Nuclear Ssr4 Is Required for the In Vitro and In Vivo Asexual Cycles and Global Gene Activity of Beauveria bassiana

et al. 2021

mSystems

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Small secreted proteins (SSPs), particularly cysteine-rich proteins secreted during fungal infection, comprise virulence effectors in plant-pathogenic fungi but remain unknown in insect-pathogenic fungi. We report here that only a small cysteine-free protein (CFP) is indispensable for insect pathogenicity of Beauveria bassiana among 10 studied SSPs (99 to 274 amino acids [aa]), including seven hypothetical proteins containing 0 to 12 Cys residues. CFP (120 aa) features an N-terminal signal peptide (residues 1 to 17), a nuclear localization signal motif (residues 24 to 57), and no predictable domain. Its homologs exist exclusively in insect-pathogenic Cordycipitaceae and Clavicipitaceae. Fluorescence-tagged CFP fusion protein was localized in the nucleus but extracellularly undetectable, suggesting an inability for CFP to be secreted out. Disruption of cfp resulted in abolished pathogenicity via normal cuticle infection, attenuated virulence via hemocoel injection, compromised conidiation capacity versus little growth defect, impaired conidial coat, blocked secretion of cuticle-degrading enzymes, impeded proliferation in vivo, disturbed cell cycle, reduced stress tolerance, and 1,818 dysregulated genes (genomic 17.54%). Hundreds of those genes correlated with phenotypic changes observed in the disruption mutant. Intriguingly, nearly 40% of those dysregulated genes encode hypothetical or unknown proteins, and another 13% encode transcription factors and enzymes or proteins collectively involved in genome-wide gene regulation. However, purified CFP showed no DNA-binding activity in an electrophoretic mobility shift assay. These findings unveil that CFP is a novel regulator of fungal insect-pathogenic life cycle and genomic expression and that cysteine richness is dispensable for distinguishing virulence effectors from putative SSPs in B. bassiana. IMPORTANCE Small cysteine-rich proteins secreted during plant-pathogenic fungal infection comprise virulence effectors. Our study confirms that only a cysteine-free protein (CFP) is determinant to insect-pathogenic fungal virulence among 10 small putatively secreted proteins containing 0 to 12 Cys residues. Disruption of cfp abolished insect pathogenicity and caused not only a series of compromised cellular events associated with host infection and disease development but also dysregulation of 1,818 genes, although no DNA-binding activity was detected in purified CFP samples. Nearly 13% of those genes encode transcription factors and enzymes or proteins collectively involved in transcriptional regulation. Altogether, CFP serves as a novel regulator of the fungal insect-pathogenic life cycle and genomic expression. Cysteine richness is dispensable for distinguishing virulence effectors from the fungal SSPs.

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A Small Cysteine-Free Protein Acts as a Novel Regulator of Fungal Insect-Pathogenic Lifecycle and Genomic Expression

et al. 2021

mSystems

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“…Meanwhile, biomass levels were measured from cellophane-overlaid SDAY cultures every 2 days from day 4 onward. Hydrophobic/hydro- philic features of such cultures were observed by dropping 10 μL aliquots of 0.02% Tween 80 onto the surfaces of 4-day-old SDAY cultures and monitoring the dispersal of water droplets for 72 h at 25 °C, as described previously [ 50 ]. In addition, the total activities of superoxide dismutases (SODs) and catalases (U/mg) were assayedfrom the protein extracts of 3-day-old SDAY cultures with an SOD Activity Assay Kit (Sigma, Shanghai, China) and a Catalase Activity Assays Kit (Jiancheng Biotech, Nanjing, China) following the manufacturers’ guides, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…The quality of conidia from the 9-day-old SDAY cultures was assessed using the indices of median germination time (GT 50 , h) at 25 °C, the median lethal dose (LD 50 , J/cm 2 ) for UVB (weighted wavelength: 312 nm) resistance and hydrophobicity in an aqueous/ organic system, and examined with scanning electronic microscopy (SEM) for microstructural features of their surfaces, as described elsewhere [ 42 , 50 ].…”

Section: Methodsmentioning

confidence: 99%

Essential Role of COP9 Signalosome Subunit 5 (Csn5) in Insect Pathogenicity and Asexual Development of Beauveria bassiana

Tong

et al. 2021

JoF

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Csn5 is a subunit ofthe COP9/signalosome complex in model fungi. Here, we report heavier accumulation of orthologous Csn5 in the nucleus than in the cytoplasm and its indispensability to insect pathogenicity and virulence-related cellular events of Beauveria bassiana. Deletion of csn5 led to a 68% increase in intracellular ubiquitin accumulation and the dysregulation of 18 genes encoding ubiquitin-activating (E1), -conjugating (E2), and -ligating (E3) enzymes and ubiquitin-specific proteases, suggesting the role of Csn5 in balanced ubiquitination/deubiquitination. Consequently, the deletion mutant displayed abolished insect pathogenicity, marked reductions in conidial hydrophobicity and adherence to the insect cuticle, the abolished secretion of cuticle penetration-required enzymes, blocked haemocoel colonisation, and reduced conidiation capacity despite unaffected biomass accumulation. These phenotypes correlated well with sharply repressed or abolished expressions of key hydrophobin genes required for hydrophobin biosynthesis/assembly and of developmental activator genes essential for aerial conidiation and submerged blastospore production. In the mutant, increased sensitivities to heat shock and oxidative stress also correlated with reduced expression levels of several heat-responsive genes and decreased activities of antioxidant enzymes. Altogether, Csn5-reliant ubiquitination/deubiquitination balance coordinates the expression of those crucial genes and the quality control of functionally important enzymes, which are collectively essential for fungal pathogenicity, virulence-related cellular events, and asexual development.

Fungal insecticidal activity elevated by non‐risky markerless overexpression of an endogenous cysteine‐free protein gene in Beauveria bassiana

Pest Management Science

Tong

et al. 2022

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BACKGROUND: Fungal insecticides are notorious for slow kill action, an intrinsic trait that can be improved by the genetic engineering of an exogenous or endogenous virulence factor. However, transgenic insecticides expressing exogenous toxin and herbicide-resistant marker genes may cause unexpected ecological risks and are hardly permitted for field release due to strict regulatory hurdles. It is necessary to improve biotechnology that can speed up fungal insect-killing action and exclude ecological risk source.RESULTS: A markerless transformation system of Beauveria bassiana, a main source of wide-spectrum fungal insecticides, was reconstructed based on the fungal uridine auxotrophy (Δura3). The system was applied for overexpression of the small cysteine-free protein (120 amino acids) gene cfp previously characterized as a regulator of the fungal virulence and gene expression. Three cfp-overexpressed strains showed much faster kill action to Galleria mellonella larvae than the parental wild-type via normal cuticle infection but no change in vegetative growth and aerial condition. The faster kill action was achieved due to not only significant increases in conidial adherence to insect cuticle and total activity of secreted cuticledegrading Pr1 proteases and of antioxidant enzymes crucial for collapse of insect immune defense but acceleration of hemocoel localization, proliferation in vivo and host death from mummification.CONCLUSION: The markerless system is free of any foreign DNA fragment as a source of ecologic risk and provides a novel biotechnological approach to enhancing fungal insecticidal activity with non-risky endogenous genes and striding over the regulatory hurdles.