Proteins that mediate cellular and subcellular membrane fusion are key factors in vesicular trafficking in all eukaryotic cells, including the secretion and transport of plant pathogen virulence factors. In this study, we identified vesicle-fusion components that included 22 soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), four Sec1/Munc18 (SM) family proteins, and 10 Rab GTPases encoded in the genome of the vascular wilt pathogen Verticillium dahliae Vd991. Targeted deletion of two SNARE-encoding genes in V. dahliae, VdSec22 and VdSso1, significantly reduced virulence of both mutants on cotton, relative to the wild-type Vd991 strain. Comparative analyses of the secreted protein content (exoproteome) revealed that many enzymes involved in carbohydrate hydrolysis were regulated by VdSec22 or VdSso1. Consistent with a role of these enzymes in plant cell-wall degradation, pectin, cellulose, and xylan utilization were reduced in the VdSec22 or VdSso1 mutant strains along with a loss of exoproteome cytotoxic activity on cotton leaves. Comparisons with a pathogenicity-related exoproteome revealed that several known virulence factors were not regulated by VdSec22 or VdSso1, but some of the proteins regulated by VdSec22 or VdSso1 displayed different characteristics, including the lack of a typical signal peptide, suggesting that V. dahliae employs more than one secretory route to transport proteins to extracellular sites during infection.
Previous studies have reported the ability of fungi to overwinter in soil or on crop debris under different environmental conditions, but how fungi adapt to chilling is still largely unknown. In this study, we have identified and characterized the RNA binding protein (RBP) (VdNop12) by screening an Agrobacterium tumefaciens-mediated transformationmediated insertional mutational library of Verticillium dahliae. We determined that this protein was essential to the pathogen for virulence on cotton plants. VdNop12 contains two tandem RNA recognition motif domains, and its orthologs are widely distributed in filamentous fungi. Mutants produced by disruption of VdNop12 showed defects in vegetative growth, conidiation and cell wall integrity. The mutant also showed an increase in sensitivity to low temperature, as compared to the wildtype and complementation strains. Yeast complementation assay showed that VdNop12 could functionally restore the growth phenotype of ΔScNop12 mutant of Saccharomyces cerevisiae at 15 C. We demonstrated that the VdNop12 is localized in the nucleus, and its loss resulted in the downregulated expression of several genes related to cAMP-PKA and MAPK pathways in V. dahliae. Our results demonstrated a crucial role of RBPs in the regulation of morphology, cold adaption, and pathogenic development in V. dahliae.
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