Functional analyses of small secreted cysteine-rich proteins identified candidate effectors in Verticillium dahliae.

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.

The Gossypium hirsutum TIR-NBS-LRR gene GhDSC1 mediates resistance against Verticillium wilt.

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.

Population genomics demystifies the defoliation phenotype in the plant pathogen Verticillium dahliae.

Verticillium dahliae is a broad host-range pathogen that causes vascular wilts in plants. Interactions between three hosts and specific V. dahliae genotypes result in severe defoliation. The underlying mechanisms of defoliation are unresolved. Genome resequencing, gene deletion and complementation, gene expression analysis, sequence divergence, defoliating phenotype identification, virulence analysis, and quantification of V. dahliae secondary metabolites were performed. Population genomics previously revealed that G-LSR2 was horizontally transferred from the fungus Fusarium oxysporum f. sp. vasinfectum to V. dahliae and is exclusively found in the genomes of defoliating (D) strains. Deletion of seven genes within G-LSR2, designated as VdDf genes, produced the nondefoliation phenotype on cotton, olive, and okra but complementation of two genes restored the defoliation phenotype. Genes VdDf5 and VdDf6 associated with defoliation shared homology with polyketide synthases involved in secondary metabolism, whereas VdDf7 shared homology with proteins involved in the biosynthesis of N-lauroylethanolamine (N-acylethanolamine (NAE) 12:0), a compound that induces defoliation. NAE overbiosynthesis by D strains also appears to disrupt NAE metabolism in cotton by inducing overexpression of fatty acid amide hydrolase. The VdDfs modulate the synthesis and overproduction of secondary metabolites, such as NAE 12:0, that cause defoliation either by altering abscisic acid sensitivity, hormone disruption, or sensitivity to the pathogen.

Genome-Wide Identification and Functional Analyses of the CRK Gene Family in Cotton Reveals GbCRK18 Confers Verticillium Wilt Resistance in Gossypium barbadense.

Cysteine-rich receptor-like kinases (CRKs) are a large subfamily of plant receptor-like kinases that play a critical role in disease resistance in plants. However, knowledge about the CRK gene family in cotton and its function against Verticillium wilt (VW), a destructive disease caused by Verticillium dahliae that significantly reduces cotton yields is lacking. In this study, we identified a total of 30 typical CRKs in a Gossypium barbadense genome (GbCRKs). Eleven of these (>30%) are located on the A06 and D06 chromosomes, and 18 consisted of 9 paralogous pairs encoded in the A and D subgenomes. Phylogenetic analysis showed that the GbCRKs could be classified into four broad groups, the expansion of which has probably been driven by tandem duplication. Gene expression profiling of the GbCRKs in resistant and susceptible cotton cultivars revealed that a phylogenetic cluster of nine of the GbCRK genes were up-regulated in response to V. dahliae infection. Virus-induced gene silencing of each of these nine GbCRKs independently revealed that the silencing of GbCRK18 was sufficient to compromise VW resistance in G. barbadense. GbCRK18 expression could be induced by V. dahliae infection or jasmonic acid, and displayed plasma membrane localization. Therefore, our expression analyses indicated that the CRK gene family is differentially regulated in response to Verticillium infection, while gene silencing experiments revealed that GbCRK18 in particular confers VW resistance in G. barbadense.

Heterologous Expression of the Cotton NBS-LRR Gene GbaNA1 Enhances Verticillium Wilt Resistance in Arabidopsis.

Verticillium wilt caused by Verticillium dahliae results in severe losses in cotton, and is economically the most destructive disease of this crop. Improving genetic resistance is the cleanest and least expensive option to manage Verticillium wilt. Previously, we identified the island cotton NBS-LRR-encoding gene GbaNA1 that confers resistance to the highly virulent V. dahliae isolate Vd991. In this study, we expressed cotton GbaNA1 in the heterologous system of Arabidopsis thaliana and investigated the defense response mediated by GbaNA1 following inoculations with V. dahliae. Heterologous expression of GbaNA1 conferred Verticillium wilt resistance in A. thaliana. Moreover, overexpression of GbaNA1 enabled recovery of the resistance phenotype of A. thaliana mutants that had lost the function of GbaNA1 ortholog gene. Investigations of the defense response in A. thaliana showed that the reactive oxygen species (ROS) production and the expression of genes associated with the ethylene signaling pathway were enhanced significantly following overexpression of GbaNA1. Intriguingly, overexpression of the GbaNA1 ortholog from Gossypium hirsutum (GhNA1) in A. thaliana did not induce the defense response of ROS production due to the premature termination of GhNA1, which lacks the encoded NB-ARC and LRR motifs. GbaNA1 therefore confers Verticillium wilt resistance in A. thaliana by the activation of ROS production and ethylene signaling. These results demonstrate the functional conservation of the NBS-LRR-encoding GbaNA1 in a heterologous system, and the mechanism of this resistance, both of which may prove valuable in incorporating GbaNA1-mediated resistance into other plant species.

A Verticillium dahliae Extracellular Cutinase Modulates Plant Immune Responses.

Cutinases have been implicated as important enzymes during the process of fungal infection of aerial plant organs. The function of cutinases in the disease cycle of fungal pathogens that invade plants through the roots has been less studied. Here, functional analysis of 13 cutinase (carbohydrate esterase family 5 domain-containing) genes (VdCUTs) in the highly virulent vascular wilt pathogen Verticillium dahliae Vd991 was performed. Significant sequence divergence in cutinase family members was observed in the genome of V. dahliae Vd991. Functional analyses demonstrated that only VdCUT11, as purified protein, induced cell death and triggered defense responses in Nicotiana benthamiana, cotton, and tomato plants. Virus-induced gene silencing showed that VdCUT11 induces plant defense responses in Nicotiana benthamania in a BAK1 and SOBIR-dependent manner. Furthermore, coinfiltration assays revealed that the carbohydrate-binding module family 1 protein (VdCBM1) suppressed VdCUT11-induced cell death and other defense responses in N. benthamiana. Targeted deletion of VdCUT11 in V. dahliae significantly compromised virulence on cotton plants. The cutinase VdCUT11 is an important secreted enzyme and virulence factor that elicits plant defense responses in the absence of VdCBM1.

Comparative genomics reveals cotton-specific virulence factors in flexible genomic regions in Verticillium dahliae and evidence of horizontal gene transfer from Fusarium.

Verticillium dahliae isolates are most virulent on the host from which they were originally isolated. Mechanisms underlying these dominant host adaptations are currently unknown. We sequenced the genome of V. dahliae Vd991, which is highly virulent on its original host, cotton, and performed comparisons with the reference genomes of JR2 (from tomato) and VdLs.17 (from lettuce). Pathogenicity-related factor prediction, orthology and multigene family classification, transcriptome analyses, phylogenetic analyses, and pathogenicity experiments were performed. The Vd991 genome harbored several exclusive, lineage-specific (LS) genes within LS regions (LSRs). Deletion mutants of the seven genes within one LSR (G-LSR2) in Vd991 were less virulent only on cotton. Integration of G-LSR2 genes individually into JR2 and VdLs.17 resulted in significantly enhanced virulence on cotton but did not affect virulence on tomato or lettuce. Transcription levels of the seven LS genes in Vd991 were higher during the early stages of cotton infection, as compared with other hosts. Phylogenetic analyses suggested that G-LSR2 was acquired from Fusarium oxysporum f. sp. vasinfectum through horizontal gene transfer. Our results provide evidence that horizontal gene transfer from Fusarium to Vd991 contributed significantly to its adaptation to cotton and may represent a significant mechanism in the evolution of an asexual plant pathogen.

The island cotton NBS-LRR gene GbaNA1 confers resistance to the non-race 1 Verticillium dahliae isolate Vd991.

Wilt caused by Verticillium dahliae significantly reduces cotton yields, as host resistance in commercially cultivated Gossypium species is lacking. Understanding the molecular basis of disease resistance in non-commercial Gossypium species could galvanize the development of Verticillium wilt resistance in cultivated species. Nucleotide-binding site leucine-rich repeat (NBS-LRR) proteins play a central role in plant defence against pathogens. In this study, we focused on the relationship between a locus enriched with eight NBS-LRR genes and Verticillium wilt resistance in G. barbadense. Independent virus-induced gene silencing of each of the eight NBS-LRR genes in G. barbadense cultivar Hai 7124 revealed that silencing of GbaNA1 alone compromised the resistance of G. barbadense to V. dahliae isolate Vd991. In cultivar Hai 7124, GbaNA1 could be induced by V. dahliae isolate Vd991 and by ethylene, jasmonic acid and salicylic acid. Nuclear protein localization of GbaNA1 was demonstrated by transient expression. Sequencing of the GbaNA1 orthologue in nine G. hirsutum accessions revealed that all carried a non-functional allele, caused by a premature peptide truncation. In addition, all 10 G. barbadense and nine G. hirsutum accessions tested carried a full-length (∼1140 amino acids) homologue of the V. dahliae race 1 resistance gene Gbve1, although some sequence polymorphisms were observed. Verticillium dahliae Vd991 is a non-race 1 isolate that lacks the Ave1 gene. Thus, the resistance imparted by GbaNA1 appears to be mediated by a mechanism distinct from recognition of the fungal effector Ave1.

Verticillium dahliae transcription factor VdFTF1 regulates the expression of multiple secreted virulence factors and is required for full virulence in cotton.

Fungal transcription factors (TFs) implicated in the regulation of virulence gene expression have been identified in a number of plant pathogens. In Verticillium dahliae, despite its agricultural importance, few regulators of transcription have been characterized. In this study, a T-DNA insertion mutant with significantly reduced virulence towards cotton was identified. The T-DNA was traced to VdFTF1, a gene encoding a TF containing a Fungal_trans domain. Transient expression in onion epidermal cells indicated that VdFTF1 is localized to the nucleus. The VdFTF1-deletion strains displayed normal vegetative growth, mycelial pigmentation and conidial morphology, but exhibited significantly reduced virulence on cotton, suggesting that VdFTF1 is required exclusively for pathogenesis. Comparisons of global transcription patterns of wild-type and VdFTF1-deletion strains indicated that VdFTF1 affected the expression of 802 genes, 233 of which were associated with catalytic processes. These genes encoded 69 potentially secreted proteins, 43 of which contained a carbohydrate enzyme domain known to participate in pathogenesis during infection of cotton. Targeted gene deletion of one VdFTF1-regulated gene resulted in significantly impaired vascular colonization, as measured by quantitative polymerase chain reaction, as well as aggressiveness and symptom severity in cotton. In conclusion, VdFTF1, which encodes a TF containing a Fungal_trans domain, regulates the gene expression of plant cell wall degradation enzymes in V. dahliae, which are required for full virulence on cotton.

Verticillium dahliae manipulates plant immunity by glycoside hydrolase 12 proteins in conjunction with carbohydrate-binding module 1.

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.

[Biodegradation characteristics of organic compounds in leachate in electrochemical oxidation and anaerobic process combined treatment system].

Degradation characteristics of typical organic compounds in landfill leachate were investigated by electrochemical oxidation and anaerobic process combined treatment system in this paper. In electrochemical oxidation system, degradation rate of polyphenolic, polyamide, benzothiazole, benzoquinone, quinoline and naphthalene compounds was higher than that of Exo-2-hydroxycineole and isoquinoline compounds, but that of the former was lower in anaerobic process. After electrochemical oxidation process, content of Volatile Fatty Acids (VFAs) markedly increased from 0.68% of raw leachate to 16.18% of effluent of electrochemical reactor. The results indicate that synergetic and antagonistic effects of complicated components on biotoxicity in leachate are notably decreased in such the combined treatment system, and biodegradability of systemic effluent is improved. These are key reasons to highly effectively treat landfill leachate by using such the combined treatment system, and can provide technical assistance to further investigate treatment technology of landfill leachate and large-scale development of electrochemical oxidation and anaerobic process combined treatment system.