Three Small Cysteine-Free Proteins (CFP1-3) Are Required for Insect-Pathogenic Lifestyle of Metarhizium robertsii.

Unique CFP (cysteine-free protein; 120 aa) has been identified as an extraordinary virulence factor in Beauveria bassiana (Cordycipitaceae), a main source of wide-spectrum fungal insecticides. Its homologs exclusively exist in wide-spectrum insect pathogens of Hypocreales, suggesting their importance for a fungal insect-pathogenic lifestyle. In this study, all three CFP homologs (CFP1-3, 128-145 aa) were proven essential virulence factors in Metarhizium robertsii (Clavicipitaceae). Despite limited effects on asexual cycles in vitro, knockout mutants of cfp1,cfp2 and cfp3 were severely compromised in their capability for normal cuticle infection, in which most tested Galleria mellonella larvae survived. The blocked cuticle infection concurred with reduced secretion of extracellular enzymes, including Pr1 proteases required cuticle penetration. Cuticle-bypassing infection by intrahemocoel injection of ~250 conidia per larva resulted in a greater reduction in virulence in the mutant of cfp1 (82%) than of cfp2 (21%) or cfp3 (25%) versus the parental wild-type. Transcriptomic analysis revealed dysregulation of 604 genes (up/down ratio: 251:353) in the Δcfp1 mutant. Many of them were involved in virulence-related cellular processes and events aside from 154 functionally unknown genes (up/down ratio: 56:98). These results reinforce the essential roles of small CFP homologs in hypocrealean fungal adaptation to insect-pathogenic lifestyle and their exploitability for the genetic improvement of fungal insecticidal activity.

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

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 cuticle-degrading 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. © 2022 Society of Chemical Industry.

Genome-Wide Insight into Profound Effect of Carbon Catabolite Repressor (Cre1) on the Insect-Pathogenic Lifecycle of Beauveriabassiana.

Carbon catabolite repression (CCR) is critical for the preferential utilization of glucose derived from environmental carbon sources and regulated by carbon catabolite repressor A (Cre1/CreA) in filamentous fungi. However, a role of Cre1-mediated CCR in insect-pathogenic fungal utilization of host nutrients during normal cuticle infection (NCI) and hemocoel colonization remains explored insufficiently. Here, we report an indispensability of Cre1 for Beauveriabassiana's utilization of nutrients in insect integument and hemocoel. Deletion of cre1 resulted in severe defects in radial growth on various media, hypersensitivity to oxidative stress, abolished pathogenicity via NCI or intrahemocoel injection (cuticle-bypassing infection) but no change in conidial hydrophobicity and adherence to insect cuticle. Markedly reduced biomass accumulation in the Δcre1 cultures was directly causative of severe defect in aerial conidiation and reduced secretion of various cuticle-degrading enzymes. The majority (1117) of 1881 dysregulated genes identified from the Δcre1 versus wild-type cultures were significantly downregulated, leading to substantial repression of many enriched function terms and pathways, particularly those involved in carbon and nitrogen metabolisms, cuticle degradation, antioxidant response, cellular transport and homeostasis, and direct/indirect gene mediation. These findings offer a novel insight into profound effect of Cre1 on the insect-pathogenic lifestyle of B. bassiana.

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

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.

P-type Na+/K+ ATPases essential and nonessential for cellular homeostasis and insect pathogenicity of Beauveria bassiana.

ENA1 and ENA2 are P-type IID/ENA Na+/K+-ATPases required for cellular homeostasis in yeasts but remain poorly understood in filamentous fungal insect pathogens. Here, we characterized seven genes encoding five ENA1/2 homologues (ENA1a-c and ENA2a/b) and two P-type IIC/NK Na+/K+-ATPases (NK1/2) in Beauveria bassiana, an insect-pathogenic fungus serving as a main source of fungal insecticides worldwide. Most of these genes were highly responsive to alkaline pH and Na+/K+ cues at transcription level. Cellular Na+, K+ and H+ homeostasis was disturbed only in the absence of ena1a or ena2b. The disturbed homeostasis featured acceleration of vacuolar acidification, elevation of cytosolic Na+/K+ level at pH 5.0 to 9.0, and stabilization of extracellular H+ level to initial pH 7.5 during a 5-day period of submerged incubation. Despite little defect in hyphal growth and asexual development, the Δena1a and Δena2b mutants were less tolerant to metal cations (Na+, K+, Li+, Zn2+, Mn2+ and Fe3+), cell wall perturbation, oxidation, non-cation hyperosmolarity and UVB irradiation, severely compromised in insect pathogenicity via normal cuticle infection, and attenuated in virulence via hemocoel injection. The deletion mutants of five other ENA and NK genes showed little change in vacuolar pH and all examined phenotypes. Therefore, only ENA1a and ENA2b evidently involved in both transmembrane and vacuolar activities are essential for cellular cation homeostasis, insect pathogenicity and multiple stress tolerance in B. bassiana. These findings provide a novel insight into ENA1a- and ENA2b-dependent vacuolar pH stability, cation-homeostatic process and fungal fitness to host insect and environment.

Identification of Genes Relevant to Pesticides and Biology from Global Transcriptome Data of Monochamus alternatus Hope (Coleoptera: Cerambycidae) Larvae.

Monochamus alternatus Hope is the main vector in China of the Pine Wilt Disease caused by the pine wood nematode Bursaphelenchus xylophilus. Although chemical control is traditionally used to prevent pine wilt disease, new strategies based in biological control are promising ways for the management of the disease. However, there is no deep sequence analysis of Monochamus alternatus Hope that describes the transcriptome and no information is available about gene function of this insect vector. We used next generation sequencing technology to sequence the whole fourth instar larva transcriptome of Monochamus alternatus Hope and successfully built a Monochamus alternatus Hope transcriptome database. In total, 105,612 unigenes were assigned for Gene Ontology (GO) terms, information for 16,730 classified unigenes was obtained in the Clusters of Orthologous Groups (COGs) database, and 13,024 unigenes matched with 224 predicted pathways in the Kyoto Encyclopedia of Genes and Genome (KEGG). In addition, genes related to putative insecticide resistance-related genes, RNAi, the Bt receptor, intestinal digestive enzymes, possible future insect control targets and immune-related molecules are described. This study provides valuable basic information that can be used as a gateway to develop new molecular tools for Monochamus alternatus Hope control strategies.