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.

In the face of carbon, nitrogen, and phosphorus starvation, microorganisms have evolved adaptive mechanisms to maintain growth. In a previous study, we identified a protein predicted to contain acyl-CoA-binding domains in the plant pathogenic fungus Verticillium dahliae. The predicted protein, designated VdAcb1, possesses an atypical signal peptide. However, the functions of this acyl-CoA-binding protein in V. dahliae are not clear. In this research, in vivo or in vitro assays confirmed that VdAcb1 is secreted extracellularly from V. dahliae, although it does not have the typical signal peptide. Furthermore, the unconventional secretion of VdAcb1 was dependent on VdGRASP, a member of the compartment for unconventional protein secretion (CUPS). The deletion mutant strain of VdAcb1 (ΔVdAcb1) exhibited significant sensitivity to carbon starvation. RNA-seq revealed that the expression of genes related to filamentous growth (MSB2 pathway) and sugar transport were regulated by VdAcb1 under conditions of carbon starvation. Yeast one-hybrid experiments further showed that the expression of VdAcb1 was positively regulated by the transcription factor VdMsn4. The ΔVdAcb1 strain showed significantly reduced virulence on Gossypium hirsutum and Nicotiana benthamiana. We hypothesize that under conditions of carbon starvation, the expression of VdAcb1 is activated by VdMsn4 and VdAcb1 is secreted into the extracellular space. In turn, this activates the downstream MAPK pathway to enhance filamentous growth and virulence of V. dahliae.