FgSsn3 kinase, a component of the mediator complex, is important for sexual reproduction and pathogenesis in Fusarium graminearum

Abstract

Fusarium graminearum is an important pathogen of wheat and barley. In addition to severe yield losses, infested grains are often contaminated with harmful mycotoxins. In this study, we characterized the functions of FgSSN3 kinase gene in different developmental and infection processes and gene regulation in F. graminearum. The FgSSN3 deletion mutant had a nutrient-dependent growth defects and abnormal conidium morphology. It was significantly reduced in DON production, TRI gene expression, and virulence. Deletion of FgSSN3 also resulted in up-regulation of HTF1 and PCS1 expression in juvenile cultures, and repression of TRI genes in DON-producing cultures. In addition, Fgssn3 was female sterile and defective in hypopodium formation and infectious growth. RNA-seq analysis showed that FgSsn3 is involved in the transcriptional regulation of a wide variety genes acting as either a repressor or activator. FgSsn3 physically interacted with C-type cyclin Cid1 and the cid1 mutant had similar phenotypes with Fgssn3, indicating that FgSsn3 and Cid1 form the CDK-cyclin pair as a component of the mediator complex in F. graminearum. Taken together, our results indicate that FgSSN3 is important for secondary metabolism, sexual reproduction, and plant infection, as a subunit of mediator complex contributing to transcriptional regulation of diverse genes.

Figure 1. Defects of the Fgssn3 mutant in growth and sexual reproduction.

(A) Colonies of wild-type (PH-1), Fgssn3 deletion mutant (M9), complemented strain (C1) cultured on PDA, OTA and 5 × YEG medium for 3 days. (B) Self-crossing plates of PH-1, M9, and C1 at 14 days post-fertilization. Arrows point to perithecia. (C) Mating cultures of the Fgssn3 mutant used as the male (left) or female (right) crossed with the mat1-1-1 mutant were examined for perithecia and ascospore formation 2 weeks post- fertilization. Bar = 20 μm.

Figure 2. Defects of the Fgssn3 mutant in conidiogenesis and conidium morphology.

(A) CMC cultures of the wild type (PH-1) and Fgssn3 mutant (M9) after incubation for 12 h. Bar = 10 μm. (B) Conidia of PH-1, M9, and the complemented transformant C1 were examined for difference in morphology (upper row) and germ tube growth after incubation in YEPD for 6 h (lower row). Bar = 10 μm. (C) Expression levels of genes related to conidiation were assayed by qRT-PCR assays. RNA samples were isolated from 12 h YEPD cultures of PH-1 and M9. For each gene, its expression level in PH-1 was arbitrarily set to 1. Mean and standard deviation were calculated with data from three biological replicates.

Figure 3. Defects of the Fgssn3 mutant in plant infection.

(A) Flowering wheat heads were drop-inoculated with conidia from the wild type (PH-1), Fgssn3 mutant (M9), and complemented strain (C1). Black dots mark the inoculated spikelets. Photographs were taken 14 days post-inoculation (dpi). (B) Corn stalks were inoculated with toothpicks dipped in conidia of the same set of strains and examined for stalk rot symptoms 14 dpi. (C) Corn silks were inoculated with blocks of cultures of the same set of strains. Photographs were taken 5 dpi. (D) Lemma from the spikelets inoculated with PH-1 and M9 were examined by SEM 24 hpi. Hyphopodia formed on the inner surface are marked with white arrows. Bar = 10 μm. (E) Infectious hyphae (IH) formed by PH-1 and M9 inside lemma tissues 48 hpi. Bar = 50 μm. (F) Thick sections of rachis tissues directly below the inoculated spikelet were examined for infectious growth 5 dpi. In samples inoculated with PH-1, abundant hyphal growth was observed. No infectious hyphae (IH) were observed in the rachis inoculated with M9. Bar = 50 μm.

Figure 4. Assays for expression levels of selected genes related to trichothecene and aurofusarin biosynthesis by qRT-PCR.

The expression level of each gene in PH-1 was arbitrarily set to 1. Mean and standard deviation were calculated with data from three biological replicates. (A) Expression of TRI4, TRI5, TRI6, TRI10, and TRI11 in the wild-type strain PH-1 and Fgssn3 mutant M9. RNA samples were isolated from DON-producing cultures (containing 5 mM arginine). (B) Expression of PKS12, GIP1, and GIP2 in PH-1 and M9. RNA samples were isolated from DON-producing cultures containing 5 mM arginine.

Figure 5. Localization and expression of FgSSN3.

(A) Conidia and hyphae of the Fgssn3/FgSSN3-GFP H1-mCherry transformant were examined by DIC and epifluorescence microscopy. Bar = 20 μm. (B) Relative expression level of FgSSN3 in conidia (C), 4 or 12 h germlings, and perithecia (P). Mean and standard deviation were calculated with data from three replicates.

Figure 6. Functions and Localization of FgSsn3^D191A–GFP and FgSsn3^K71R-GFP.

(A) Two-day-old 5 × YEG cultures of the wide type (PH-1), Fgssn3 mutant (M9), Fgssn3/FgSSN3^D191A-GFP transformant (D24), and Fgssn3/FgSSN3^K71R-GFP transformant (K4). (B) Conidia of PH-1, D24, and K4 in 4-day-old CMC cultures. Bar = 10 μm. (C) Corn silks inoculated with PH-1, D24, and K4 were examined 5 dpi. (D) Mating cultures of PH-1, D24 and K4 were examined 2 weeks post-induction for sexual reproduction. (E) Hyphae of D24 and K4 were examined by DIC and epifluorescence microscopy. Bar = 10 μm.

Figure 7. Yeast two-hybrid assays for the interaction between FgSsn3 and Cid1 or FgMed8.

Different concentrations (cells/ml) of the yeast transformants expressing the FgSSN3 prey and Cid1 or FgMed8 bait constructs were assayed for growth on SD-Leu-Trp-His plates and β-galactosidase (LacZ) activities. Positive and negative controls were provided in the BD Matchmaker library construct kit.