The transcription factor GhWRKY70 from gossypium hirsutum enhances resistance to verticillium wilt via the jasmonic acid pathway.

BACKGROUND: The WRKY transcription factors play significant roles in plant growth, development, and defense responses. However, in cotton, the molecular mechanism of most WRKY proteins and their involvement in Verticillium wilt tolerance are not well understood. RESULTS: GhWRKY70 is greatly up-regulated in cotton by Verticillium dahliae. Subcellular localization suggests that GhWRKY70 is only located in the nucleus. Transcriptional activation of GhWRKY70 further demonstrates that GhWRKY70 function as a transcriptional activator. Transgenic Arabidopsis plants overexpressing GhWRKY70 exhibited better growth performance and higher lignin content, antioxidant enzyme activities and jasmonic acid (JA) levels than wild-type plants after infection with V. dahliae. In addition, the transgenic Arabidopsis resulted in an enhanced expression level of AtAOS1, a gene related to JA synthesis, further leading to a higher JA accumulation compared to the wild type. However, the disease index (DI) values of the VIGS-treated cotton plants with TRV:WRKY70 were also significantly higher than those of the VIGS-treated cotton plants with TRV:00. The chlorophyll and lignin contents of TRV:WRKY70 plants were significantly lower than those of TRV:00 plants. GhAOS1 expression and JA abundance in TRV:WRKY70 plants were decreased. The GhWRKY70 protein was confirmed to bind to the W-box element in the promoter region of GhAOS by yeast one-hybrid assay and transient expression. CONCLUSION: These results indicate that the GhWRKY70 transcription factor is a positive regulator in Verticillium wilt tolerance of cotton, and may promote the production of JA via regulation of GhAOS1 expression.

Verticillium dahliae chromatin remodeling facilitates the DNA damage repair in response to plant ROS stress.

Reactive oxygen species (ROS) production is one of the earliest responses when plants percept pathogens and acts as antimicrobials to block pathogen entry. However, whether and how pathogens tolerate ROS stress remains elusive. Here, we report the chromatin remodeling in Verticillium dahliae, a soil-borne pathogenic fungus that causes vascular wilts of a wide range of plants, facilitates the DNA damage repair in response to plant ROS stress. We identified VdDpb4, encoding a histone-fold protein of the ISW2 chromatin remodeling complex in V. dahliae, is a virulence gene. The reduced virulence in wild type Arabidopsis plants arising from VdDpb4 deletion was impaired in the rbohd mutant plants that did not produce ROS. Further characterization of VdDpb4 and its interacting protein, VdIsw2, an ATP-dependent chromatin-remodeling factor, we show that while the depletion of VdIsw2 led to the decondensing of chromatin, the depletion of VdDpb4 resulted in a more compact chromatin structure and affected the VdIsw2-dependent transcriptional effect on gene expression, including genes involved in DNA damage repair. A knockout mutant of either VdDpb4 or VdIsw2 reduced the efficiency of DNA repair in the presence of DNA-damaging agents and virulence during plant infection. Together, our data demonstrate that VdDpb4 and VdIsw2 play roles in maintaining chromatin structure for positioning nucleosomes and transcription regulation, including genes involved in DNA repair in response to ROS stress during development and plant infection.

The Cotton Apoplastic Protein CRR1 Stabilizes Chitinase 28 to Facilitate Defense against the Fungal Pathogen Verticillium dahliae.

The apoplast serves as the first battlefield between the plant hosts and invading microbes; therefore, work on plant-pathogen interactions has increasingly focused on apoplastic immunity. In this study, we identified three proteins in the apoplast of cotton (Gossypium sp) root cells during interaction of the plant with the fungal pathogen Verticillium dahliae Among these proteins, cotton host cells secrete chitinase 28 (Chi28) and the Cys-rich repeat protein 1 (CRR1), while the pathogen releases the protease VdSSEP1. Biochemical analysis demonstrated that VdSSEP1 hydrolyzed Chi28, but CRR1 protected Chi28 from cleavage by Verticillium dahliae secretory Ser protease 1 (VdSSEP1). In accordance with the in vitro results, CRR1 interacted with Chi28 in yeast and plant cells and attenuated the observed decrease in Chi28 level that occurred in the apoplast of plant cells upon pathogen attack. Knockdown of CRR1 or Chi28 in cotton plants resulted in higher susceptibility to V. dahliae infection, and overexpression of CRR1 increased plant resistance to V dahliae, the fungus Botrytis cinerea, and the oomycete Phytophthora parasitica var nicotianae By contrast, knockout of VdSSEP1 in V. dahliae destroyed the pathogenicity of this fungus. Together, our results provide compelling evidence for a multilayered interplay of factors in cotton apoplastic immunity.

The use of ribosome-nascent chain complex-seq to reveal the translated mRNA profile and the role of ASN1 in resistance to Verticillium wilt in cotton.

We combined traditional mRNA-seq and RNC-seq together to reveal post-transcriptional regulation events impacting gene expression and interactions between the serious fungal pathogen Verticillium dahliae and a susceptible host, Gossypium hirsutum TM-1. After screening the differentially expressed and translated genes, V. dahliae infection was observed to influence gene transcription and translation in its host. Interestingly, the asparagine synthase (ASN1) gene transcripts increased significantly with the increase of infection time, while the rate of ASN1 protein accumulation in host TM-1 was distinctly lower than that in resistant hosts. We knocked down the ASN1 gene in resistant plants (ZZM2), and found that Verticillium-resistance was significantly reduced upon knockdown of ASN1. Our study revealed both transcriptional and post-transcriptional regulation of gene expression in TM-1 cotton plants infected by V. dahliae, and showed that ASN1 functions in the V. dahliae resistance process. These insights support breeding of disease resistance in cotton.

Core RNA Interference Genes Involved in miRNA and Ta-siRNA Biogenesis in Hops and Their Expression Analysis after Challenging with Verticillium nonalfalfae.

RNA interference is an evolutionary conserved mechanism by which organisms regulate the expression of genes in a sequence-specific manner to modulate defense responses against various abiotic or biotic stresses. Hops are grown for their use in brewing and, in recent years, for the pharmaceutical industry. Hop production is threatened by many phytopathogens, of which Verticillium, the causal agent of Verticillium wilt, is a major contributor to yield losses. In the present study, we performed identification, characterization, phylogenetic, and expression analyses of three Argonaute, two Dicer-like, and two RNA-dependent RNA polymerase genes in the susceptible hop cultivar Celeia and the resistant cultivar Wye Target after infection with Verticillium nonalfalfae. Phylogeny results showed clustering of hop RNAi proteins with their orthologues from the closely related species Cannabis sativa, Morus notabilis and Ziziphus jujuba which form a common cluster with species of the Rosaceae family. Expression analysis revealed downregulation of argonaute 2 in both cultivars on the third day post-inoculation, which may result in reduced AGO2-siRNA-mediated posttranscriptional gene silencing. Both cultivars may also repress ta-siRNA biogenesis at different dpi, as we observed downregulation of argonaute 7 in the susceptible cultivar on day 1 and downregulation of RDR6 in the resistant cultivar on day 3 after inoculation.

A newly identified cluster of glutathione S-transferase genes provides Verticillium wilt resistance in cotton.

As the largest cultivated fiber crop in the world, cotton (Gossypium hirsutum) is often exposed to various biotic stresses during its growth periods. Verticillium wilt caused by Verticillium dahliae is a severe disease in cotton, and the molecular mechanism of cotton resistance for Verticillium wilt needs to be further investigated. Here, we revealed that the cotton genome contains nine types of GST genes. An evolutionary analysis showed that a newly identified cluster (including Gh_A09G1508, Gh_A09G1509 and Gh_A09G1510) located on chromosome 09 of the A-subgenome was under positive selection pressure during the formation of an allotetraploid. Transcriptome analysis showed that this cluster participates in Verticillium wilt resistance. Because the Gh_A09G1509 gene showed the greatest differential expression in the resistant cultivar under V. dahliae stress, we overexpressed this gene in tobacco and found that its overexpression resulted in enhanced Verticillium wilt resistance. Suppression of the gene cluster via virus-induced gene silencing made cotton plants of the resistant cultivar Nongda601 significantly susceptible. These results demonstrated that the GST cluster played an important role in Verticillium wilt resistance. Further investigation showed that the encoded enzymes of the cluster were essential for the delicate equilibrium between the production and scavenging of H2 O2 during V. dahliae stress.

Melatonin enhances cotton immunity to Verticillium wilt via manipulating lignin and gossypol biosynthesis.

Plants endure challenging environments in which they are constantly threatened by diverse pathogens. The soil-borne fungus Verticillium dahliae is a devastating pathogen affecting many plant species including cotton, in which it significantly reduces crop yield and fiber quality. Melatonin involvement in plant immunity to pathogens has been reported, but the mechanisms of melatonin-induced plant resistance are unclear. In this study, the role of melatonin in enhancing cotton resistance to V. dahliae was investigated. At the transcriptome level, exogenous melatonin increased the expression of genes in phenylpropanoid, mevalonate (MVA), and gossypol pathways after V. dahliae inoculation. As a result, lignin and gossypol, the products of these metabolic pathways, significantly increased. Silencing the serotonin N-acetyltransferase 1 (GhSNAT1) and caffeic acid O-methyltransferase (GhCOMT) melatonin biosynthesis genes compromised cotton resistance, with reduced lignin and gossypol levels after V. dahliae inoculation. Exogenous melatonin pre-treatment prior to V. dahliae inoculation restored the level of cotton resistance reduced by the above gene silencing effects. Melatonin levels were higher in resistant cotton cultivars than in susceptible cultivars after V. dahliae inoculation. The findings indicate that melatonin affects lignin and gossypol synthesis genes in phenylpropanoid, MVA, and gossypol pathways, thereby enhancing cotton resistance to V. dahliae.

Molecular Mechanisms Controlling the Disease Cycle in the Vascular Pathogen Verticillium dahliae Characterized Through Forward Genetics and Transcriptomics.

The soil-borne pathogen Verticillium dahliae has a worldwide distribution and a plethora of hosts of agronomic value. Molecular analysis of virulence processes can identify targets for disease control. In this work, we compared the global gene transcription profile of random T-DNA insertion mutant strain D-10-8F, which exhibits reduced virulence and alterations in microsclerotium formation and polar growth, with that of the wild-type strain. Three genes identified as differentially expressed were selected for functional characterization. To produce deletion mutants, we developed an updated version of one-step construction of Agrobacterium-recombination-ready plasmids (OSCAR) that included the negative selection marker HSVtk (herpes simplex virus thymidine kinase gene) to prevent ectopic integration of the deletion constructs. Deletion of VdRGS1 (VDAG_00683), encoding a regulator of G protein signaling (RGS) protein and highly upregulated in the wild type versus D-10-8F, resulted in phenotypic alterations in development and virulence that were indistinguishable from those of the random T-DNA insertion mutant. In contrast, deletion of the other two genes selected, vrg1 (VDAG_07039) and vvs1 (VDAG_01858), showed that they do not play major roles in morphogenesis or virulence in V. dahliae. Taken together the results presented here on the transcriptomic analysis and phenotypic characterization of D-10-8F and ∆VdRGS1 strains provide evidence that variations in G protein signaling control the progression of the disease cycle in V. dahliae. We propose that G protein-mediated signals induce the expression of multiple virulence factors during biotrophic growth, whereas massive production of microsclerotia at late stages of infection requires repression of G protein signaling via upregulation of VdRGS1 activity.

VdOGDH is involved in energy metabolism and required for virulence of Verticillium dahliae.

Verticillium dahliae, a soil-borne fungus, can invade plant vascular tissue and cause Verticillium wilt. The enzyme α-oxoglutarate dehydrogenase (OGDH), catalyzing the oxidation of α-oxoglutarate in the tricarboxylic acid cycle (TCA), is vital for energy metabolism in the fungi. Here, we identified the OGDH gene in V. dahliae (VdOGDH, VDAG_10018) and investigated its function in virulence by generating gene deletion mutants (ΔVdOGDH) and complementary mutants (ΔVdOGDH-C). When the ΔVdOGDH mutants were supplemented with different carbon sources, vegetative growth on Czapek Dox medium was significantly impaired, suggesting that VdOGDH is crucial for vegetative growth and carbon utilization. Conidia of the ΔVdOGDH mutants were atypically rounded or spherical, and hyphae were irregularly branched and lacked typical whorled branches. Mutants ΔVdOGDH-1 and ΔVdOGDH-2 were highly sensitive to H2O2 in the medium plates and had higher intracellular ROS levels. ΔVdOGDH mutants also had elevated expression of oxidative response-related genes, indicating that VdOGDH is involved in response to oxidative stress. In addition, the disruption of VdOGDH caused a significant increase in the expression of energy metabolism-related genes VdICL, VdICDH, VdMDH, and VdPDH and melanin-related genes Vayg1, VdSCD, VdLAC, VT4HR, and VaflM in the ΔVdOGDH mutants; thus, VdOGDH is also important for energy metabolism and melanin accumulation. Cotton plants inoculated with ΔVdOGDH mutants exhibited mild leaf chlorosis and the disease index was lower compared with wild type and ΔVdOGDH-C strains. These results together show that VdOGDH involved in energy metabolism of V. dahliae, is also essential for full virulence by regulating multiple fungal developmental factors.

GbCYP86A1-1 from Gossypium barbadense positively regulates defence against Verticillium dahliae by cell wall modification and activation of immune pathways.

Suberin acts as stress-induced antipathogen barrier in the root cell wall. CYP86A1 encodes cytochrome P450 fatty acid ω-hydroxylase, which has been reported to be a key enzyme for suberin biosynthesis; however, its role in resistance to fungi and the mechanisms related to immune responses remain unknown. Here, we identified a disease resistance-related gene, GbCYP86A1-1, from Gossypium barbadense cv. Hai7124. There were three homologs of GbCYP86A1 in cotton, which are specifically expressed in roots and induced by Verticillium dahliae. Among them, GbCYP86A1-1 contributed the most significantly to resistance. Silencing of GbCYP86A1-1 in Hai7124 resulted in severely compromised resistance to V. dahliae, while heterologous overexpression of GbCYP86A1-1 in Arabidopsis improved tolerance. Tissue sections showed that the roots of GbCYP86A1-1 transgenic Arabidopsis had more suberin accumulation and significantly higher C16-C18 fatty acid content than control. Transcriptome analysis revealed that overexpression of GbCYP86A1-1 not only affected lipid biosynthesis in roots, but also activated the disease-resistant immune pathway; genes encoding the receptor-like kinases (RLKs), receptor-like proteins (RLPs), hormone-related transcription factors, and pathogenesis-related protein genes (PRs) were more highly expressed in the GbCYP86A1-1 transgenic line than control. Furthermore, we found that when comparing V. dahliae -inoculated and noninoculated plants, few differential genes related to disease immunity were detected in the GbCYP86A1-1 transgenic line; however, a large number of resistance genes were activated in the control. This study highlights the role of GbCYP86A1-1 in the defence against fungi and its underlying molecular immune mechanisms in this process.

An Overview of the Molecular Genetics of Plant Resistance to the Verticillium Wilt Pathogen Verticillium dahliae.

Verticillium dahliae is a soil-borne hemibiotrophic fungus that can lead to plant vascular disease and significant economic loss worldwide. Its hosts include over 400 dicotyledon plant species, such as annual herbs, perennials, and woody plants. The average yield loss of cotton crop caused by Verticillium wilt is approximately 10-35%. As the control of this disease is an urgent task for many countries, further understanding of the interaction between plants and V. dahliae is essential. Fungi can promote or inhibit plant growth, which is important; however, the most important relationship between plants and fungi is the host-pathogen relationship. Plants can become resistant to V. dahliae through diverse mechanisms such as cell wall modifications, extracellular enzymes, pattern recognition receptors, transcription factors, and salicylic acid (SA)/jasmonic acid (JA)/ethylene (ET)-related signal transduction pathways. Over the last decade, several studies on the physiological and molecular mechanisms of plant resistance to V. dahliae have been undertaken. In this review, many resistance-related genes are summarised to provide a theoretical basis for better understanding of the molecular genetic mechanisms of plant resistance to V. dahliae. Moreover, it is intended to serve as a resource for research focused on the development of genetic resistance mechanisms to combat Verticillium wilt.

Structural Diversity and Highly Specific Host-Pathogen Transcriptional Regulation of Defensin Genes Is Revealed in Tomato.

Defensins are small and rather ubiquitous cysteine-rich anti-microbial peptides. These proteins may act against pathogenic microorganisms either directly (by binding and disrupting membranes) or indirectly (as signaling molecules that participate in the organization of the cellular defense). Even though defensins are widespread across eukaryotes, still, extensive nucleotide and amino acid dissimilarities hamper the elucidation of their response to stimuli and mode of function. In the current study, we screened the Solanum lycopersicum genome for the identification of defensin genes, predicted the relating protein structures, and further studied their transcriptional responses to biotic (Verticillium dahliae, Meloidogyne javanica, Cucumber Mosaic Virus, and Potato Virus Y infections) and abiotic (cold stress) stimuli. Tomato defensin sequences were classified into two groups (C8 and C12). Our data indicate that the transcription of defensin coding genes primarily depends on the specific pathogen recognition patterns of V. dahliae and M. javanica. The immunodetection of plant defensin 1 protein was achieved only in the roots of plants inoculated with V. dahliae. In contrast, the almost null effects of viral infections and cold stress, and the failure to substantially induce the gene transcription suggest that these factors are probably not primarily targeted by the tomato defensin network.

Verticillium dahliae VdTHI20, Involved in Pyrimidine Biosynthesis, Is Required for DNA Repair Functions and Pathogenicity.

Verticillium dahliae (V. dahliae) infects roots and colonizes the vascular vessels of host plants, significantly reducing the economic yield of cotton and other crops. In this study, the protein VdTHI20, which is involved in the thiamine biosynthesis pathway, was characterized by knocking out the corresponding VdTHI20 gene in V. dahliae via Agrobacterium tumefaciens-mediated transformation (ATMT). The deletion of VdTHI20 resulted in several phenotypic defects in vegetative growth and conidiation and in impaired virulence in tobacco seedlings. We show that VdTHI20 increases the tolerance of V. dahliae to UV damage. The impaired vegetative growth of ΔVdTHI20 mutant strains was restored by complementation with a functional copy of the VdTHI20 gene or by supplementation with additional thiamine. Furthermore, the root infection and colonization of the ΔVdTHI20 mutant strains were suppressed, as indicated by green fluorescent protein (GFP)-labelling under microscope observation. When the RNAi constructs of VdTHI20 were used to transform Nicotiana benthamiana, the transgenic lines expressing dsVdTHI20 showed elevated resistance to V. dahliae. Together, these results suggest that VdTHI20 plays a significant role in the pathogenicity of V. dahliae. In addition, the pathogenesis-related gene VdTHI20 exhibits potential for controlling V. dahliae in important crops.

GhCyP3 improves the resistance of cotton to Verticillium dahliae by inhibiting the E3 ubiquitin ligase activity of GhPUB17.

KEY MESSAGE: A U-box E3 ubiquitin ligase GhPUB17 is inhibited by GhCyP3 with antifungal activity and acts as a negative regulator involved in cotton resistance to Verticillium dahliae. E3 ubiquitin ligases, the key component enzymes of the ubiquitin-proteasome system, which contains the most diverse structural and functional members involved in the determination of target specificity and the regulation of metabolism, have been well documented in previous studies. Here, we identify GhPUB17, a U-box E3 ligase in cotton that has ubiquitination activity and is involved in the cotton immune response to Verticillium dahliae. The expression level of GhPUB17 is downregulated in the ssn mutant with a constitutively activated immune response (Sun et al., Nat Commun 5:5372, 2014). Infection with V. dahliae or exogenous hormone treatment, including jasmonic acid and salicylic acid, significantly upregulated GhPUB17 in cotton roots, which suggested a possible role for this E3 ligase in the plant immune response to pathogens. Moreover, GhPUB17-knockdown cotton plants are more resistant to V. dahliae, whereas GhPUB17-overexpressing plants are more susceptible to the pathogen, which indicated that GhPUB17 is a negative regulator of cotton resistance to V. dahliae. A yeast two-hybrid (Y2H) assay identified GhCyP3 as a protein that interacts with GhPUB17, and this finding was confirmed by further protein interaction assays. The downregulation of GhCyP3 in cotton seedlings attenuated the plants' resistance to V. dahliae. In addition, GhCyP3 showed antifungal activity against V. dahliae, and the E3 ligase activity of GhPUB17 was repressed by GhCyP3 in vitro. These results suggest that GhPUB17 negatively regulates cotton immunity to V. dahliae and that the antifungal protein GhCyP3 likely interacts with and inhibits the ligase activity of GhPUB17 and plays an important role in the cotton-Verticillium interaction.

The Verticillium dahliae Sho1-MAPK pathway regulates melanin biosynthesis and is required for cotton infection.

Verticillium dahliae is a soil-borne fungus that causes vascular wilt on numerous plants worldwide. The fungus survives in the soil for up to 14 years by producing melanized microsclerotia. The protective function of melanin in abiotic stresses is well documented. Here, we found that the V. dahliae tetraspan transmembrane protein VdSho1, a homolog of the Saccharomyces cerevisiae Sho1, acts as an osmosensor, and is required for plant penetration and melanin biosynthesis. The deletion mutant ΔSho1 was incubated on a cellophane membrane substrate that mimics the plant epidermis, revealing that the penetration of ΔSho1 strain was reduced compared to the wild-type strain. Furthermore, VdSho1 regulates melanin biosynthesis by a signalling mechanism requiring a kinase-kinase signalling module of Vst50-Vst11-Vst7. Strains, ΔVst50, ΔVst7 and ΔVst11 also displayed defective penetration and melanin production like the ΔSho1 strain. Defects in penetration and melanin production in ΔSho1 were restored by overexpression of Vst50, suggesting that Vst50 lies downstream of VdSho1 in the regulatory pathway governing penetration and melanin biosynthesis. Data analyses revealed that the transmembrane portion of VdSho1 was essential for both membrane penetration and melanin production. This study demonstrates that Vst50-Vst11-Vst7 module regulates VdSho1-mediated plant penetration and melanin production in V. dahliae, contributing to virulence.

Transposons passively and actively contribute to evolution of the two-speed genome of a fungal pathogen.

Genomic plasticity enables adaptation to changing environments, which is especially relevant for pathogens that engage in "arms races" with their hosts. In many pathogens, genes mediating virulence cluster in highly variable, transposon-rich, physically distinct genomic compartments. However, understanding of the evolution of these compartments, and the role of transposons therein, remains limited. Here, we show that transposons are the major driving force for adaptive genome evolution in the fungal plant pathogen Verticillium dahliae We show that highly variable lineage-specific (LS) regions evolved by genomic rearrangements that are mediated by erroneous double-strand repair, often utilizing transposons. We furthermore show that recent genetic duplications are enhanced in LS regions, against an older episode of duplication events. Finally, LS regions are enriched in active transposons, which contribute to local genome plasticity. Thus, we provide evidence for genome shaping by transposons, both in an active and passive manner, which impacts the evolution of pathogen virulence.

Transcriptomic and biochemical analysis of upland cotton (Gossypium hirsutum) and a chromosome segment substitution line from G. hirsutum × G. barbadense in response to Verticillium dahliae infection.

BACKGROUND: Verticillium wilt (VW), also known as "cotton cancer," is one of the most destructive diseases in global cotton production that seriously impacts fiber yield and quality. Despite numerous attempts, little significant progress has been made in improving the VW resistance of upland cotton. The development of chromosome segment substitution lines (CSSLs) from Gossypium hirsutum × G. barbadense has emerged as a means of simultaneously developing new cotton varieties with high-yield, superior fiber, and resistance to VW. RESULTS: In this study, VW-resistant investigations were first conducted in an artificial greenhouse, a natural field, and diseased nursery conditions, resulting in the identification of one stably VW-resistant CSSL, MBI8255, and one VW-susceptible G. hirsutum, CCRI36, which were subsequently subjected to biochemical tests and transcriptome sequencing during V991 infection (0, 1, and 2 days after inoculation). Eighteen root samples with three replications were collected to perform multiple comparisons of enzyme activity and biochemical substance contents. The findings indicated that VW resistance was positively correlated with peroxidase and polyphenol oxidase activity, but negatively correlated with malondialdehyde content. Additionally, RNA sequencing was used for the same root samples, resulting in a total of 77,412 genes, of which 23,180 differentially expressed genes were identified from multiple comparisons between samples. After Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis on the expression profiles identified using Short Time-series Expression Miner, we found that the metabolic process in the biological process, as well as the pathways of phenylpropanoid biosynthesis and plant hormone signal transduction, participated significantly in the response to VW. Gene functional annotation and expression quantity analysis indicated the important roles of the phenylpropanoid metabolic pathway and oxidation-reduction process in response to VW, which also provided plenty of candidate genes related to plant resistance. CONCLUSIONS: This study concentrates on the preliminary response to V991 infection by comparing the VW-resistant CSSL and its VW-susceptible recurrent parent. Not only do our findings facilitate the culturing of new resistant varieties with high yield and superior performance, but they also broaden our understanding of the mechanisms of cotton resistance to VW.

Secretory proteins are delivered to the septin-organized penetration interface during root infection by Verticillium dahliae.

Successful infection of the host requires secretion of effector proteins to evade or suppress plant immunity. Secretion of effectors in root-infecting fungal pathogens, however, remains unexplored. We previously reported that Verticillium dahliae, a root-infecting phytopathogenic fungus, develops a penetration peg from a hyphopodium to infect cotton roots. In this study, we report that a septin ring, requiring VdSep5, partitions the hyphopodium and the invasive hypha and form the specialized fungus-host interface. The mutant strain, VdΔnoxb, in which NADPH oxidase B (VdNoxB) is deleted, impaired formation of the septin ring at the hyphal neck, indicating that NADPH oxidases regulate septin ring organization. Using GFP tagging and live cell imaging, we observed that several signal peptide containing secreted proteins showed ring signal accumulation/secretion at the penetration interface surrounding the hyphal neck. Targeted mutation for VdSep5 reduced the delivery rate of secretory proteins to the penetration interface. Blocking the secretory pathway by disrupting the vesicular trafficking factors, VdSec22 and VdSyn8, or the exocyst subunit, VdExo70, also arrested delivery of the secreted proteins inside the hyphopodium. Reduced virulence was observed when cotton roots were infected with VdΔsep5, VdΔsec22, VdΔsyn8 and VdΔexo70 mutants compared to infection with the isogenic wild-type V592. Taken together, our data demonstrate that the hyphal neck is an important site for protein secretion during plant root infection, and that the multiple secretory routes are involved in the secretion.

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.

From pathogen to endophyte: an endophytic population of Verticillium dahliae evolved from a sympatric pathogenic population.

The fungus Verticillium dahliae causes wilts of several hundred plant species, including potato and mint. Verticillium spp. also colonize sympatric hosts such as mustards and grasses as endophytes. The evolutionary history of and interactions between pathogenic and endophytic of this fungus are unknown. Verticillium dahliae isolates recovered from sympatric potato, mint, mustard and grasses were characterized genotypically with microsatellite markers and phenotypically for pathogenicity. The evolutionary history of pathogenic and endophytic populations was reconstructed and gene flow between populations quantified. Verticillium dahliae was recovered from all hosts. Endophytic populations were genetically and genotypically similar to but marginally differentiated from the potato population, from which they evolved. Bidirectional migration was detected between these populations and endophytic isolates were pathogenic to potato and behaved as endophytes in mustard and barley. Verticillium dahliae colonizes plants as both endophytes and pathogens. A historical host-range expansion together with endophytic and pathogenic capabilities are likely to have enabled infection of and gene flow between asymptomatic and symptomatic host populations despite minor differentiation. The ability of hosts to harbor asymptomatic infections and the stability of asymptomatic infections over time warrants investigation to elucidate the mechanisms involved in the maintenance of endophytism and pathogenesis.