Functional characterization of BbEaf6 in Beauveria bassiana: Implications for fungal virulence and stress response.

The Eaf6 protein, a conserved component of the NuA4 and NuA3 complexes in yeast and MOZ/MORF complexes in humans, plays crucial roles in transcriptional activation, gene regulation, and cell cycle control. Despite its significance in other organisms, the functional role of Eaf6 in entomopathogenic fungi (EPF) remained unexplored. Here, we investigate the function of BbEaf6, the Eaf6 homolog in the entomopathogenic fungus Beauveria bassiana. We demonstrate that BbEaf6 is predominantly localized in nuclei, similar to its counterpart in other fungi. Deletion of BbEaf6 resulted in delayed conidiation, reduced conidial yield, and altered conidial properties. Transcriptomic analysis revealed dysregulation of the genes involved in asexual development and cell cycle progression in the ΔBbEaf6 mutant. Furthermore, the ΔBbEaf6 mutant exhibited decreased tolerance to various stresses, including ionic stress, cell wall perturbation, and DNA damage stress. Notably, the ΔBbEaf6 mutant displayed attenuated virulence in insect bioassays, accompanied by dysregulation of genes associated with cuticle penetration and haemocoel infection. Overall, our study elucidates the multifaceted role of BbEaf6 in stress response, development, and virulence in B. bassiana, providing valuable insights into the molecular mechanisms governing fungal pathogenesis and potential targets for pest management strategies.

Two sirtuin proteins, Hst3 and Hst4, modulate asexual development, stress tolerance, and virulence by affecting global gene expression in Beauveria bassiana.

Beauveria bassiana is a widely used entomopathogenic fungus in insect biological control applications. In this study, we investigated the role of two sirtuin homologs, BbHst3 and BbHst4, in the biological activities and pathogenicity of B. bassiana. Our results showed that deletion of BbHst3 and/or BbHst4 led to impaired sporulation, reduced (~50%) conidial production, and decreased tolerance to various stresses, including osmotic, oxidative, and cell wall-disturbing agents. Moreover, BbHst4 plays dominant roles in histone H3-K56 acetylation and DNA damage response, while BbHst3 is more responsible for maintaining cell wall integrity. Transcriptomic analyses revealed significant changes (>1,500 differentially expressed genes) in gene expression patterns in the mutant strains, particularly in genes related to secondary metabolism, detoxification, and transporters. Furthermore, the ΔBbHst3, ΔBbHst4, and ΔBbHst3ΔBbHst4 strains exhibited reduced virulence in insect bioassays, with decreased (~20%) abilities to kill insect hosts through topical application and intra-hemocoel injection. These findings highlight the crucial role of BbHst3 and BbHst4 in sporulation, DNA damage repair, cell wall integrity, and fungal infection in B. bassiana. Our study provides new insights into the regulatory mechanisms underlying the biological activities and pathogenicity of B. bassiana and emphasizes the potential of targeting sirtuins for improving the efficacy of fungal biocontrol agents.IMPORTANCESirtuins, as a class of histone deacetylases, have been shown to play important roles in various cellular processes in fungi, including asexual development, stress response, and pathogenicity. By investigating the functions of BbHst3 and BbHst4, we have uncovered their critical contributions to important phenotypes in Beauveria bassiana. Deletion of these sirtuin homologs led to reduced conidial yield, increased sensitivity to osmotic and oxidative stresses, impaired DNA damage repair processes, and decreased fungal virulence. Transcriptomic analyses showed differential expression of numerous genes involved in secondary metabolism, detoxification, transporters, and virulence-related factors, potentially uncovering new targets for manipulation and optimization of fungal biocontrol agents. Our study also emphasizes the significance of sirtuins as key regulators in fungal biology and highlights their potential as promising targets for the development of novel antifungal strategies.

Contributions of a Histone Deacetylase (SirT2/Hst2) to Beauveria bassiana Growth, Development, and Virulence.

Sirtuins are a class of histone deacetylases that promote heterochromatin formation to repress transcription. The entomopathogenic fungus Beauveria bassiana contains six sirtuin homologs. The class III histone deacetylase, BbSir2, has been previously shown to affect the regulation of carbon/nitrogen metabolism and asexual development, with only moderate effects on virulence. Here, we examine another class III histone deacetylase (BbSirT2) and show that it contributes to deacetylation of lysine residues on histone H4-K16ac. Directed gene-knockout of BbSirT2 dramatically reduced conidiation, the ability of the fungus to metabolize a range of carbon and nitrogen sources, and tolerances to oxidative, heat, and UV stress and significantly attenuated virulence in both intrahemocoel injection and topical bioassays using the Greater wax moth (Galleria mellonella) as the insect host. ΔBbSirT2 cells showed alterations in cell cycle development and hyphal septation and produced morphologically aberrant conidia. Comparative transcriptomic analyses of wild type versus ΔBbSirT2 cells indicated differential expression of 1148 genes. Differentially expressed genes were enriched in pathways involved in cell cycle and rescue, carbon/nitrogen metabolism, and pathogenesis. These included changes in the expression of polyketide synthases (PKSs) and LysM effector proteins that contribute to degradation of host toxins and target host pathways, respectively. These data indicate contributions of BbSirT2 in helping to mediate fungal stress and development, with the identification of affected gene targets that can help account for the observed reduced virulence phenotype.

The Spt10 GNAT Superfamily Protein Modulates Development, Cell Cycle Progression and Virulence in the Fungal Insect Pathogen, Beauveria bassiana.

Chromatin remodeling is mediated in part by post-translational acetylation/deacetylation modifications of histones. Histone acetyltransferases (HATs), e.g., members of the GNAT/MYST superfamily, activate gene transcription via promotion of euchromatin formation. Here, we characterized a GNAT family HAT, Spt10 (BbSpt10), in the environmentally and economically important fungal insect pathogen, Beauveria bassiana. Targeted gene knockout of BbSpt10 resulted in impaired asexual development and morphogenesis; reduced abilities to utilize various carbon/nitrogen sources; reduced tolerance to heat, fungicides, and DNA damage stress; and attenuated virulence. The ΔBbSpt10 mutant showed disrupted cell cycle development and abnormal hyphal septation patterns. Transcriptome analyses of wild type and ΔBbSpt10 cells revealed the differential expression of 373 genes, including 153 downregulated and 220 upregulated genes. Bioinformatic analyses revealed downregulated genes to be enriched in pathways involved in amino acid metabolism, cellular transportation, cell type differentiation, and virulence, while upregulated genes were enriched in carbon/nitrogen metabolism, lipid metabolism, DNA process, and cell rescue, defense, and virulence. Downregulated virulence genes included hydrophobins, cellular transporters (ABC and MFS multidrug transporters) and cytochrome P450 detoxification genes. These data indicated broad effects of BbSpt10 on fungal development, multi-stress response, and virulence.

Rice false smut fungus hijacks the rice nutrients supply by blocking and mimicking the fertilization of rice ovary.

Rice false smut disease is an increasing threat to rice production in the world. Despite of best efforts, research for the infection of the fungus has yielded equivocal and conflicting results about where and how the infection is initiated and developed. Here we show a stepwise infection pattern and sophisticated regulation during this process. Initial infection occurred on the filaments, which prevented the production of mature pollen thus blocked the pollination. In the following days, the pathogen invaded the stigmas and styles, occasionally the ovaries. Expression analysis indicated that the fungus mimicked a successful fertilization process and enabled the continuous supply of nutrients for fungus to produce false smut balls. The stepwise infection of flower organs and mimicry of ovary fertilization unveiled in this study guided the rice plant into supplying nutrients for false smut ball development and represents a new and unique biological process of host pathogen interactions.

Exploring mechanisms of resistance to dimethachlone in Sclerotinia sclerotiorum.

BACKGROUND: The dicarboximide fungicide dimethachlone has been widely used in China for more than 12 years to control the Sclerotinia stem rot caused by Sclerotinia sclerotiorum disease. First signs of resistance in the field are reported at low frequency. In this study, four resistant isolate/mutants were used to explore still unknown mechanisms leading to dimethachlone resistance. RESULTS: The resistant isolate/mutants had significantly higher EC50 values compared with the sensitive control isolates. Cross-resistance was confirmed between dimethachlone and procymidone, iprodione and fludioxonil. The resistant isolate/mutants revealed a decreased mycelial growth rate, were less pathogenic on leaves of oilseed rape, were more sensitive to osmotic pressure and oxidative stress and released more electrolytes compared with the sensitive isolates. Only in one lab mutant did we find a point mutation (V238A) in the SsOs1 gene of the high-osmolarity glycerol (HOG) signalling pathway. The expression of this gene was lost in the field resistant isolate HN456-1-JBJ and decreased in mycelium that was subjected to either high osmotic pressure or dimethachlone; however, another key gene in the HOG pathway, SsHog1, could be induced in the resistant isolate and mutants with NaCl treatment. CONCLUSION: This study demonstrates that resistance to dicarboximide fungicide dimethachlone in S. sclerotiorum is emerging in China. Several fitness parameters, including mycelial growth rate, sclerotia formed in vitro, aggressiveness on leaves and osmotic and H2 O2 sensitivity, indicate that the resistant strains may not effectively compete with sensitive isolates in the field in the absence of selection pressure. Lost expression or the V238A point mutation in the SsOs1 gene may confer resistance to dicarboximide fungicide dimethachlone in S. sclerotiorum, but this study illustrates that other, yet unknown mechanisms also exist.

Degradation of oxalic acid by the mycoparasite Coniothyrium minitans plays an important role in interacting with Sclerotinia sclerotiorum.

Coniothyrium minitans (Cm) is a mycoparasite of the phytopathogenic fungus Sclerotinia sclerotiorum (Ss). Ss produces a virulence factor oxalic acid (OA) which is toxic to plants and also to Cm, and Cm detoxifies OA by degradation. In this study, two oxalate decarboxylase genes, Cmoxdc1 and Cmoxdc2, were cloned from Cm strain Chy-1. OA and low pH induced expression of Cmoxdc1, but not Cmoxdc2. Cmoxdc1 was partially responsible for OA degradation, whereas Cmoxdc2 had no effect on OA degradation. Disruption of Cmoxdc1 in Cm reduced its ability to infect Ss in dual cultures where OA accumulated. Compared with Chy-1, the Cmoxdc1-disrupted mutants had reduced expression levels of two mycoparasitism-related genes chitinase (Cmch1) and ß-1,3-glucanase (Cmg1), and had no detectable activity of extracellular proteases in the presence of OA. On the other hand, the cultural filtrates of the Cmoxdc1-disrupted mutants in OA-amended media showed enhanced antifungal activity, possibly because of increased production of antifungal substances under acidic pH condition resulted from reduced Cmoxdc1-mediated OA degradation. This study provides direct genetic evidence of OA degradation regulating mycoparasitism and antibiosis of Cm against Ss, and sheds light on the sophisticated strategies of Cm in interacting with metabolically active mycelia and dormant sclerotia of Ss.

DsRNA-free transmissible hypovirulence associated with formation of intra-hyphal hyphae in Botrytis cinerea.

A spontaneous mutant CanBc-3HV and its parental strain CanBc-3 of Botrytis cinerea were investigated in terms of pathogenicity, colony morphology, hypovirulence transmissibility, presence of double-stranded RNA (dsRNA), and formation of intra-hyphal hyphae (IH). Results showed that inoculation of CanBc-3HV on detached leaves of Brassica napus did not produce any visible necrotic lesions (20°C, 72h), whereas inoculation of CanBc-3 caused necrotic leaf lesions. Compared to CanBc-3, CanBc-3HV grew slowly, formed numerous mycelial sectors, sporulated sporadically and failed to produce sclerotia on potato dextrose agar (PDA) (20°C, 15d). Hypovirulence and the abnormal cultural characteristics of CanBc-3HV were transmissible from CanBc-3HV to CanBc-3 in pair cultures on PDA. However, the transmission was unsuccessful from CanBc-3HV to another virulent strain CanBc-2 of B. cinerea. These results suggest that transmission of the hypovirulence and the abnormal cultural characteristics of CanBc-3HV are strain-specific. No dsRNA was detected in mycelia of either CanBc-3HV or CanBc-3, implying that the hypovirulence of CanBc-3HV is caused by a transmissible element (TE) of non-RNA mycoviral origin. Formation of IH through self-infection was observed in CanBc-3HV, CanBc-3T1 (a hypovirulent derivative of CanBc-3 trans-infected by TE in CanBc-3HV), but was not observed in CanBc-3, suggesting that IH formation is associated with the hypovirulence of CanBc-3HV. To our knowledge, this is the first report of dsRNA-free transmissible hypovirulence associated with IH formation in B. cinerea.

Botrytis fabiopsis, a new species causing chocolate spot of broad bean in central China.

The current study was conducted to identify Botrytis spp. isolated from symptomatic broad bean plants grown in Hubei Province, China. Among 184 Botrytis strains, three distinct species, B. cinerea, B. fabae and a previously undescribed Botrytis sp., were identified based on morphology of colonies, sclerotia and conidia. The novel Botrytis sp. is described herein as a new species, Botrytis fabiopsis sp. nov. At 20 C B. fabiopsis grew on potato dextrose agar (PDA) at 12-13 mm d(-1), similar to B. fabae (13 mm d(-1)), but slower than B. cinerea (17-19 mm d(-1)). It formed pale gray colonies with short aerial mycelia and produced gray to black sclerotia in concentric rings on PDA. B. fabiopsis produced greater numbers of sclerotia than B. cinerea but fewer than B. fabae. Conidia produced by B. fabiopsis on broad bean leaves are hyaline to pale brown, elliptical to ovoid, wrinkled on the surface and are larger than conidia of B. fabae and B. cinerea. Phylogenetic analysis based on combined DNA sequence data of three nuclear genes (G3PDH, HSP60 and RPB2) showed that B. fabiopsis is closely related to B. galanthina, the causal agent of gray mold disease of Galanthus sp., but distantly related to B. fabae and B. cinerea. Sequence analysis of genes encoding necrosis and ethylene-inducing proteins (NEPs) indicated that B. fabiopsis is distinct from B. galanthina. Inoculation of broad bean leaves with conidia of B. fabiopsis caused typical chocolate spot symptoms with a similar disease severity to that caused by B. fabae but significantly greater than that caused by B. cinerea. This study suggests that B. fabiopsis is a new causal agent for chocolate spot of broad bean.