The biosynthesis of mycotoxin deoxynivalenol (DON) in Fusarium graminearum is regulated by two pathway-specific transcription factors Tri6 and Tri10 and affected by various host and environmental factors. In this study, we showed that cyclic adenosine monophosphate (cAMP) treatment induced DON production by stimulating TRI gene expression and DON-associated cellular differentiation in F. graminearum. Interestingly, exogenous cAMP had no effects on the tri6 mutant but partially recovered the defect of tri10 mutant in DON biosynthesis. Although the two cAMP phosphodiesterase genes PDE1 and PDE2 had overlapping functions in vegetative growth, conidiation, sexual reproduction and plant infection, deletion of PDE2 but not PDE1 activated intracellular PKA activities and increased DON production. Whereas the tri6 pde2 mutant failed to produce DON, the tri10 pde2 double mutant produced a significantly higher level of DON than the tri10 mutant. Cellular differentiation associated with DON production was stimulated by exogenous cAMP or deletion of PDE2 in both tri10 and tri6 mutants. These data indicate that TRI6 is essential for the regulation of DON biosynthesis by cAMP signalling but elevated PKA activities could partially bypass the requirement of TRI10 for TRI gene-expression and DON production, and Pde2 is the major cAMP phosphodiesterase to negatively regulate DON biosynthesis in F. graminearum.

Histone acetylation, balanced by histone acetyltransferase (HAT) and histone deacetylase (HDAC) complexes, affects dynamic transitions of chromatin structure to regulate transcriptional accessibility. However, little is known about the interplay between HAT and HDAC complexes in Fusarium graminearum, a causal agent of Fusarium Head Blight (FHB) that uniquely contains chromosomal regions enriched for house-keeping or infection-related genes. In this study, we identified the ortholog of the human inhibitor of growth (ING1) gene in F. graminearum (FNG1) and found that it specifically interacts with the FgEsa1 HAT of the NuA4 complex. Deletion of FNG1 led to severe growth defects and blocked conidiation, sexual reproduction, DON production, and plant infection. The fng1 mutant was normal in H3 acetylation but significantly reduced in H4 acetylation. A total of 34 spontaneous suppressors of fng1 with faster growth rate were isolated. Most of them were still defective in sexual reproduction and plant infection. Thirty two of them had mutations in orthologs of yeast RPD3, SIN3, and SDS3, three key components of the yeast Rpd3L HDAC complex. Four mutations in these three genes were verified to suppress the defects of fng1 mutant in growth and H4 acetylation. The rest two suppressor strains had a frameshift or nonsense mutation in a glutamine-rich hypothetical protein that may be a novel component of the FgRpd3 HDAC complex in filamentous fungi. FgRpd3, like Fng1, localized in euchromatin. Deletion of FgRPD3 resulted in severe growth defects and elevated H4 acetylation. In contract, the Fgsds3 deletion mutant had only a minor reduction in growth rate but FgSIN3 appeared to be an essential gene. RNA-seq analysis revealed that 48.1% and 54.2% of the genes with altered expression levels in the fng1 mutant were recovered to normal expression levels in two suppressor strains with mutations in FgRPD3 and FgSDS3, respectively. Taken together, our data showed that Fng1 is important for H4 acetylation as a component of the NuA4 complex and functionally related to the FgRpd3 HDAC complex for transcriptional regulation of genes important for growth, conidiation, sexual reproduction, and plant infection in F. graminearum.

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Deletion of Prp4, the only kinase among spliceosome components, is not lethal in Fusarium graminearum but Fgprp4 mutants have severe growth defects and produced spontaneous suppressors. To identify novel suppressor mutations of Fgprp4, we sequenced the genome of suppressor S37 that was normal in growth but only partially recovered for intron splicing and identified a tandem duplication of 9-aa in the tri-snRNP component FgSNU66. Among the 19 additional suppressor strains found to have mutations in FgSNU66 (out of 260 screened), five had the same 9-aa duplication event with S37 and another five had the R477H/C mutation. The rest had nonsense or G-to-D mutations in the C-terminal 27-aa (CT27) region of FgSnu66, which is absent in its yeast ortholog. Truncation of this C-terminal region reduced the interaction of FgSnu66 with FgHub1 but increased its interaction with FgPrp8 and FgPrp6. Five phosphorylation sites were identified in FgSnu66 by phosphoproteomic analysis and the T418A-S420A-S422A mutation was shown to reduce virulence. Overall, our results showed that mutations in FgSNU66 can suppress deletion of Fgprp4, which has not been reported in other organisms, and the C-terminal tail of FgSnu66 plays a role in its interaction with key tri-snRNP components during spliceosome activation.

Summary The wheat head blight fungus Fusarium graminearum has two highly similar beta‐tubulin genes with overlapping functions during vegetative growth but only TUB1 is important for sexual reproduction. To better understand their functional divergence during ascosporogenesis, in this study we characterized the sequence elements important for stage‐specific functions of TUB1. Deletion of TUB1 blocked the late but not initial stages of perithecium formation. Perithecia formed by tub1 mutant had limited ascogenous hyphae and failed to develop asci. Silencing of TUB1 by MSUD also resulted in defects in ascospore formation. Interestingly, the 3′‐UTR of TUB1 was dispensable for growth but essential for its function during sexual reproduction. RIP mutations that specifically affected Tub1 functions during sexual reproduction also were identified in two ascospore progeny. Furthermore, site‐directed mutagenesis showed that whereas the non‐editable mutations at three A‐to‐I RNA editing sites had no effects, the N347D (not T362D or I368V) edited mutation affected ascospore development. In addition, the F167Y, but not E198K or F200Y, mutation in TUB1 conferred tolerance to carbendazim and caused a minor defect in sexual reproduction. Taken together, our data indicate TUB1 plays an essential role in ascosporogenesis and sexual‐specific functions of TUB1 require stage‐specific RNA processing and Tub1 expression.