnt1 increased its interaction with Hdf1, a major HDAC in the Set3 complex. Although Pkr did not, the Cpk1 catalytic subunit of PKA directly interacted with the N-terminal region of FgSnt1 and phosphorylated it at the conserved S443 site. Nevertheless, Pkr is important for protecting the Cpk1 proteins from degradation by the 26S proteosome. The S443D mutation mimicking phosphorylation of FgSnt1 by Cpk1 enabled the likely self-inhibitory interaction between its N-terminal and C-terminal regions and partially rescued the defects of pkr and H4 acetylation. Overall, this work described the comprehensive genetic interaction of the Set3 complex with the cAMP-PKA pathway. Our work extends and deepens our understanding of cAMP-PKA pathway in histone acetylation and virulence of plant pathogenic fungi

FgSnt1 of the Set3 HDAC complex plays a key role in mediating the regulation of histone acetylation by the cAMP-PKA pathway in Fusarium graminearum.

The cAMP-PKA pathway is critical for regulating growth, differentiation, and pathogenesis in fungal pathogens. In Fusarium graminearum, mutants deleted of PKR regulatory-subunit of PKA had severe defects but often produced spontaneous suppressors. In this study eleven pkr suppressors were found to have mutations in FgSNT1, a component of the Set3C histone deacetylase (HDAC) complex, that result in the truncation of its C-terminal region. Targeted deletion of the C-terminal 98 aa (CT98) in FgSNT1 suppressed the defects of pkr in growth and H4 acetylation. CT98 truncation also increased the interaction of FgSnt1 with Hdf1, a major HDAC in the Set3 complex. The pkr mutant had no detectable expression of the Cpk1 catalytic subunit and PKA activities, which was not suppressed by mutations in FgSNT1. Cpk1 directly interacted with the N-terminal region of FgSnt1 and phosphorylated it at S443, a conserved PKA-phosphorylation site. CT98 of FgSnt1 carrying the S443D mutation interacted with its own N-terminal region. Expression of FgSNT1S443D rescued the defects of pkr in growth and H4 acetylation. Therefore, phosphorylation at S443 and suppressor mutations may relieve self-inhibitory binding of FgSnt1 and increase its interaction with Hdf1 and H4 acetylation, indicating a key role of FgSnt1 in crosstalk between cAMP signaling and Set3 complex

FgSfl1 and Its Conserved PKA Phosphorylation Sites Are Important for Conidiation, Sexual Reproduction, and Pathogenesis in Fusarium graminearum

The fungal plant pathogen, Fusarium graminearum, contains two genes, FgCPK1 and FgCPK2, encoding the catalytic subunits of cAMP-dependent protein kinase A. FgCPK1 and FgCPK2 are responsible for most of the PKA activities and have overlapping functions in various cellular processes in F. graminearum. The cpk1 cpk2 double mutant was significantly reduced in growth, rarely produced conidia, and was non-pathogenic. In this study, we found that the cpk1 cpk2 double mutant was unstable and produced fast-growing spontaneous sectors that were defective in plant infection. All spontaneous suppressor strains had mutations in FgSFL1, a transcription factor gene orthologous to SFL1 in yeast. Thirteen suppressor strains had non-sense mutations at Q501, three suppressor strains had frameshift mutations at W198, and five suppressor strains had mutations in the HSF binding domain of FgSfl1. Only one suppressor strain had both a non-synonymous mutation at H225 and a non-sense mutation at R490. We generated the SFL1 deletion mutant and found that it produced less than 2% of conidia than that of the wild-type strain PH-1. The sfl1 mutant was significantly reduced in the number of perithecia on carrot agar plates at 7 days post-fertilization (dpf). When incubated for more than 12 days, ascospore cirrhi were observed on the sfl1 mutant perithecia. The infection ability of the sfl1 deletion mutant was also obviously defective. Furthermore, we found that in addition to the S223 and S559 phosphorylation sites, FgSFL1 had another predicted phosphorylation site: T452. Interestingly, the S223 phosphorylation site was responsible for sexual reproduction, and the T452 phosphorylation site was responsible for growth and sexual reproduction. Only the S559 phosphorylation site was found to play an important role in conidiation, sexual reproduction, and infection. Overall, our results indicate that FgSFL1 and its conserved PKA phosphorylation sites are important for vegetative growth, conidiation, sexual reproduction, and pathogenesis in F. graminearum