Manipulation of host ecdysteroid hormone levels facilitates infection by the fungal insect pathogen, Metarhizium rileyi.

Entomopathogenic fungi such as Metarhizium rileyi and Beauveria bassiana are widely used insect biological control agents. Little, however, is known concerning genetic or enzymatic factors that differentiate the mechanisms employed by these two fungal pathogens to infect target hosts. Infection by either of these organisms is known to increase levels of the growth and molting hormone, ecdysone, which also regulates the expression of a number of innate immune pathways. M. rileyi, but not B. bassiana, has apparently evolved an ecdysteroid-22-oxidase (MrE22O) that inactivate ecdysone. We show that deletion of MrE22O impaired virulence compared with the wild-type strain, with an increase in ecdysone titer seen in hosts that was coupled to an increase in the expression of antimicrobial genes. An M. rileyi strain engineered to overexpress MrE22O (MrE22OOE ), as well as trans-expression in B. bassiana (Bb::MrE220OE ) resulted, in strains displaying enhanced virulence and dampening of host immune responses compared with their respective wild-type parental strains. These results indicate that ecdysone plays an important role in mediating responses to fungal infection and that some insect pathogenic fungi have evolved mechanisms for targeting this hormone as a means for facilitating infection.

A polyketide synthase, BbpksP, contributes to conidial cell wall structure and UV tolerance in Beauveria bassiana.

Conidial pigments of filamentous fungi play vital roles in fungal biotic/abiotic stress tolerance and are usually synthesized by polyketide synthases or other pigment synthesis proteins. Beauveria bassiana, an important insect pathogenic fungus used worldwide for pest biocontrol, produces white conidia on artificial media, while no conidial pigment has been observed or reported in it. However, real-time PCR and promoter-report analyses reveal a polyketide gene of B. bassiana (named BbpksP), homologous to melanin synthesis genes, is specifically expressed in aerial conidia. We show that deletion of BbpksP does not result in changes in conidial yield, germination rate or colony radial growth; however, the defect impairs conidial cell wall structure. A dense electron layer appears in the outer edge of the cell envelope in wild-type conidia, as observed by TEM, but this dense layer is absent in the ΔBbpksP mutant. The lack of BbpksP gene also reduces the UV-B tolerance of B. bassiana conidia. Bioassay reveals that deletion of BbpksP decreased virulence of B. bassiana against Galleria mellonella larvae via topical infection. These data indicate that the product(s) of BbpksP contributes to the integrity of the B. bassiana conidial cell wall and further affects the tolerance of UV-B stress and insecticidal activity.

Rhizobium hedysari sp. nov., a novel species isolated from a root nodule of Hedysarum multijugum in China.

A strain 5-1-2T was isolated from a root nodule of Hedysarum multijugum collected from Zhangye city, Gansu province, north-west China. Phylogenetic analysis based on the 16S rRNA gene sequence and other housekeeping genes (recA and atpD) indicated that the strain represents a novel species in the genus Rhizobium close to the strain Rhizobium subbaraonis JC85T with similarities of 98.27, 88.92 and 89.62%, respectively. Strain 5-1-2T contained Q-10 as the predominant ubiquinone. Our results showed that the major fatty acids were feature 8 (C18:1 ω7c and/or C18:1 ω6c; 38.90%). In addition, the DNA-DNA hybridizations with the type strains R. subbaraonis JC85T and Rhizobium halophytocola YC6881T were 39.2 ± 2.1 and 44.3 ± 1.9, respectively. Therefore, a novel species Rhizobium hedysari sp. nov. is proposed, and 5-1-2T (=CGMCC1.15677T = NBRC112532T) is designated as the type strain.

An efficient visual screening of gene knockout mutants in the insect pathogenic fungus Beauveria bassiana.

Beauveria bassiana is an entomopathognic fungus, which is widely employed in the biological control of pests. Gene disruption is a common method for studying the functions of genes involved in fungal development or its interactions with hosts. However, generating gene deletion mutants was a time-consuming work. The transcriptional factor OpS3 has been identified as a positive regulator of a red secondary metabolite oosporein in B. bassiana. In this study, we have designed a new screening system by integrating a constitutive OpS3 expression cassette outside one of the homologous arms of target gene. Ectopic transformants predominantly exhibit a red colour with oosporein production, while knockout mutants appear as white colonies due to the loss of the OpS3 expression cassette caused by recombinant events. This screening strategy was used to obtain the deletion mutants of both tenS and NRPS genes. Correct mutants were obtained by screening fewer than 10 mutants with a positive efficiency ranging from 50% to 75%. This system significantly reduces the workload associated with DNA extraction and PCR amplification, thereby enhancing the efficiency of obtaining correct transformants in B. bassiana.

Expression of a viral ecdysteroid UDP-glucosyltransferase enhanced the insecticidal activity of the insect pathogenic fungus Beauveria bassiana.

BACKGROUND: Entomopathogenic fungi, such as Beauveria bassiana, hold promise as biological control agents against insect pests. However, the efficacy of these fungi can be hindered by insect immune responses. One strategy to enhance fungal virulence is to manipulate host immune by targeting key regulatory molecules like 20-hydroxyecdysone (20E). RESULTS: In this study, we engineered B. bassiana strains to constitutively express the enzyme ecdysteroid UDP-glucosyltransferase (EGT), which inactivates 20E, a crucial insect molting hormone. The engineered strain Bb::EGT-1 exhibited robust expression of EGT, leading to a significant reduction in insect 20E levels upon infection. Moreover, infection with Bb::EGT-1 resulted in accelerated larval mortality. Immune responses analysis revealed repression of insect immune response genes and decreased phenoloxidase (PO) activity in larvae infected with Bb::EGT-1. Microbiome analysis indicated alterations in bacterial composition within infected insects, with increased abundance observed during infection with Bb::EGT-1. Additionally, the presence of bacteria hindered hyphal emergence from insect cadavers, suggesting a role for microbial competition in fungal dissemination. CONCLUSIONS: Constitutive expression of EGT in B. bassiana enhances fungal virulence by reducing insect 20E levels, suppressing immune responses, and altering the insect microbiome. These findings highlighted the potential of engineered fungi as effective biocontrol agents against insect pests and provide insights into the complex interactions between entomopathogenic fungi, their hosts, and associated microbes. © 2024 Society of Chemical Industry.

Multifaceted roles of NADPH oxidases in the growth and pathogenicity of Beauveria bassiana.

Reactive oxygen species (ROS), synthesized by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) complex, are vital molecules in biological cells, influencing various physiological processes such as fungal growth, development, and virulence. Beauveria bassiana, an entomopathogenic fungus, is a promising biopesticide for agricultural, forestry, and urban pest control. This study focuses on the characterization of NADPH oxidases (Noxs) in B. bassiana. Gene expression profiles of Noxs in B. bassiana (BbNoxs) were analysed using RT-qPCR. Knockout strains of single BbNoxA, BbNoxB, BbNoxR, and double BbNoxA and BbNoxB were constructed via homologous recombination, and their phenotypic characteristics were examined. Fungal virulence was evaluated using Galleria mellonella larvae, and infection structures formation and penetration ability were assessed on cicada wings. ROS production and actin assembly during fungal growth and infection were detected using staining and marker methods. Expression analysis revealed significant upregulation of BbNoxs during fungal growth and infection. Compared to the wild-type strain, single knockouts (ΔBbNoxA/B/R) and double knockout (ΔBbNoxAB) of BbNoxs exhibited reduced conidial yields, accelerated conidial germination rates. Deletion of BbNoxB or BbNoxR decreased fungal virulence compared to the WT strain in topical inoculation experiments. Additionally, loss of BbNoxB or BbNoxR impaired infection structures formation, penetration ability, ROS production, and actin aggregation during fungal infection. BbNoxs are crucial for fungal growth, development, and virulence in B. bassiana, playing essential roles in infection structures formation, penetration, ROS production, and actin assembly. Understanding their functions provides insights into B. bassiana's pathogenic mechanisms.

Transcription Factors BbPacC and Bbmsn2 Jointly Regulate Oosporein Production in Beauveria bassiana.

The entomopathogenic fungus Beauveria bassiana can produce the secondary metabolite oosporein under alkaline conditions or in fungus-killed cadavers. However, the regulatory mechanism of oosporein synthesis is not fully understood. In thisstudy, we found that the pH signaling transcription factor BbPacC is involved in the regulation of oosporein production. Overexpression of BbPacC promotes oosporein production in B. bassiana at pH 6.0 or under alkaline conditions (pH 8.0), but deletion of this gene abolished oosporein production. Under acidic conditions (pH 4.0), no oosporein production was observed in the wild-type and BbPacC overexpression strains. Yeast one-hybrid assays and electrophoretic mobility shift assay (EMSA) confirmed the binding ability of BbPacC with 4 putative PacC-binding sites in the promoter region of BbOpS3, a transcription factor located in the oosporein synthetic gene cluster regulating the expression of oosporein synthetic genes. Overexpression of Bbmsn2, a previously reported negative regulator of oosporein synthesis, in OEPacC or wild-type strains abolished oosporein production in all tested conditions. However, deletion of Bbmsn2 in the BbPacC overexpression strain significantly improved oosporein production even at pH 4.0. These results indicated that BbPacC is a positive regulator of oosporein production and functions jointly with Bbmsn2 to regulate oosporein production in different environments and particularly under alkaline conditions. IMPORTANCE B. bassiana produces the red dibenzoquinone pigment oosporein under certain specific conditions, such as alkaline conditions and fungus-killed cadavers. Ooporein possesses antibiotic and insect immune inhibition activities and plays multiple roles during the infection process of B. bassiana against insect hosts. Several negative regulators involved in oosporein synthesis have been reported; however, we know little about the positive regulators outside the biosynthetic gene cluster. Here, we found that the pH signaling transcription factor BbPacC positively regulates oosporein production by binding to several PacC-binding sites. In addition, our results also indicate that BbPacC jointly acts with the negative regulator Bbmsn2 to regulate oosporein synthesis. Our results provide insight into understanding the regulatory mechanism of oosporein production as well as targets to engineer B. bassiana strains producing high levels of oosporein.

A Perilipin Affects Lipid Droplet Homeostasis and Aerial Hyphal Growth, but Has Only Small Effects on Virulence in the Insect Pathogenic Fungus Beauveria bassiana.

Lipid assimilation, storage, and turnover impact growth, development, and virulence in many microbial pathogens including fungi. Perilipins are proteins associated with lipid droplets (LDs) that mediate their assembly and turnover. Here, we characterized the Beauveria bassiana (BbPlin1) perilipin. BbPlin1 expression was higher in minimal media than in rich media, and, using a BbPlin1::eGFP fusion protein, the protein was shown to be co−localized to LDs, with the high expression seen during infection and proliferation within the insect (Galleria mellonella) host that dramatically decreased to almost no expression during fungal outgrowth on cadavers including in conidia, but that BbPlin1 production resumed in the conidia once placed in nutrient−containing media allowing for germination and growth. Characterization of a targeted gene deletion strain (ΔBbPlin1) revealed a dramatic (>30%) reduction in cellular LD content, promotion of aerial hyphal growth, and a small decrease in virulence, with little to no effects on vegetative growth and stress responses. However, in the ΔBbPlin1 strain, expression of the complementary LD−associated caleosin gene, BbCal1, was enhanced under nutrient−poor conditions, although no changes in BbPlin1 expression were seen in a ΔBbCal1 strain and the expression of BbPlin1 in the ΔBbCal1 strain did not change LD patterns in cells. Transcriptome and RT−PCR analyses indicated increased expression of lipid metabolism−related genes, including triacylglyercol lipase 3, enoyl−CoA isomerase, and diacylglycerol−O−acetyl transferase in the BbPlin1 deletion mutant. Lipid profile analyses confirmed that the loss of BbPlin1 significantly reduced the cellular levels of contents of triacylglycerol, diacylglycerol, and phosphatidylethanolamine as compared to the wild−type strain. These results demonstrate the involvement of the B. bassiana perilipin in mediating lipid homeostasis, fungal aerial hyphal growth, and virulence, revealing critical cycling from high expression during nutrient utilization within host cadavers to low expression during growth on the surface of the cadaver during the infection process.