Broad taxonomic characterization of Verticillium wilt resistance genes reveals an ancient origin of the tomato Ve1 immune receptor

Plant‐pathogenic microbes secrete effector molecules to establish themselves on their hosts, whereas plants use immune receptors to try and intercept such effectors in order to prevent pathogen colonization. The tomato cell surface‐localized receptor Ve1 confers race‐specific resistance against race 1 strains of the soil‐borne vascular wilt fungus Verticillium dahliae which secrete the Ave1 effector. Here, we describe the cloning and characterization of Ve1 homologues from tobacco (Nicotiana glutinosa), potato (Solanum tuberosum), wild eggplant (Solanum torvum) and hop (Humulus lupulus), and demonstrate that particular Ve1 homologues govern resistance against V. dahliae race 1 strains through the recognition of the Ave1 effector. Phylogenetic analysis shows that Ve1 homologues are widely distributed in land plants. Thus, our study suggests an ancient origin of the Ve1 immune receptor in the plant kingdom.     

Allele-specific CAPS marker in a <Emphasis Type="Italic">Ve1</Emphasis> homolog of <Emphasis Type="Italic">Capsicum annuum</Emphasis> for improved selection of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> resistance

Verticillium wilt (Verticillium dahliae) is an economically important disease for many high-value crops. The pathogen is difficult to manage due to the long viability of its resting structures, wide host range, and the inability of fungicides to affect the pathogen once in the plant vascular system. In chile pepper (Capsicum annuum), breeding for resistance to Verticillium wilt is especially challenging due to the limited resistance sources. The dominant Ve locus in tomato (Solanum lycopersicum) contains two closely linked and inversely oriented genes, Ve1 and Ve2. Homologs of Ve1 have been characterized in diverse plant species, and interfamily transfer of Ve1 confers race-specific resistance. Queries in the chile pepper WGS database in NCBI with Ve1 and Ve2 sequences identified one open reading frame (ORF) with homology to the tomato Ve genes. Comparison of the candidate CaVe (Capsicum annuum Ve) gene sequences from susceptible and resistant accessions revealed 16 single nucleotide polymorphisms (SNPs) and several haplotypes. A homozygous haplotype was identified for the susceptible accessions and for resistant accessions. We developed a cleaved amplified polymorphic sequence (CAPS) molecular marker within the coding region of CaVe and screened diverse germplasm that has been previously reported as being resistant to Verticillium wilt in other regions. Based on our phenotyping using the New Mexico V. dahliae isolate, the marker could select resistance accessions with 48% accuracy. This molecular marker is a promising tool towards marker-assisted selection for Verticillium wilt resistance and has the potential to improve the efficacy of chile pepper breeding programs, but does not eliminate the need for a bioassay. Furthermore, this work provides a basis for future research in this important pathosystem.     

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.     

Development of a molecular marker associated with Verticillium wilt resistance in diploid interspecific potato hybrids

Verticillium wilt (VW) is a widespread and serious potato (Solanum tuberosum) disease caused by the soilborne fungi Verticillium dahliae and V. albo-atrum. Breeding for VW resistance in potato is challenging due to ambiguous symptom expression, a lack of high throughput screening techniques, and variability in colonization by the fungus among and within plants. Genetic studies have identified major genes that confer resistance in diploid Solanum chacoense (V _c ) and interspecific hybrids (V _w and V _t ). However, to date, these genes have not been used to develop molecular markers for the identification of resistant clones. Tomato Ve1 and Ve2 gene sequence information was used to amplify candidate Ve gene orthologs from both resistant and susceptible diploid potato hybrids. A CAPS marker was generated to track VW resistance in a backcross population segregating for resistance. The marker was also tested for its usefulness in other breeding lines. Our results indicate that this marker is effective for selection of the V _w gene in segregating breeding populations.     

Island Cotton Gbve1 Gene Encoding A Receptor-Like Protein Confers Resistance to Both Defoliating and Non-Defoliating Isolates of Verticillium dahliae

Verticillium wilt caused by soilborne fungus Verticillium dahliae could significantly reduce cotton yield. Here, we cloned a tomato Ve homologous gene, Gbve1, from an island cotton cultivar that is resistant to Verticillium wilt. We found that the Gbve1 gene was induced by V. dahliae and by phytohormones salicylic acid, jasmonic acid, and ethylene, but not by abscisic acid. The induction of Gbve1 in resistant cotton was quicker and stronger than in Verticillium-susceptible upland cotton following V. dahliae inoculation. Gbve1 promoter-driving GUS activity was found exclusively in the vascular bundles of roots and stems of transgenic Arabidopsis. Virus-induced silencing of endogenous genes in resistant cotton via targeting a fragment of the Gbve1 gene compromised cotton resistance to V. dahliae. Furthermore, we transformed the Gbve1 gene into Arabidopsis and upland cotton through Agrobacterium-mediated transformation. Overexpression of the Gbve1 gene endowed transgenic Arabidopsis and upland cotton with resistance to high aggressive defoliating and non-defoliating isolates of V. dahliae. And HR-mimic cell death was observed in the transgenic Arabidopsis. Our results demonstrate that the Gbve1 gene is responsible for resistance to V. dahliae in island cotton and can be used for breeding cotton varieties that are resistant to Verticillium wilt.     

Mapping genes for resistance to<Emphasis Type="Italic"> Verticillium albo-atrum</Emphasis> in tetraploid and diploid potato populations using haplotype association tests and genetic linkage analysis

Verticillium wilt disease of potato is caused predominantly by Verticillium albo-atrum and V. dahliae. StVe1 —a putative QTL for resistance against V. dahliae —was previously mapped to potato chromosome 9. To develop allele-specific, SNP-based markers within the locus, the StVe1 fragment from a set of 30 North American potato cultivars was analyzed. Three distinct and highly diverse haplotypes can be distinguished at the StVe1 locus. These were detected in 97%, 33%, and 10% of the cultivars analyzed. We tested for haplotype association and for genetic linkage between the StVe1 haplotypes and resistance of tetraploid potato to V. albo-atrum. Moreover, field resistance was assessed in diploid populations with known molecular linkage maps in order to identify novel QTLs. Resistance QTLs against V. albo-atrum were detected on four chromosomes (2, 6, 9, and 12) at the diploid level, with one QTL on chromosome 2 contributing over 40% to the total phenotypic variation of the trait. At the tetraploid level, a significant association between the StVe1-839-C haplotype and susceptibility to the disease was detected, suggesting that resistance-related genes directed against V. albo-atrum and V. dahliae are located in the same genomic region of chromosome 9. However, on the basis of the present analysis, we cannot determine whether these genes are closely linked or if a single gene provides resistance against both Verticillium species. To assess the usefulness of the StVe1-839-C haplotype for marker-assisted selection, we subjected the resistance data to Bayesian analysis, and calculated positive (0.65) and negative (0.75) predictive values, and overall predictive accuracy (0.72). Our results indicate that tagging of additional genes for resistance to Verticillium with molecular markers will be required for efficient marker-assisted selection.Communicated by M.-A. Grandbastien     

Host Specialization among Vegetative Compatibility Groups of Verticillium dahliae in Relation to Verticillium longisporum

A collection of 24 isolates of Verticillium dahliae and 10 isolates of Verticillium longisporum originating from nine different host plants and from several geographic regions was tested for host specificity on 11 economically important crops such as potato, tomato, strawberry, linseed, three legumes and four Brassica species. In order to reveal host specificity the potential of each isolate to induce disease and affect plant yield was recorded for all isolate–host combinations. The collected data were statistically processed by means of a cluster analysis. As a result, the host range of individual isolates was found to be more dependent on the vegetative compatibility group (VCG) of the isolate than on its original host plant provenance. Twenty‐two out of 24 V. dahliae isolates belonged to either VCG 2B or 4B. VCG 2B isolates showed specificity for legumes, strawberry, potato and linseed, whereas VCG 4B was specifically virulent on potato, strawberry and linseed. Subgroups within VCG 2B and 4B almost lacking any host preference were designated 2B* and 4B*. Three isolates from VCG 2B*, however, severely attacked tomato which is a host outside the authentic host range of VCG 2B. The pathogenicity of V. longisporum isolates was restricted to cruciferous hosts. Conversely, cruciferous plants were not affected by isolates from VCGs 2B and 4B of V. dahliae. This lack of cross‐infectivity of certain subpopulations of V. dahliae and of V. longisporum may be useful in the management of this soil‐borne wilt 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.     

Modeling of Yield Loss in Potato Early Dying Caused by Pratylenchus penetrans and Verticillium dahliae

Yield-loss models were developed for potato early dying, caused by an interaction between Verticillium dahliae and Pratylenchus penetrans. Yield data were collected over 5 years (1985-1989) from potato plants grown in microplots infested with V. dahliae and (or) P. penetrans. The model y = b₀ + (1 - b₀)/(1 + [VD/36.7]), where y was the relative yield (with uninfested controls = 1.0) and VD was the preplant density of V. dahliae microsclerotia per cm³ soil, was fitted to the data set. When P. penetrans = 0, b₀ = 0.55 (SE = 0.099), and when P. penetrans > 0, b₀ = 0.23 (SE = 0.035). This model assumed that yield loss was proportional to the concentration of preplant microsclerotia of V. dahliae, and only qualitatively related to presence or absence of P. penetrans. This study contrasts with previous reports that predict yield loss being proportional to preplant population densities of both P. penetrans and V. dahliae.     

The symptoms and causal agents of early-dying disease (Verticillium wilt) of potatoes

The only characteristic symptom produced by Verticillium albo-atrum and V. dahliae in infected potato plants is unilateral chlorosis and necrosis: this was not shown until the approach of host maturity, and was distinguishable from symptoms of natural senescence only in its slightly earlier expression. Of six species of Verticillium tested against potato (King Edward), V. albo-atrum, V. dahliae, V. nigrescens and V. nubilum were pathogenic (all produced ‘wilt’ symptoms and relative virulence was in that decreasing order) but V. tricorpus and V. lateritium did not induce disease. Isolates of V. albo-atrum and V. dahliae, obtained from a number of other host plants, were also pathogenic to potato. Possible reasons are given for the fewness of records of ‘Early dying’ disease (Verticillium wilt) of potatoes in the field.     

Interactions Among Pratylenchus penetrans,P. scribneri,and Verticillium dahliae in the Potato Early Dying Disease Complex

Microplots were infested with combinations of the fungus Verticillium dahliae and Pratylenchus penetrans and P. scribneri to test for individual and combined effects of these organisms on potato yield and nematode reproduction. Verticillium dahliae alone caused yield losses in all 3 years of the experiment, and the interaction between P. penetrans and V. dahliae was significant (P ≤ 0.05) in 2 years. Pratylenchus penetrans alone caused yield losses in 2 years and P. scribneri alone caused yield losses in 1 year. No two-way or three-way interaction was found involving P. scribneri. In 1987, reproduction for low densities of P. penetrans was 5 times higher when P. scribneri was also present than when it was absent, and 3.5 times higher in 1988. In nematode species mixtures, reproduction of P. scribneri was decreased by V. dahliae in 1987-88. The final population density of P. scribneri was negatively affected by V. dahliae and positively related to the initial proportion of P. scribneri to P. penetrans. In species mixtures with proportions of P. penetrans ranging from 0.1 to 0.5, reproduction of P. penetrans was negatively affected by V. dahliae and decreased linearly in relation to the increase in the initial proportion of P. penetrans in both years. The final population density of P. penetrans was affected only by V. dahliae.     

Interfamily transfer of tomato Ve1 mediates Verticillium resistance in Arabidopsis

Vascular wilts caused by soil-borne fungal species of the Verticillium genus are devastating plant diseases. The most common species, Verticillium dahliae and Verticillium albo-atrum, have broad host ranges and are notoriously difficult to control. Therefore, genetic resistance is the preferred method for disease control. Only from tomato (Solanum lycopersicum) has a Verticillium resistance locus been cloned, comprising the Ve1 gene that encodes a receptor-like protein-type cell surface receptor. Due to lack of a suitable model for receptor-like protein (RLP)-mediated resistance signaling in Arabidopsis (Arabidopsis thaliana), so far relatively little is known about RLP signaling in pathogen resistance. Here, we show that Ve1 remains fully functional after interfamily transfer to Arabidopsis and that Ve1-transgenic Arabidopsis is resistant to race 1 but not to race 2 strains of V. dahliae and V. albo-atrum, nor to the Brassicaceae-specific pathogen Verticillium longisporum. Furthermore, we show that signaling components utilized by Ve1 in Arabidopsis to establish Verticillium resistance overlap with those required in tomato and include SERK3/BAK1, EDS1, and NDR1, which strongly suggests that critical components for resistance signaling are conserved. We subsequently investigated the requirement of SERK family members for Ve1 resistance in Arabidopsis, revealing that SERK1 is required in addition to SERK3/BAK1. Using virus-induced gene silencing, the requirement of SERK1 for Ve1-mediated resistance was confirmed in tomato. Moreover, we show the requirement of SERK1 for resistance against the foliar fungal pathogen Cladosporium fulvum mediated by the RLP Cf-4. Our results demonstrate that Arabidopsis can be used as model to unravel the genetics of Ve1-mediated resistance.     

Identification and Differentiation of Verticillium Species and V. longisporum Lineages by Simplex and Multiplex PCR Assays

Accurate species identification is essential for effective plant disease management, but is challenging in fungi including Verticillium sensu stricto (Ascomycota, Sordariomycetes, Plectosphaerellaceae), a small genus of ten species that includes important plant pathogens. Here we present fifteen PCR assays for the identification of all recognized Verticillium species and the three lineages of the diploid hybrid V. longisporum. The assays were based on DNA sequence data from the ribosomal internal transcribed spacer region, and coding and non-coding regions of actin, elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase and tryptophan synthase genes. The eleven single target (simplex) PCR assays resulted in amplicons of diagnostic size for V. alfalfae, V. albo-atrum, V. dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii, V. nonalfalfae, V. nubilum, V. tricorpus, V. zaregamsianum, and Species A1 and Species D1, the two undescribed ancestors of V. longisporum. The four multiple target (multiplex) PCR assays simultaneously differentiated the species or lineages within the following four groups: Verticillium albo-atrum, V. alfalfae and V. nonalfalfae; Verticillium dahliae and V. longisporum lineages A1/D1, A1/D2 and A1/D3; Verticillium dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii and V. tricorpus; Verticillium isaacii, V. klebahnii and V. tricorpus. Since V. dahliae is a parent of two of the three lineages of the diploid hybrid V. longisporum, no simplex PCR assay is able to differentiate V. dahliae from all V. longisporum lineages. PCR assays were tested with fungal DNA extracts from pure cultures, and were not evaluated for detection and quantification of Verticillium species from plant or soil samples. The DNA sequence alignments are provided and can be used for the design of additional primers.     

Expression of a wild eggplant ribosomal protein L13a in potato enhances resistance to Verticillium dahliae

The ribosomal protein L13a was recently found to play a role not only in protein synthesis, but also in modulating translation. We reported in previous study that L13a was responsive to Verticillium dahliae (V. dahliae) infection in a highly resistant eggplant species (Solanum torvum, SW). To elucidate the possible role of L13a in V. dahliae infection, we cloned and characterized its cDNA (designated StoL13a) in this study. StoL13a encodes a protein of 23.46 kDa and shows a high similarity to the L13a from other plants. Phylogenetic analysis revealed that StoL13a clearly grouped with L13a-like sequences. Expression level of StoL13a altered in response to V. dahliae infection and phytohormone treatment. We expressed StoL13a in V. dahliae sensitive potato, and found that the transgenic potato plants were more resistant to V. dahliae infection than the control plants with disease index of 15–25.2. The transgenic plants showed a lower quantity of reactive oxygen species and attenuated oxidative injury. Also, six defense and antioxidant enzyme genes were up-regulated in the StoL13a ectopic expression plants. These results suggest that the StoL13a plays a role in plant defense to V. dahliae infection.