A secreted ribonuclease effector from Verticillium dahliae localizes in the plant nucleus to modulate host immunity

The arms race between fungal pathogens and plant hosts involves recognition of fungal effectors to induce host immunity. Although various fungal effectors have been identified, the effector functions of ribonucleases are largely unknown. Herein, we identified a ribonuclease secreted by Verticillium dahliae (VdRTX1) that translocates into the plant nucleus to modulate immunity. The activity of VdRTX1 causes hypersensitive response (HR)‐related cell death in Nicotiana benthamiana and cotton. VdRTX1 possesses a signal peptide but is unlikely to be an apoplastic effector because its nuclear localization in the plant is necessary for cell death induction. Knockout of VdRTX1 significantly enhanced V. dahliae virulence on tobacco while V. dahliae employs the known suppressor VdCBM1 to escape the immunity induced by VdRTX1. VdRTX1 homologs are widely distributed in fungi but transient expression of 24 homologs from other fungi did not yield cell death induction, suggesting that this function is specific to the VdRTX1 in V. dahliae. Expression of site‐directed mutants of VdRTX1 in N. benthamiana leaves revealed conserved ligand‐binding sites that are important for VdRTX1 function in inducing cell death. Thus, VdRTX1 functions as a unique HR‐inducing effector in V. dahliae that contributes to the activation of plant immunity.     

Verticillium dahliae LysM effectors differentially contribute to virulence on plant hosts

Chitin‐binding lysin motif (LysM) effectors contribute to the virulence of various plant‐pathogenic fungi that are causal agents of foliar diseases. Here, we report the LysM effectors of the soil‐borne fungal vascular wilt pathogen Verticillium dahliae. Comparative genomics revealed three core LysM effectors that are conserved in a collection of V. dahliae strains. Remarkably, and in contrast with the previously studied LysM effectors of other plant pathogens, no expression of core LysM effectors was monitored in planta in a taxonomically diverse panel of host plants. Moreover, targeted deletion of the individual LysM effector genes in V. dahliae strain JR2 did not compromise virulence in infections on Arabidopsis, tomato or Nicotiana benthamiana. Interestingly, an additional lineage‐specific LysM effector is encoded in the genome of V. dahliae strain VdLs17, but not in any other V. dahliae strain sequenced to date. Remarkably, this lineage‐specific effector is expressed in planta and contributes to the virulence of V. dahliae strain VdLs17 on tomato, but not on Arabidopsis or N. benthamiana. Functional analysis revealed that this LysM effector binds chitin, is able to suppress chitin‐induced immune responses and protects fungal hyphae against hydrolysis by plant hydrolytic enzymes. Thus, in contrast with the core LysM effectors of V. dahliae, this lineage‐specific LysM effector of strain VdLs17 contributes to virulence in planta.     

iTRAQ-based proteomics analysis of autophagy-mediated immune responses against the vascular fungal pathogen Verticillium dahliae in Arabidopsis

The mechanisms underlying the functional link between autophagy and plant innate immunity remain largely unknown. In this study, we investigated the autophagy-mediated plant defense responses against Verticillium dahliae (V. dahliae) infection by comparative proteomics and cellular analyses. An assessment of the autophagy activity and disease development showed that autophagic processes were tightly related to the tolerance of Arabidopsis plant to Verticillium wilt. An isobaric tags for relative and absolute quantification (iTRAQ)-based proteomics analysis was performed, and we identified a total of 780 differentially accumulated proteins (DAPs) between wild-type and mutant atg10-1 Arabidopsis plants upon V. dahliae infection, of which, 193 ATG8-family-interacting proteins were identified in silico and their associations with autophagy were verified for several selected proteins. Three important aspects of autophagy-mediated defense against V. dahliae infection were revealed: 1) autophagy is required for the activation of upstream defense responses; 2) autophagy-mediated mitochondrial degradation (mitophagy) occurs and is an important player in the defense process; and 3) autophagy promotes the transdifferentiation of perivascular cells and the formation of xylem hyperplasia, which are crucial for protection against this vascular disease. Together, our results provide several novel insights for understanding the functional association between autophagy and plant immune responses.     

Molecular characterization and functional analysis of a specific secreted protein from highly virulent defoliating Verticillium dahliae

Verticillium dahliae Kleb. is a phytopathogenic fungus that causes wilt diseases in hundreds of dicotyledonous plant species. Previous research has demonstrated that the secretome plays an important role in the pathogenicity of V. dahliae. In this study, the specific secreted protein gene (VdSSP1) in highly virulent defoliating V. dahliae strain VDG1 was cloned, and considered to be a secreted protein by signal peptide activity assay. VdSSP1 deletion mutants in VDG1 significantly compromised virulence, and the fungal growth decreased in media with pectin and starch as carbon sources. Pathogenicity and carbon utilization were restored upon complementation of the VdSSP1 deletion strains or low virulence non-defoliating strain VDG2, which lacks VdSSP1. It is indicated that the virulence role of VdSSP1 is associated with plant cell wall degradation. In conclusion, our data suggested that VdSSP1 is a secreted protein that is engaged in the pathogenicity of the highly virulent defoliating V. dahliae.     

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.     

Biological control of Verticillium wilt of olive by Paenibacillus alvei,strain K165

In the present study, the efficiency of the biocontrol agent Paenibacillus alvei (strain K165) to suppress Verticillium wilt of olive tree was evaluated in greenhouse and field experiments. In planta bioassays were conducted under greenhouse conditions and revealed that K165 significantly decreased symptoms on the susceptible cultivar ‘Amfissis’ by 44.5 and 51.6 % of the final disease severity index and relative area under disease progress curve (AUDPC), respectively. Thereafter, the suppressive effect of K165 against Verticillium dahliae was studied for two consecutive years (2007 and 2008) in a newly established olive orchard of the susceptible cv Amfissis and the resistant cv Kalamon, naturally infested with V. dahliae. The evaluation of K165 was carried out by recording symptoms, isolations and qPCR quantification of the pathogen in olive tissues. In both years, ‘Amfissis’ trees treated with K165 showed significantly lower final disease severity and relative AUDPC values compared to the non treated controls, whereas, in 2008 decreased symptom severity was associated with significantly lower V. dahliae DNA levels in plant tissues, indicating the suppressive effect of the biocontrol agent. However, no significant suppression was observed in ‘Kalamon’. Pathogen isolations along with qPCR quantification revealed a seasonal fluctuation of V. dahliae biomass in olive tissues with higher amounts occurring in May, and lower amounts in February, August and November. This is the first report of biological control of Verticillium wilt of olive tree under field conditions, associated with reduced pathogen levels inside the xylem tissues.     

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.     

Live-cell imaging elaborating epidermal invasion and vascular proliferation/colonization strategy of Verticillium dahliae in host plants

The soilborne ascomycete fungus Verticillium dahliae causes destructive vascular wilt disease in hundreds of dicotyledonous plant species. However, our understanding of the early invasion from the epidermis to the vasculature and the prompt proliferation and colonization in the xylem tissues remains poor. To elaborate the detailed infection strategy of V. dahliae in host plants, we traced the whole infection process of V. dahliae by live-cell imaging combined with high-resolution scanning electron microscopy. The 4D image series demonstrated that the apex of invading hyphae becomes tapered and directly invades the intercellular space of root epidermal cells at the initial infection. Following successful epidermal invasion, the invading hyphae extend in the intercellular space of the root cortex toward the vascular tissues. Importantly, the high-resolution microscopic and live-cell images demonstrated (a) that conidia are formed via budding at the apex of the hyphae in the xylem vessels to promote systemic propagation vertically, and (b) that the hyphae freely cross adjacent xylem vessels through the intertracheary pits to achieve horizontal colonization. Our findings provide a solid cellular basis for future studies on both intracellular invasion and vascular colonization/proliferation during V. dahliae infection and pathogenesis in host plants.     

嫁接茄子根系分泌物变化及其对黄萎菌的影响

采用番茄为砧木嫁接茄子,经过GC-MS检测,研究了黄萎菌胁迫前后嫁接对茄子植株根系次生代谢的影响。通过比较黄萎菌胁迫前后茄子根系次生代谢物质的变化,探讨了嫁接在胁迫前后对植株根系次生代谢的调节作用,并对嫁接茄根系分泌物中丁二酸二甲酯对茄子黄萎菌及茄子种子萌发、幼苗生长的化感效应进行了研究。结果表明,黄萎菌胁迫前,嫁接影响了根系的次生代谢物质的分泌,表现为物质种类和数量的增加,各类物质相对含量改变。嫁接茄子根系分泌物中检测出9大类、66种物质,比自根茄处理多出4大类、33种物质。黄萎菌胁迫时,嫁接茄子田间表现出明显的抗病性;进一步对根系分泌物进行检测发现,嫁接茄根系分泌物中物质种类和相对含量与自根茄处理相比均有显著差别;嫁接茄根系分泌物中烃和酚醇类物质相对含量分别增加了3.25%和0.07%,苯类、茚类和脂肪酸酯类物质相对含量降低,降幅分别为2.62%、0.26%和0.07%。新出现了胺类物质,芴类物质。黄萎菌胁迫前后茄子根系次生代谢物质成分同样发生了变化。与接菌前嫁接茄子植株根系分泌物相比,接菌后嫁接处理的苯类、茚类、酚醇类和胺类物质相对含量增加,增幅分别为22.07%、1.72%、1.21%和0.34%;烃类和脂肪酸酯类物质相对含量降低了1.28%和21.75%;酮类、咔唑类和芴类物质消失。新增物质中以丁二酸二甲酯的相对含量最高,达14.38%。随后的生物检测结果显示,丁二酸二甲酯能够提高茄子田间抗病性,对黄萎菌菌丝生长起化感抑制作用,并促进了茄子种子的萌发和幼苗的生长,随着处理浓度升高作用效果增强,并在1 mmol/L处理时达到最佳作用效果。     

Interactions between Verticillium dahliae and its host: vegetative growth,pathogenicity, plant immunity

Verticillium dahliae is a soil-borne phytopathogenic fungus that causes vascular wilt diseases in a wide variety of crop plants, resulting in extensive economic losses. In the past 5 years, progress has been made in elaborating the interaction between this hemibiotrophic fungus and its host plants. Some genes responsible for the vegetative growth and/or pathogenicity in V. dahliae have been identified. Plants have accrued a series of defense mechanisms, including inducible defense signaling pathways and some resistant genes to combat V. dahliae infection. Here, we have reviewed the progress in V. dahliae–plant interaction research.     

MiR395 Overexpression Increases Eggplant Sensibility to <Emphasis Type="Italic">Verticillium dahliae</Emphasis> Infection

Verticillium wilt (V. wilt), a notorious wilt disease caused by Verticillium dahliae, often leads to the reduction of eggplant (Solanum melongena L.) production. MiRNAs, as a class of small RNAs, can regulate gene expression and then affect growth and development in plants. MiR395 has been proven to respond to sulfate-deficient stress in Arabidopsis thaliana and sulfate is well known to have a close relationship with plant disease resistance. To explore the function of eggplant miR395, we examined its expression in V. dahliae-infected eggplant by qRT-PCR and found miR395 exhibited a gradual reduction trend with time after infection. We then expressed pre-miR395 from Arabidopsis thaliana in Suqi eggplant and resistance analysis showed that miR395 overexpressed plants were hypersensitive to V. dahliae infection. We further measured the content of GSH and activities of POD and SOD and the results indicated that the index of GSH/POD/SOD in the overexpressed plants was lower than that of the wild-type control under V. dahliae infection. These results suggest that miR395 plays a negative role in eggplant response to V. dahliae infection.     

Soil Temperature Determines the Reaction of Olive Cultivars to Verticillium dahliae Pathotypes

Background

Development of Verticillium wilt in olive, caused by the soil-borne fungus Verticillium dahliae, can be influenced by biotic and environmental factors. In this study we modeled i) the combined effects of biotic factors (i.e., pathotype virulence and cultivar susceptibility) and abiotic factors (i.e., soil temperature) on disease development and ii) the relationship between disease severity and several remote sensing parameters and plant stress indicators.

Methodology

Plants of Arbequina and Picual olive cultivars inoculated with isolates of defoliating and non-defoliating V. dahliae pathotypes were grown in soil tanks with a range of soil temperatures from 16 to 32°C. Disease progression was correlated with plant stress parameters (i.e., leaf temperature, steady-state chlorophyll fluorescence, photochemical reflectance index, chlorophyll content, and ethylene production) and plant growth-related parameters (i.e., canopy length and dry weight).

Findings

Disease development in plants infected with the defoliating pathotype was faster and more severe in Picual. Models estimated that infection with the defoliating pathotype was promoted by soil temperatures in a range of 16 to 24°C in cv. Picual and of 20 to 24°C in cv. Arbequina. In the non-defoliating pathotype, soil temperatures ranging from 16 to 20°C were estimated to be most favorable for infection. The relationship between stress-related parameters and disease severity determined by multinomial logistic regression and classification trees was able to detect the effects of V. dahliae infection and colonization on water flow that eventually cause water stress.

Conclusions

Chlorophyll content, steady-state chlorophyll fluorescence, and leaf temperature were the best indicators for Verticillium wilt detection at early stages of disease development, while ethylene production and photochemical reflectance index were indicators for disease detection at advanced stages. These results provide a better understanding of the differential geographic distribution of V. dahliae pathotypes and to assess the potential effect of climate change on Verticillium wilt development.     

Characterization of a Novel Cotton Subtilase Gene GbSBT1 in Response to Extracellular Stimulations and Its Role in Verticillium Resistance

Verticillium wilt is a disastrous vascular disease in plants caused by Verticillium dahliae. Verticillium pathogens secrete various disease-causing effectors in cotton. This study identified a subtilase gene GbSBT1 from Gossypium babardense and investigated the roles against V. dahliae infection. GbSBT1 gene expression is responsive to V. dahliae defense signals, jasmonic acid, and ethylene treatments. Moreover, the GbSBT1 protein is mainly localized in the cell membrane and moves into the cytoplasm following jasmonic acid and ethylene treatments. Silencing GbSBT1 gene expression through virus-induced GbSBT1 gene silencing reduced the tolerance of Pima-90 (resistant genotype), but not facilitated the infection process of V. dahliae in Coker-312 (sensitive genotype). Moreover, the ectopically expressed GbSBT1 gene enhanced the resistance of Arabidopsis to Fusarium oxysporum and V. dahliae infection and activated the expression levels of defense-related genes. Furthermore, pull-down, yeast two-hybrid assay, and BiFC analysis revealed that GbSBT1 interacts with a prohibitin (PHB)-like protein expressed in V. dahliae pathogens during infection. In summary, GbSBT1 recognizes the effector PHB protein secreted from V. dahliae and is involved in Verticillium-induced resistance in cotton.     

Effect of Verticillium dahliae on Photosynthesis, Leaf Expansion and Senescence of Field-grown Sunflower

On the basis of known sunflower (Helianthus annuus L.) responsesto soil water deficit, it is proposed that the effect of thefungus Verticillium dahliae Klebahn on plant leaf area precedesand is greater than its effect on leaf photosynthesis and stomatalconductance. To test this hypothesis, we measured shoot andleaf area growth, leaf photosynthetic rate, stomatal conductanceand disease symptoms in a field experiment including hybridsof high (Sankol) and low (Dekasol 3900) susceptibility to V.dahliae. Plants inoculated with V. dahliae and controls werecompared. We also investigated the effect of V. dahliae on keycomponents of plant leaf area, leaf expansion and senescence,in inoculated and control plants of Sankol and Toba, a hybridof intermediate susceptibility to V. dahliae. Reduction in plantleaf area caused by V. dahliae was first detected 31 d afterinoculation (DAI), when visual symptoms of disease in inoculatedplants were slight (Sankol) or absent (Dekasol 3900). Reductionin leaf photosynthesis was first observed 66 DAI; stomatal conductanceand leaf dark respiration were both unaffected by V. dahliaeduring the whole experiment. In comparison with controls, V.dahliae reduced seasonal duration of plant leaf area by 25%in Dekalb 3900 and by 55% in Sankol, whereas the average reductionin leaf photosynthetic rate was 9%. In correspondence with thereduction in leaf area duration, inoculation reduced shoot drymatter of mature Sankol by 50%. In both experiments, less leafexpansion accounted for most of the early reduction in plantleaf area; as the disease progressed, increasing senescencealso contributed to reduced plant leaf area. It is concludedthat the response of sunflower to V. dahliae resembled the responseof the plant to soil water deficit: (1) plant leaf area, ratherthan leaf photosynthetic rate, accounted for the reduction ingrowth in mass; and (2) reduced leaf expansion early in theseason and faster leaf senescence in older plants accountedfor the decrease in plant leaf area. Copyright 2000 Annals ofBotany Company Helianthus annuus, Verticillium dahliae, allometry, apical dominance, drought, leaf expansion, leaf senescence, photosynthesis, stomatal conductance, growth     

VdMsb regulates virulence and microsclerotia production in the fungal plant pathogen Verticillium dahliae

The vascular wilt fungus Verticillium dahliae infects the roots of cotton plants and can seriously diminish the yield and quality of this and other dicotyledons. However, the key genes involved in V. dahliae infection and pathogenesis in cotton remain unclear. Msb encodes a transmembrane mucin that is highly conserved in the MAPK signal pathway. Msb has been implicated previously in pathogenicity in various aerial plant fungi. In this study, V. dahliae Msb (VdMsb) was found to be required for fungal virulence and microsclerotia production. Strains lacking VdMsb exhibited reduced conidiation and microsclerotia formation. Compared with wild-type and gene-complemented strains, the invasive growth and adhesive capacity of VdMsb deletion mutants were significantly decreased. These results suggest that VdMsb plays a role in development and virulence in V. dahliae.     

Two CAPS markers predict Verticillium wilt resistance in wild Solanum species

Verticillium wilt of potato is a persistent problem in the USA and worldwide. The disease, which is caused primarily by the fungus Verticillium dahliae, is difficult to manage, causes yield losses, and contaminates soil for subsequent plantings. Control strategies based on host resistance are seen as long-term, stable solutions, but difficult to achieve given the genetic nature of the host and the challenges associated with resistance evaluations. To provide breeders with marker-assisted selection opportunities, we generated a pair of cleaved amplified polymorphic sequence molecular markers within the coding region of Ve2, a potato gene with homology to the tomato Ve1 gene that confers resistance to V. dahliae. The position of the marker was determined according to the consensus sequences of Ve2 homologs of wild Solanum species with resistance to V. dahliae. Marker testing indicated their broad applicability, being able to track the resistance to V. dahliae in progeny containing genetic information derived from species S. chacoense, S. brevicaule, S. berthaultii, S. tarijense, and S. tuberosum. Furthermore, the two isolates of V. dahliae used in our inoculation experiments differed in virulence and demonstrated specificity for some wild potato species. Experimentation leading to the development of the markers and tests of their usefulness against a wide range of diploid potato germplasm is presented.