Summary: Glycoside hydrolase 12 (GH12) proteins act as virulence factors and pathogen‐associated molecular patterns (PAMPs) in oomycetes. However, the pathogenic mechanisms of fungal GH12 proteins have not been characterized. In this study, we demonstrated that two of the six GH12 proteins produced by the fungus Verticillium dahliae Vd991, VdEG1 and VdEG3 acted as PAMPs to trigger cell death and PAMP‐triggered immunity (PTI) independent of their enzymatic activity in Nicotiana benthamiana . A 63‐amino‐acid peptide of VdEG3 was sufficient for cell death‐inducing activity, but this was not the case for the corresponding peptide of VdEG1. Further study indicated that VdEG1 and VdEG3 trigger PTI in different ways: BAK1 is required for VdEG1‐ and VdEG3‐triggered immunity, while SOBIR1 is specifically required for VdEG1‐triggered immunity in N. benthamiana . Unlike oomycetes, which employ RXLR effectors to suppress host immunity, a carbohydrate‐binding module family 1 (CBM1) protein domain suppressed GH12 protein‐induced cell death. Furthermore, during infection of N. benthamiana and cotton, VdEG1 and VdEG3 acted as PAMPs and virulence factors, respectively indicative of host‐dependent molecular functions. These results suggest that VdEG1 and VdEG3 associate differently with BAK1 and SOBIR1 receptor‐like kinases to trigger immunity in N. benthamiana , and together with CBM1‐containing proteins manipulate plant immunity.

Verticillium wilt, caused by the Verticillium dahliae phytopathogen, is a devastating disease affecting many economically important crops. Previous studies have shown that the exoproteome of V. dahliae plays a significant role in this pathogenic process, but the components and mechanisms that underlie this remain unclear. In this study, the exoproteome of V. dahliae was induced in a cotton-containing C’zapek-Dox (CCD) medium and quantified using the high-throughput isobaric tag technique for relative and absolute quantification (iTRAQ). Results showed that the abundance of 271 secreted proteins was affected by the CCD medium, of which 172 contain typical signal peptides generally produced by the Golgi/endoplasmic reticulum (ER). These enhanced abundance proteins were predominantly enriched in carbohydrate hydrolases; 126 were classified as carbohydrate-active (CAZymes) and almost all were significantly up-regulated in the CCD medium. Results showed that CAZymes proteins 30 and 22 participate in pectin and cellulose degradation pathways, corresponding with the transcription levels of several genes encoded plant cell wall degradation enzyme activated significantly during cotton infection. In addition, targeted deletion of two pectin lyase genes (VdPL3.1 and VdPL3.3) impaired wilt virulence to cotton. This study demonstrates that the V. dahliae exoproteome plays a crucial role in the development of symptoms of wilting and necrosis, predominantly via the pathogenic mechanisms of plant cell wall degradation as part of host plant infection.

The accumulation of reactive oxygen species (ROS) is a widespread defence mechanism in higher plants against pathogen attack and sometimes is the cause of cell death that facilitates attack by necrotrophic pathogens. Plant pathogens use superoxide dismutase (SOD) to scavenge ROS derived from their own metabolism or generated from host defence. The significance and roles of SODs in the vascular plant pathogen Verticillium dahliae are unclear. Our previous study showed a significant upregulation of Cu/Zn‐SOD1 (VdSOD1) in cotton tissues following V. dahliae infection, suggesting that it may play a role in pathogen virulence. Here, we constructed VdSOD1 deletion mutants (ΔSOD1) and investigated its function in scavenging ROS and promoting pathogen virulence. ΔSOD1 had normal growth and conidiation but exhibited significantly higher sensitivity to the intracellular ROS generator menadione. Despite lacking a signal peptide, assays in vitro by western blot and in vivo by confocal microscopy revealed that secretion of VdSOD1 is dependent on the Golgi reassembly stacking protein (VdGRASP). Both menadione‐treated ΔSOD1 and cotton roots infected with ΔSOD1 accumulated more and less H₂O₂ than with the wildtype strain. The absence of a functioning VdSOD1 significantly reduced symptom severity and pathogen colonization in both cotton and Nicotiana benthamiana. VdSOD1 is nonessential for growth or viability of V. dahliae, but is involved in the detoxification of both intracellular ROS and host‐generated extracellular ROS, and contributes significantly to virulence in V. dahliae.

Summary: Improving genetic resistance is a preferred method to manage Verticillium wilt of cotton and other hosts. Identifying host resistance is difficult because of the dearth of resistance genes against this pathogen. Previously, a novel candidate gene involved in Verticillium wilt resistance was identified by a genome‐wide association study using a panel of Gossypium hirsutum accessions. In this study, we cloned the candidate resistance gene from cotton that encodes a protein sharing homology with the TIR‐NBS‐LRR receptor‐like defence protein DSC1 in Arabidopsis thaliana (hereafter named GhDSC1 ). GhDSC1 expressed at higher levels in response to Verticillium wilt and jasmonic acid (JA) treatment in resistant cotton cultivars as compared to susceptible cultivars and its product was localized to nucleus. The transfer of GhDSC1 to Arabidopsis conferred Verticillium resistance in an A. thaliana dsc1 mutant. This resistance response was associated with reactive oxygen species (ROS) accumulation and increased expression of JA‐signalling‐related genes. Furthermore, the expression of GhDSC1 in response to Verticillium wilt and JA signalling in A. thaliana displayed expression patterns similar to GhCAMTA3 in cotton under identical conditions, suggesting a coordinated DSC1 and CAMTA3 response in A. thaliana to Verticillium wilt. Analyses of GhDSC1 sequence polymorphism revealed a single nucleotide polymorphism (SNP) difference between resistant and susceptible cotton accessions, within the P‐loop motif encoded by GhDSC1 . This SNP difference causes ineffective activation of defence response in susceptible cultivars. These results demonstrated that GhDSC1 confers Verticillium resistance in the model plant system of A. thaliana , and therefore represents a suitable candidate for the genetic engineering of Verticillium wilt resistance in cotton.

Background: Verticillium dahliae is a fungal pathogen that causes a vascular wilt on many economically important crops. Common fungal extracellular membrane (CFEM) domain proteins including secreted types have been implicated in virulence, but their roles in this pathogen are still unknown. Results: Nine secreted small cysteine-rich proteins (VdSCPs) with CFEM domains were identified by bioinformatic analyses and their differential suppression of host immune responses were evaluated. Two of these proteins, VdSCP76 and VdSCP77, localized to the plant plasma membrane owing to their signal peptides and mediated broad-spectrum suppression of all immune responses induced by typical effectors. Deletion of either VdSCP76 or VdSCP77 significantly reduced the virulence of V. dahliae on cotton. Furthermore, VdSCP76 and VdSCP77 suppressed host immunity through the potential iron binding site conserved in CFEM family members, characterized by an aspartic acid residue in seven VdSCPs (Asp-type) in contrast with an asparagine residue (Asn-type) in VdSCP76 and VdSCP77. V. dahliae isolates carrying the Asn-type CFEM members were more virulent on cotton than those carrying the Asp-type. Conclusions: In the iron-insufficient xylem, V. dahliae is likely to employ the Asp-type CFEM members to chelate iron, and Asn-type CFEM members to suppress immunity, for successful colonization and propagation in host plants.