The role of autophagy in necrotrophic fungal physiology and infection biology is poorly understood. We have studied autophagy in the necrotrophic plant pathogen Fusarium graminearum in relation to development of nonassimilating structures and infection. We identified an ATG8 homolog F. graminearum ATG8 whose first 116 amino acids before the predicted ATG4 cleavage site are 100% identical to Podospora anserina ATG8. We generated a ΔFgatg8 mutant by gene replacement and showed that this cannot form autophagic compartments. The strain forms no perithecia, has reduced conidia production and the aerial mycelium collapses after a few days in culture. The collapsing aerial mycelium contains lipid droplets indicative of nitrogen starvation and/or an inability to use storage lipids. The capacity to use carbon/energy stored in lipid droplets after a shift from carbon rich conditions to carbon starvation is severely inhibited in the ΔFgatg8 strain demonstrating autophagy-dependent lipid utilization, lipophagy, in fungi. Radial growth rate of the ΔFgatg8 strain is reduced compared with the wild type and the mutant does not grow over inert plastic surfaces in contrast to the wild type. The ability to infect barley and wheat is normal but the mutant is unable to spread from spikelet to spikelet in wheat. Complementation by inserting the F. graminearum atg8 gene into a region adjacent to the actin gene in ΔFgatg8 fully restores the WT phenotype. The results showed that autophagy plays a pivotal role for supplying nutrients to nonassimilating structures necessary for growth and is important for plant colonization. This also indicates that autophagy is a central mechanism for fungal adaptation to nonoptimal C/N ratios.

Redox sensing is of primary importance for fungi to cope with oxidant compounds found in their environment. Plant pathogens are particularly subject to the oxidative burst during the primary steps of infection. In the budding yeast Saccharomyces cerevisiae, it is the transcription factor Yap1 that mediates the response to oxidative stress via activation of genes coding for detoxification enzymes. In the cereal pathogen Fusarium graminearum, Fgap1 a homologue of Yap1 was identified and its role was investigated. During infection, this pathogen produces mycotoxins belonging to the trichothecenes family that accumulate in the grains. The global regulation of toxin biosynthesis is not completely understood. However, it is now clearly established that an oxidative stress activates the production of toxins by F. graminearum. The involvement of Fgap1 in this activation was investigated. A deleted mutant and a strain expressing a truncated constitutive form of Fgap1 were constructed. None of the mutants was affected in pathogenicity. The deleted mutant showed higher level of trichothecenes production associated with overexpression of Tri genes. Moreover activation of toxin accumulation in response to oxidative stress was no longer observed. Regarding the mutant with the truncated constitutive form of Fgap1, toxin production was strongly reduced. Expression of oxidative stress response genes was not activated in the deleted mutant and expression of the gene encoding the mitochondrial superoxide dismutase MnSOD1 was up-regulated in the mutant with the truncated constitutive form of Fgap1. Our results demonstrate that Fgap1 plays a key role in the link between oxidative stress response and F. graminearum secondary metabolism.

Fusarium graminearum is a necrotrophic plant pathogen of cereals that produces mycotoxins such as deoxynivalenol (DON) and zearalenone (ZEA) in grains. The stress-activated mitogen-activated protein kinase FgOS-2 is a central regulator in F. graminearum and controls, among others, virulence and DON and ZEA production. Here, we characterized the ATF/CREB-activating transcription factor FgAtf1, a regulator that functions downstream of FgOS-2. We created deletion and overexpression mutants of Fgatf1, the latter being also in an FgOS-2 deletion mutant. FgAtf1 localizes to the nucleus and appears to interact with FgOS-2 under osmotic stress conditions. Deletion mutants in Fgatf1 (ΔFgatf1) are more sensitive to osmotic stress and less sensitive to oxidative stress compared with the wild type. Furthermore, sexual reproduction is delayed. ΔFgatf1 strains produced higher amounts of DON under in vitro induction conditions than that of the wild type. However, during wheat infection, DON production by ΔFgatf1 is strongly reduced. The ΔFgatf1 strains displayed strongly reduced virulence to wheat and maize. Interestingly, constitutive expression of Fgatf1 in the wild type led to hypervirulence on wheat, maize, and Brachypodium distachyon. Moreover, constitutive expression of Fgatf1 in the ΔFgOS-2 mutant background almost complements ΔFgOS-2-phenotypes. These data suggest that FgAtf1 may be the most important transcription factor regulated by FgOS-2.

Fusarium graminearum is one of the most destructive pathogens of cereals and a threat to food and feed production worldwide. It is an ascomycetous plant pathogen and the causal agent of Fusarium head blight disease in small grain cereals and of cob rot disease in maize. Infection with F. graminearum leads to yield losses and mycotoxin contamination. Zearalenone (ZEA) and deoxynivalenol (DON) are hazardous mycotoxins; the latter is necessary for virulence toward wheat. Deletion mutants of the F. graminearum orthologue of the Saccharomyces cerevisiae Hog1 stress-activated protein kinase, FgOS-2 (ΔFgOS-2), showed drastically reduced in planta DON and ZEA production. However, ΔFgOS-2 produced even more DON than the wild type under in vitro conditions, whereas ZEA production was similar to that of the wild type. These deletion strains are dramatically reduced in pathogenicity toward maize and wheat. We constitutively expressed the fluorescent protein dsRed in the deletion strains and the wild type. Microscopic analysis revealed that ΔFgOS-2 is unable to reach the rachis node at the base of wheat spikelets. During vegetative growth, ΔFgOS-2 strains exhibit increased resistance against the phenylpyrrole fludioxonil. Growth of mutant colonies on agar plates supplemented with NaCl is reduced but conidia formation remained unchanged. However, germination of mutant conidia on osmotic media is severely impaired. Germ tubes are swollen and contain multiple nuclei. The deletion mutants completely fail to produce perithecia and ascospores. Furthermore, FgOS-2 also plays a role in reactive oxygen species (ROS)-related signaling. The transcription and activity of fungal catalases is modulated by FgOS-2. Among the genes regulated by FgOS-2, we found a putative calcium-dependent NADPH-oxidase (noxC) and the transcriptional regulator of ROS metabolism, atf1. The present study describes new aspects of stress-activated protein kinase signaling in F. graminearum.