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The PKR regulatory subunit of protein kinase A (PKA) is involved in the regulation of growth, sexual and asexual development, and pathogenesis in Fusarium graminearum.

ABSTRACT: Fusarium graminearum is a causal agent of wheat scab disease and a producer of deoxynivalenol (DON) mycotoxins. Treatment with exogenous cyclic adenosine monophosphate (cAMP) increases its DON production. In this study, to better understand the role of the cAMP-protein kinase A (PKA) pathway in F. graminearum, we functionally characterized the PKR gene encoding the regulatory subunit of PKA. Mutants deleted of PKR were viable, but showed severe defects in growth, conidiation and plant infection. The pkr mutant produced compact colonies with shorter aerial hyphae with an increased number of nuclei in hyphal compartments. Mutant conidia were morphologically abnormal and appeared to undergo rapid autophagy-related cell death. The pkr mutant showed blocked perithecium development, but increased DON production. It had a disease index of less than unity and failed to spread to neighbouring spikelets. The mutant was unstable and spontaneous suppressors with a faster growth rate were often produced on older cultures. A total of 67 suppressor strains that grew faster than the original mutant were isolated. Three showed a similar growth rate and colony morphology to the wild-type, but were still defective in conidiation. Sequencing analysis with 18 candidate PKA-related genes in three representative suppressor strains identified mutations only in the CPK1 catalytic subunit gene. Further characterization showed that 10 of the other 64 suppressor strains also had mutations in CPK1. Overall, these results showed that PKR is important for the regulation of hyphal growth, reproduction, pathogenesis and DON production, and mutations in CPK1 are partially suppressive to the deletion of PKR in F. graminearum.

SUBMITTER: Li C

PROVIDER: S-EPMC6638095 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:Sexual spores (ascospores) of Fusarium graminearum, a homothallic ascomycetous fungus, are believed to be the primary inocula for epidemics of the diseases caused by this species in cereal crops. Based on the light requirement for the formation of fruiting bodies (perithecia) of F. graminearum under laboratory conditions, we explored whether photoreceptors play an important role in sexual development. Here, we evaluated the roles of three genes encoding putative photoreceptors [a phytochrome gene (FgFph) and two white collar genes (FgWc-1 and FgWc-2)] during sexual development in F. graminearum. For functional analyses, we generated transgenic strains lacking one or two genes from the self-fertile Z3643 strain. Unlike the wild-type (WT) and add-back strains, the single deletion strains (ΔFgWc-1 and ΔFgWc-2) produced fertile perithecia under constant light on complete medium (CM, an unfavorable medium for sexual development) as well as on carrot agar (a perithecial induction condition). The expression of mating-type (MAT) genes increased significantly in the gene deletion strains compared to the WT under both conditions. Deletion of FgFph had no significant effect on sexual development or MAT gene expression. In contrast, all of the deletion strains examined did not show significant changes in other traits such as hyphal growth, mycotoxin production, and virulence. A split luciferase assay confirmed the in vivo protein-protein interactions among three photoreceptors along with FgLaeA, a global regulator of secondary metabolism and fungal development. Introduction of an intact copy of the A. nidulans LreA and LreB genes, which are homologs of FgWc-1 and FgWc-2, into the ΔFgWc-1 and ΔFgWc-2 strains, respectively, failed to repress perithecia formation on CM in the gene deletion strains. Taken together, these results demonstrate that FgWc-1 and FgWc-2, two central components of the blue-light sensing system, negatively regulate sexual development in F. graminearum, which differs from the regulation pattern in A. nidulans.

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The PKR regulatory subunit of protein kinase A (PKA) is involved in the regulation of growth, sexual and asexual development, and pathogenesis in Fusarium graminearum.

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