Functional analysis of the BbSEC22 gene related to insecticidal and stress resistance of Beauveria bassiana

Functional analysis of the BbSEC22 gene related to insecticidal and stress resistance in Beauveria bassiana

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are core components that mediate cell membrane fusion and intracellular vesicle transport mechanisms in eukaryotic cells, playing important roles in the growth, development, and pathogenic mechanisms of filamentous fungal plant pathogens. However, little is known about the contribution of SNAREs to the physiology and biocontrol potential of insect pathogenic filamentous fungi. Insect fungal pathogens such as Beauveria spp. and Metarhizium spp. have been successfully used as biocontrol agents against plant-feeding and blood-sucking pests. Beauveria bassiana is a classical insect pathogenic fungus that has been successfully developed as a biopesticide for pest control. However, their slow killing speed and poor tolerance to adverse environmental factors have limited their application and commercial development. Since SNAREs are highly conserved and functionally diverse in fungi, elucidating the contribution of SNAREs to the physiology and biocontrol potential of insect pathogens is necessary. Researchers utilized whole-genome information to explore the SNARE superfamily genes in the whole-genome sequence of Beauveria bassiana and further characterized their functions by constructing disruption and complementation mutants.

Based on comparative genomics, 22 genes possibly encoding SNAREs were identified from the whole genome of Beauveria bassiana. According to the conservative characteristics of the encoded proteins, they were divided into 4 groups (Qa- 7, Qb- 4, Qc- 6, R-SNAREs 5). Structural domain prediction showed that BbSEC22 contains a conservative SNARE domain and a transmembrane motif located at the C-terminus. The conservative amino acid residue of the SNARE domain is R, indicating that BbSEC22 belongs to the R-SNARE superfamily. The R-SNARE encoding gene BbSEC22 was further functionally characterized through gene deletion mutation and complementation. Under cultivation conditions at 25°C, significant changes in cell morphology were observed after deleting BbSEC22. Compared to the wild-type strain and the ∆BbSEC22 complemented strain, the ∆BbSEC22 mutant exhibited reduced colony size, thinner hyphal edges, and a more fluffy appearance. After 8 days of growth on plates, the colony size of the ∆BbSEC22 mutant decreased by 28.5% compared to the control strain (~7.0 cm2), and the dry weight of the hyphae of the ∆BbSEC22 mutant also significantly decreased by 15.3%. During the incubation period, there was a significant difference in the spore yield measured from colonies between the ∆BbSEC22 mutant and the control strain. Both the wild-type strain and the complemented strain began sporulation on the 3rd day, with significantly more spores produced on the 8th day compared to the deletion mutant. The final spore yield of the ∆BbSEC22 mutant was severely reduced by 43.1% compared to the control strain. Additionally, the ∆BbSEC22 mutant showed a significant delay in spore germination. This study found that the deletion of BbSEC22 severely impaired the stress tolerance of hyphae, significantly reduced the tolerance of the ∆BbSEC22 colony to two fungicides, and increased the relative growth inhibition rates of the ∆BbSEC22 colonies grown on media supplemented with 1 µg mL−1 carbendazim and 0.1 mg mL−1 dichlorvos by 19.1% and 9.8%, respectively. The study on the effect of high temperature on the spore germination and survival rate of the deletion mutant and the control strain after 24 h of germination found that the spores of ∆BbSEC22 were 18.7% less tolerant to 45°C heat stress compared to the control strain's LT50s. Furthermore, the study found that disrupting BbSEC22 significantly reduced the pathogenicity of Beauveria bassiana against Spodoptera litura larvae.

Overall, it has been revealed that the R-SNARE protein BbSEC22 of Beauveria bassiana plays an important role in the regulation of nutrient growth, asexual spore formation, conidial germination, multi-stress tolerance, and insecticidal activity in Beauveria bassiana. Future work should focus on characterizing other SNAREs and the target proteins that these SNARE complexes deliver, which may more easily elucidate the relationship between SNAREs-mediated fungal development and vesicle transport, and may provide new avenues for generating Beauveria bassiana strains with great potential for biological pest control.

The impact of the deletion of BbSEC22 on hyphal growth, sporulation ability, and conidial germination of Beauveria bassiana.

The impact of the deletion of the BbSEC22 gene in Beauveria bassiana on tolerance and insecticidal activity.