Secreted small cysteine-rich proteins (SCPs) play a critical role in modulating host immunity in plant-pathogen interactions. Bioinformatic analyses showed that the fungal pathogen Verticillium dahliae encodes more than 100 VdSCPs, but their roles in host-pathogen interactions have not been fully characterized. Transient expression of 123 VdSCP-encoding genes in Nicotiana benthamiana identified three candidate genes involved in host-pathogen interactions. The expression of these three proteins, VdSCP27, VdSCP113, and VdSCP126, in N. benthamiana resulted in cell death accompanied by a reactive oxygen species burst, callose deposition, and induction of defence genes. The three VdSCPs mainly localized to the periphery of the cell. BAK1 and SOBIR1 (associated with receptor-like protein) were required for the immunity triggered by these three VdSCPs in N. benthamiana. Site-directed mutagenesis showed that cysteine residues that form disulphide bonds are essential for the functioning of VdSCP126, but not VdSCP27 and VdSCP113. VdSCP27, VdSCP113, and VdSCP126 individually are not essential for V. dahliae infection of N. benthamiana and Gossypium hirsutum, although there was a significant reduction of virulence on N. benthamiana and G. hirsutum when inoculated with the VdSCP27/VdSCP126 double deletion strain. These results illustrate that the SCPs play a critical role in the V. dahliae-plant interaction via an intrinsic virulence function and suppress immunity following infection.

Characterizing and engineering microbial communities for lignocellulosic biofuel production has received widespread attention. Previous research has established that Clostridium thermocellum JN4 and Thermoanaerobacterium thermosaccharolyticum GD17 coculture significantly improves overall cellulosic biofuel production efficiency. Here, we investigated this interaction and revealed the mechanism underlying the improved efficiency observed. In contrast to the previously reported mutualistic relationship, a harmful effect toward C. thermocellum JN4 was observed in these microbial consortia. Although T. thermosaccharolyticum GD17 relieves the carbon catabolite repression of C. thermocellum JN4 regarding obtaining more cellobiose or glucose released from lignocellulose, T. thermosaccharolyticum GD17 significantly hampers the growth of C. thermocellum JN4 in coculture. The increased formation of end products is due to the strong competitive metabolic advantage of T. thermosaccharolyticum GD17 over C. thermocellum JN4 in the conversion of glucose or cellobiose into final products. The possibility of controlling and rebalancing these microbial consortia to modulate cellulose degradation was achieved by adding T. thermosaccharolyticum GD17 stimulants into the system. As cellulolytic bacteria are usually at a metabolic disadvantage, these discoveries may apply to a large proportion of cellulosic biofuel-producing microbial consortia. These findings provide a reference for engineering efficient and modular microbial consortia for modulating cellulosic conversion.

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-ethyhnaleimide-sensitive factor attachment protein receptors (SNAREs), four Secl/Muncl8 (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 VdSsol, 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 VdSsol. Consistent with a role of these enzymes in plant cell-wall degradation, pectin, cellulose, and xylan utilization were reduced in the VdSec22 or VdSsol 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 VdSsol, but some of the proteins regulated by VdSec22 or VdSsol 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.

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.