Identification and Functional Characterization of β-Glucosidase Genes in Verticillium dahliae for Pathogenicity on Cotton

Verticillium dahliae is a major soil-borne pathogenic fungus that can infect more than 200 plant species, causing Verticillium wilt and leading to severe economic losses in global agricultural production. Cell wall-degrading enzymes (CWDEs) play a critical role in fungal pathogenesis. This study aimed to identify β-glucosidase genes in V. dahliae and explore their functions, thereby providing new molecular targets for cotton disease-resistant breeding.

β-glucosidase genes were identified from the whole genome of V. dahliae through bioinformatic analysis, and their evolutionary relationships, conserved domains, and expression patterns were systematically analyzed. Host-induced gene silencing (HIGS) technology was used to silence Vdbg4 and Vdbg6 in cotton to evaluate the effects of this silencing on cotton disease resistance. Artificial small interfering RNAs (asiRNAs) targeting Vdbg4 (asiR1364) and Vdbg6 (asiR1444) were designed and co-cultured with V. dahliae, respectively. The growth and development, carbon source utilization and stress response abilities, and pathogenicity of V. dahliae in co-culture were investigated. The secretory activity of Vdbg6 was verified using a yeast signal peptide trapping assay, and whether the gene could trigger plant immune responses was detected using an Agrobacterium-mediated tobacco transient expression assay.

A total of 18 β-glucosidase genes were identified from V. dahliae. Among them, the expression levels of Vdbg4 and Vdbg6 were significantly up-regulated after induction by root exudates of a susceptible cotton cultivar. Silencing Vdbg4, Vdbg6 alone, or silencing both genes simultaneously via HIGS, significantly alleviated the disease symptoms of cotton, reduced the disease index, and decreased the fungal biomass. The asiRNAs targeting Vdbg4 and Vdbg6 (asiR1364, asiR1444, and asiR1364+1444) could inhibit the fungal colony and mycelial growth, reduce the sporulation and spore germination rates, and impair the carbon source utilization and stress response abilities, and pathogenicity of V. dahliae. Vdbg6 exhibited secretory activity, but it could neither induce programmed cell death (PCD) nor suppress BAX-induced PCD in tobacco cells.

Both Vdbg4 and Vdbg6 are involved in the growth and development, carbon source utilization, stress response, and pathogenic processes of V. dahliae.