Abstract
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.
Highlights
Fusarium head blight (FHB), caused by Fusarium graminearum, is one of the most important diseases of various cereal crops [1]
These results indicate that spontaneous mutations in these suppressor strains rescued vegetative growth but did not positively impact conidiation
All other strains were not significantly defective in conidiation (Table 3; Figure 7B). These results indicate that the S559 phosphorylation site of the FgSfl1 affects conidiation
Summary
Fusarium head blight (FHB), caused by Fusarium graminearum, is one of the most important diseases of various cereal crops [1]. In Magnaporthe oryzae, the CPKA and CPK2 genes encode catalytic subunits of PKA. In F. graminearum, the deletion of both CPKA and CPK2 results in severe defects in growth and conidiation, and the double mutant is sterile in sexual reproduction and is nonpathogenic [14]. MoSFL1 can rescue growth defects in the cpkA cpk double mutant. Only S211 suppressed the defects in growth but not the appressorium formation of the cpkA cpk double mutant [17]. Three phosphorylation sites were responsible for the growth, sexual reproduction, and infection progress though precise regulation of phosphorylation in F. graminearum. These data indicate that FgSFL1 and its phosphorylation sites play important roles in conidiation, sexual reproduction, and infection. After single-spore isolation, each subculture of spontaneous suppressors was assayed for defects in growth, conidiation, and plant infection [17]
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