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.     

CGTase,a novel antimicrobial protein from Bacillus cereus YUPP-10, suppresses Verticillium dahliae and mediates plant defence responses

Verticillium wilt is a plant vascular disease caused by the soilborne fungus Verticillium dahliae that severely limits cotton production. In a previous study, we screened Bacillus cereus YUPP-10, an efficient antagonistic bacterium, to uncover mechanisms for controlling verticillium wilt. Here, we report a novel antimicrobial cyclodextrin glycosyltransferase (CGTase) from YUPP-10. Compared to other CGTases, six different conserved domains were identified, and six mutants were constructed by gene splicing with overlap extension PCR. Functional analysis showed that domain D was important for hydrolysis activity and domains A1 and C were important for inducing disease resistance. Direct effects of recombinant CGTase on V. dahliae included reduced mycelial growth, spore germination, spore production, and microsclerotia germination. In addition, CGTase also elicited cotton's innate defence reactions. Transgenic Arabidopsis thaliana lines that overexpress CGTase showed higher resistance to verticillium wilt. Transgenic CGTase A. thaliana plants grew faster and resisted disease better. CGTase overexpression enabled a burst of reactive oxygen species production and activated pathogenesis-related gene expression, indicating that the transgenic cotton was better prepared to protect itself from infection. Our work revealed that CGTase could inhibit the growth of V. dahliae, activate innate immunity, and play a major role in the biocontrol of fungal pathogens.     

Transfer of tomato immune receptor Ve1 confers Ave1‐dependent Verticillium resistance in tobacco and cotton

Verticillium wilts caused by soilborne fungal species of the Verticillium genus are economically important plant diseases that affect a wide range of host plants and are notoriously difficult to combat. Perception of pathogen(‐induced) ligands by plant immune receptors is a key component of plant innate immunity. In tomato, race‐specific resistance to Verticillium wilt is governed by the cell surface‐localized immune receptor Ve1 through recognition of the effector protein Ave1 that is secreted by race 1 strains of Verticillium spp. It was previously demonstrated that transgenic expression of tomato Ve1 in the model plant Arabidopsis thaliana leads to Verticillium wilt resistance. Here, we investigated whether tomato Ve1 can confer Verticillium resistance when expressed in the crop species tobacco (Nicotiana tabcum) and cotton (Gossypium hirsutum). We show that transgenic tobacco and cotton plants constitutively expressing tomato Ve1 exhibit enhanced resistance against Verticillium wilt in an Ave1‐dependent manner. Thus, we demonstrate that the functionality of tomato Ve1 in Verticillium wilt resistance through recognition of the Verticillium effector Ave1 is retained after transfer to tobacco and cotton, implying that the Ve1‐mediated immune signalling pathway is evolutionary conserved across these plant species. Moreover, our results suggest that transfer of tomato Ve1 across sexually incompatible plant species can be exploited in breeding programmes to engineer Verticillium wilt resistance.     

Cotton GhBAK1 Mediates Verticillium Wilt Resistance and Cell Death

Virus-induced gene silencing (VIGS) offers a powerful approach for functional analysis of individual genes by knocking down their expression. We have adopted this approach to dissect gene functions in cotton resistant to Verticillium wilt, one of the most devastating diseases worldwide. We showed here that highly efficient VIGS was obtained in a cotton breeding line (CA4002) with partial resistance to Verticillium wilt, and GhMKK2 and GhVe1 are required for its resistance to Verticillium wilt. Arabidopsis AtBAK1/SERK3, a central regulator in plant disease resistance, belongs to a subfamily of somatic embryogenesis receptor kinases (SERKs) with five members, AtSERK1 to AtSERK5. Two BAK1 orthologs and one SERK1 ortholog were identified in the cotton genome. Importantly, GhBAK1 is required for CA4002 resistance to Verticillium wilt. Surprisingly, silencing of GhBAK1 is sufficient to trigger cell death accompanied with production of reactive oxygen species in cotton. This result is distinct from Arabidopsis in which AtBAK1 and AtSERK4 play redundant functions in cell death control. Apparently, cotton has only evolved SERK1 and BAK1 whereas AtSERK4/5 are newly evolved genes in Arabidopsis. Our studies indicate the functional importance of BAK1 in Verticillium wilt resistance and suggest the dynamic evolution of SERK family members in different plant species.     

Ethylene perception via ETR1 is required in Arabidopsis infection by Verticillium dahliae

Vascular wilts caused by Verticillium spp. are very difficult to control and, as a result, are the cause of severe yield losses in a wide range of economically important crops. The responses of Arabidopsis thaliana mutant plants impaired in known pathogen response pathways were used to explore the components in defence against Verticillium dahliae . Analysis of the mutant responses revealed enhanced resistance in etr1-1 [ethylene (ET) receptor mutant] plants, but not in salicylic acid-, jasmonic acid- or other ET-deficient mutants, indicating a crucial role of ETR1 in defence against this pathogen. Quantitative polymerase chain reaction analysis revealed that the decrease in symptom severity shown in etr1-1 plants was associated with significant reductions in the growth of the pathogen in the vascular tissues of the plants, suggesting that impaired perception of ET via ETR1 results in increased disease resistance. Furthermore, the activation and increased accumulation of the PR-1 , PR-2 , PR-5 , GSTF12 , GSTU16 , CHI-1 , CHI-2 and Myb75 genes, observed in etr1-1 plants after V. dahliae inoculation, indicate that the outcome of the induced defence response of etr1-1 plants seems to be dependent on a set of defence genes activated on pathogen attack.     

A genome-wide functional investigation into the roles of receptor-like proteins in Arabidopsis

Receptor-like proteins (RLPs) are cell surface receptors that typically consist of an extracellular leucine-rich repeat domain, a transmembrane domain, and a short cytoplasmatic tail. In several plant species, RLPs have been found to play a role in disease resistance, such as the tomato (Solanum lycopersicum) Cf and Ve proteins and the apple (Malus domestica) HcrVf2 protein that mediate resistance against the fungal pathogens Cladosporium fulvum, Verticillium spp., and Venturia inaequalis, respectively. In addition, RLPs play a role in plant development; Arabidopsis (Arabidopsis thaliana) TOO MANY MOUTHS (TMM) regulates stomatal distribution, while Arabidopsis CLAVATA2 (CLV2) and its functional maize (Zea mays) ortholog FASCINATED EAR2 regulate meristem maintenance. In total, 57 RLP genes have been identified in the Arabidopsis genome and a genome-wide collection of T-DNA insertion lines was assembled. This collection was functionally analyzed with respect to plant growth and development and sensitivity to various stress responses, including susceptibility toward pathogens. A number of novel developmental phenotypes were revealed for our CLV2 and TMM insertion mutants. In addition, one AtRLP gene was found to mediate abscisic acid sensitivity and another AtRLP gene was found to influence nonhost resistance toward Pseudomonas syringae pv phaseolicola. This genome-wide collection of Arabidopsis RLP gene T-DNA insertion mutants provides a tool for future investigations into the biological roles of RLPs.     

Identification of VdASP F2-interacting protein as a regulator of microsclerotial formation in Verticillium dahliae

Verticillium dahliae, a notorious phytopathogenic fungus, causes vascular wilt diseases in many plant species. The melanized microsclerotia enable V. dahliae to survive for years in soil and are crucial for its disease cycle. In a previous study, we characterized the secretory protein VdASP F2 from V. dahliae and found that VdASP F2 deletion significantly affected the formation of microsclerotia under adverse environmental conditions. In this study, we clarified that VdASP F2 is localized to the cell wall. However, the underlying mechanism of VdASP F2 in microsclerotial formation remains unclear. Transmembrane ion channel protein VdTRP was identified as a candidate protein that interacts with VdASP F2 using pull-down assays followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, and interaction of VdASP F2 and VdTRP was confirmed by bimolecular fluorescence complementary and coimmunoprecipitation assays. The deletion mutant was analysed to reveal that VdTRP is required for microsclerotial production, but it is not essential for stress resistance, carbon utilization and pathogenicity of V. dahliae. RNA-seq revealed some differentially expressed genes related to melanin synthesis and microsclerotial formation were significantly downregulated in the VdTRP deletion mutants. Taken together, these results indicate that VdASP F2 regulates the formation of melanized microsclerotia by interacting with VdTRP.     

Proteomic analysis of the sea-island cotton roots infected by wilt pathogen Verticillium dahliae

Verticillium wilt of cotton is a vascular disease mainly caused by the soil-born filamentous fungus Verticillium dahliae. To study the mechanisms associated with defense responses in wilt-resistant sea-island cotton (Gossypium barbadense) upon V. dahliae infection, a comparative proteomic analysis between infected and mock-inoculated roots of G. barbadense var. Hai 7124 (a cultivar showing resistance against V. dahliae) was performed by 2-DE combined with local EST database-assisted PMF and MS/MS analysis. A total of 51 upregulated and 17 downregulated proteins were identified, and these proteins are mainly involved in defense and stress responses, primary and secondary metabolisms, lipid transport, and cytoskeleton organization. Three novel clues regarding wilt resistance of G. barbadense are gained from this study. First, ethylene signaling was significantly activated in the cotton roots attacked by V. dahliae as shown by the elevated expression of ethylene biosynthesis and signaling components. Second, the Bet v 1 family proteins may play an important role in the defense reaction against Verticillium wilt. Third, wilt resistance may implicate the redirection of carbohydrate flux from glycolysis to pentose phosphate pathway (PPP). To our knowledge, this study is the first root proteomic analysis on cotton wilt resistance and provides important insights for establishing strategies to control this disease.     

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.     

Functional correlation between a novel amino acid insertion at codon 19 in the protease of human immunodeficiency virus type 1 and polymorphism in the p1/p6 Gag cleavage site in drug resistance and replication fitness

Population-based sequence analysis revealed the presence of a variant of human immunodeficiency virus type 1 (HIV-1) containing an insertion of amino acid Ile in the protease gene at codon 19 (19I) and amino acid substitutions in the protease at codons 21 (E21D) and 22 (A22V) along with multiple mutations associated with drug resistance, M46I/P63L/A71V/I84V/I93L, in a patient who had failed protease inhibitor (PI) therapy. Longitudinal analysis revealed that the P63L/A71V/I93L changes were present prior to PI therapy. Polymorphisms in the Gag sequence were only seen in the p1/p6 cleavage site at the P1' position (Leu to Pro) and the P5' position (Pro to Leu). To characterize the role of these mutations in drug susceptibility and replication capacity, a chimeric HIV-1 strain containing the 19I/E21D/A22V mutations with the M46I/P63L/A71V/I84V/I93L and p1/p6 mutations was constructed. The chimera displayed high-level resistance to multiple PIs, but not to lopinavir, and grew to 30% of that of the wild type. To determine the relative contribution of each mutation to the phenotypic characteristic of the virus, a series of mutants was constructed using site-directed mutagenesis. A high level of resistance was only seen in mutants containing the 19I/A22V and p1/p6 mutations. The E21D mutation enhanced viral replication. These results suggest that the combination of the 19I/E21D/A22V mutations may emerge and lead to high-level resistance to multiple PIs. The combination of the 19I/A22V mutations may be associated with PI resistance; however, the drug resistance may be caused by the presence of a unique set of mutations in the p1/p6 mutations. The E21D mutation contributes to replication fitness rather than drug resistance.     

Early responses in the Arabidopsis-Verticillium longisporum pathosystem are dependent on NDR1, JA- and ET-associated signals via cytosolic NPR1 and RFO1

The responses of Arabidopsis accessions and characterized genotypes were used to explore components in the early defense responses to the soilborne fungus Verticillium longisporum. V. longisporum susceptibility was found to be a complex trait, in which different disease phenotypes, such as stunting, altered flowering time, weight loss, and chlorosis were perceived differently across genotypes. A Bay-0 x Shahdara recombinant inbred line population was used to identify two loci on chromosomes 2 and 3 of Bay-0 origin that caused enhanced chlorosis after V. longisporum challenge. Furthermore, the observation that a mutation in RFO1 in Col-0 resulted in susceptibility whereas the natural rfo1 allele in Ty-0 showed a high degree of resistance to the pathogen supports the hypothesis that several resistance quantitative trait loci reside among Arabidopsis accessions. Analysis of mutants impaired in known pathogen response pathways revealed an enhanced susceptibility in ein2-1, ein4-1, ein6-1, esa1-1, and pad1-1, but not in other jasmonic acid (JA)-, ethylene (ET)-, or camalexin-deficient mutants, suggesting that V. longisporum resistance is regulated via a hitherto unknown JA- and ET-associated pathway. Pretreatments with the ET precursor 1-aminocyclo-propane-1-carboxylic acid (ACC) or methyl jasmonate (MeJA) caused enhanced resistance to V. longisporum. Mutants in the salicylic acid (SA) pathway (eds1-1, NahG, npr1-3, pad4-1, and sid2-1) did not show enhanced susceptibility to V. longisporum. In contrast, the more severe npr1-1 allele displayed enhanced V. longisporum susceptibility and decreased responses to ACC or MeJA pretreatments. This shows that cytosolic NPR1, in addition to SA responses, is required for JA- and ET-mediated V. longisporum resistance. Expression of the SA-dependent PR-1 and PR-2 and the ET-dependent PR-4 were increased 7 days postinoculation with V. longisporum. This indicates increased levels of SA and ET in response to V. longisporum inoculation. The R-gene signaling mutant ndr1-1 was found to be susceptible to V. longisporum, which could be complemented by ACC or MeJA pretreatments, in contrast to the rfo1 T-DNA mutant, which remained susceptible, suggesting that RFO1 (Fusarium oxysporum resistance) and NDR1 (nonrace specific disease resistance 1) activate two distinct signaling pathways for V. longisporum resistance.     

Overexpression of potato miR482e enhanced plant sensitivity to Verticillium dahliae infection

Verticillium wilt of potato is caused by the fungus pathogen Verticillium dahliae. Present sRNA sequencing data revealed that miR482 was in response to V. dahliae infection, but the function in potato is elusive. Here, we characterized potato miR482 family and its putative role resistance to Verticillium wilt. Members of the potato miR482 superfamily are variable in sequence, but all variants target a class of disease‐resistance proteins with nucleotide binding site (NBS) and leucine‐rich repeat (LRR) motifs. When potato plantlets were infected with V. dahliae, the expression level of miR482e was downregulated, and that of several NBS‐LRR targets of miR482e were upregulated. Transgenic potato plantlets overexpressing miR482e showed hypersensitivity to V. dahliae infection. Using sRNA and degradome datasets, we validated that miR482e targets mRNAs of NBS‐LRR disease‐resistance proteins and triggers the production of trans‐acting (ta)‐siRNAs, most of which target mRNAs of defense‐related proteins. Thus, the hypersensitivity of transgenic potato could be explained by enhanced miR482e and miR482e‐derived ta‐siRNA‐mediated silencing on NBS‐LRR‐disease‐resistance proteins. It is speculated that a miR482‐mediated silencing cascade mechanism is involved in regulating potato resistance against V. dahliae infection and could be a counter defense action of plant in response to pathogen infection.     

Identification of Pathogenicity-Related Genes in the Vascular Wilt Fungus <Emphasis Type="Italic">Verticillium dahliae</Emphasis> by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-Mediated T-DNA Insertional Mutagenesis

Verticillium dahliae is the causal agent of vascular wilt in many economically important crops worldwide. Identification of genes that control pathogenicity or virulence may suggest targets for alternative control methods for this fungus. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was applied for insertional mutagenesis of V. dahliae conidia. Southern blot analysis indicated that T-DNAs were inserted randomly into the V. dahliae genome and that 69% of the transformants were the result of single copy T-DNA insertion. DNA sequences flanking T-DNA insertion were isolated through inverse PCR (iPCR), and these sequences were aligned to the genome sequence to identify the genomic position of insertion. V. dahliae mutants of particular interest selected based on culture phenotypes included those that had lost the ability to form microsclerotia and subsequently used for virulence assay. Based on the virulence assay of 181 transformants, we identified several mutant strains of V. dahliae that did not cause symptoms on lettuce plants. Among these mutants, T-DNA was inserted in genes encoding an endoglucanase 1 (VdEg-1), a hydroxyl-methyl glutaryl-CoA synthase (VdHMGS), a major facilitator superfamily 1 (VdMFS1), and a glycosylphosphatidylinositol (GPI) mannosyltransferase 3 (VdGPIM3). These results suggest that ATMT can effectively be used to identify genes associated with pathogenicity and other functions in V. dahliae.     

Genetic analysis of an interspecific cross in ornamental Viburnum (Viburnum)

A genetic analysis was performed on a population derived from crosses between Viburnum lantana and Viburnum carlesii. Linkage maps were developed for each species using AFLP, random amplified polymorphic DNA (RAPD), and sequence-tagged site markers and a half-sib approach that took advantage of both the polymorphism between the species and the heterozygosity within each parent. The map for V. lantana consisted of 153 DNA markers and spanned approximately 750 cM, whereas that for V. carlesii contained 133 markers and covered 700 cM. These maps were used to determine the location of several major genes influencing leaf spot resistance, Verticillium wilt resistance, bud color, and flower scent. Both species contained moderate levels of heterozygosity. Flow cytometric analysis revealed that the genome of V. lantana was 40% larger than that of V. carlesii, and this difference was paralleled by a proportionally greater number of intercross markers (markers segregating 3:1) from V. lantana than from V. carlesii. In addition, V. lantana (n = 9) displayed a 10th linkage group for which no homolog in V. carlesii (n = 9) could be found and which contained only markers present in the former species and absent in the latter. These results suggest that Viburnum could be an interesting genetic model for Caprifoliaceae sensu lato.     

The ascomycete Verticillium longisporum is a hybrid and a plant pathogen with an expanded host range

Hybridization plays a central role in plant evolution, but its overall importance in fungi is unknown. New plant pathogens are thought to arise by hybridization between formerly separated fungal species. Evolution of hybrid plant pathogens from non-pathogenic ancestors in the fungal-like protist Phytophthora has been demonstrated, but in fungi, the most important group of plant pathogens, there are few well-characterized examples of hybrids. We focused our attention on the hybrid and plant pathogen Verticillium longisporum, the causal agent of the Verticillium wilt disease in crucifer crops. In order to address questions related to the evolutionary origin of V. longisporum, we used phylogenetic analyses of seven nuclear loci and a dataset of 203 isolates of V. longisporum, V. dahliae and related species. We confirmed that V. longisporum was diploid, and originated three different times, involving four different lineages and three different parental species. All hybrids shared a common parent, species A1, that hybridized respectively with species D1, V. dahliae lineage D2 and V. dahliae lineage D3, to give rise to three different lineages of V. longisporum. Species A1 and species D1 constituted as yet unknown taxa. Verticillium longisporum likely originated recently, as each V. longisporum lineage was genetically homogenous, and comprised species A1 alleles that were identical across lineages.