Host-induced gene silencing of the Verticillium dahliae thiamine transporter protein gene(VdThit)confers resistance to Verticillium wilt in cotton

Verticillium wilt(VW),induced by the soil-borne fungus Verticillium dahliae(Vd),poses a substantial threat to a diverse array of plant species.Employing molecular breeding technology for the development of cotton varieties with heightened resistance to VW stands out as one of the most efficacious protective measures.In this study,we successfully generated two stable transgenic lines of cotton(Gossypium hirsutum L.),VdThitRNAi-1 and VdThit-RNAi-2,using host-induced gene silencing(HIGS)technology to introduce double-stranded RNA(dsRNA)targeting the thiamine transporter protein gene(VdThit).Southern blot analysis confirmed the presence of a single-copy insertion in each line.Microscopic examination showed marked reductions in the colonization and spread of Vd-mCherry in the roots of VdThit-RNAi cotton compared to wild type(WT).The corresponding disease index and fungal biomass of VdThit-RNAi-1/2 also exhibited significant reductions.Real-time quantitative PCR(qRT-PCR)analysis demonstrated a substantial inhibition of VdThit expression following prolonged inoculation of VdThit-RNAi cotton.Small RNA sequencing(sRNA-Seq)analysis revealed the generation of a substantial number of VdThit-specific siRNAs in the VdThit-RNAi transgenic lines.Additionally,the silencing of VdThit by the siVdThit produced by VdThit-RNAi-1/2 resulted in the elevated expression of multiple genes involved in the thiamine biosynthesis pathway in Vd.Under field conditions,VdThit-RNAi transgenic cotton exhibited significantly enhanced disease resistance and yield compared with WT.In summary,our findings underscore the efficacy of HIGS targeting VdThit in restraining the infection and spread of Vd in cotton,thereby potentially enabling the development of cotton breeding as a promising strategy for managing VW.

A glycine-rich nuclear effector VdCE51 of Verticillium dahliae suppresses plant immune responses by inhibiting the accumulation of GhTRXH2

Verticillium dahliae is an important soil-borne fungal pathogen that causes great yield losses in many cash crops.Effectors of this fungus are known to regulate plant immunity but the mechanism much remains unclear.A glycine-rich nuclear effector,VdCE51,was able to suppress immune responses in tobacco against Botrytis cinerea and Sclerotinia sclerotiorum.This effector was a required factor for full virulence of V.dahliae,and its nuclear localization was a requisite for suppressing plant immunity.The thioredoxin GhTRXH2,identified as a positive regulator of plant immunity,was a host target of VdCE51.Our findings show a virulence regulating mechanism whereby the secreted nuclear effector VdCE51 interferes with the transcription of PR genes,and the SA signaling pathway by inhibiting the accumulation of GhTRXH2,thus suppressing plant immunity.

GhWRKY33 negatively regulates jasmonate-mediated plant defense to Verticillium dahliae

Verticillium wilt,caused by Verticillium dahliae,seriously restricts the yield and quality improvement of cotton.Previous studies have revealed the involvement of WRKY members in plant defense against V.dahliae,but the underlying mechanisms involved need to be further elucidated.Here,we demonstrated that Gossypium hirsutum WRKY DNA-binding protein 33(GhWRKY33) functions as a negative regulator in plant defense against V.dahliae.GhWRKY33 expression is induced rapidly by V.dahliae and methyl jasmonate,and overexpression of GhWRKY33 reduces plant tolerance to V.dahliae in Arabidopsis.Quantitative RT-PCR analysis revealed that expression of several JA-associated genes was significantly repressed in GhWRKY33 overexpressing transgenic plants.Yeast one-hybrid analysis revealed that GhWRKY33 may repress the transcription of both AtERF1 and GhERF2 through its binding to their promoters.Protein-protein interaction analysis suggested that GhWRKY33 interacts with G.hirsutum JASMONATE ZIM-domain protein 3(GhJAZ3).Similarly,overexpression of GhJAZ3 also decreases plant tolerance to V.dahliae.Furthermore,GhJAZ3 acts synergistically with GhWRKY33 to suppress both AtERF1 and GhERF2 expression.Our results imply that GhWRKY33 may negatively regulate plant tolerance to V.dahliae via the JA-mediated signaling pathway.

Increase of β -1, 3-Glucanase and Chitinase Activities in Cotton Callus Cells Treated by Salicylic Acid and Toxin of Verticillium dahliae

The different resistance of cotton (Gossypium hirsutum L.) cultivars to crude toxin of Verticillium dah/iae(VD) was correlated with the activities of chitinase and β-1, 3-glucanase in callus cells. The activities of chitinase and β-1, 3-glucanase in the callus cells treated with the VD-toxin were increased to the higher level at earlier time point in resistant cultivars than these in the susceptible cultivars. Exogenous salicylic acid (SA) induced the accumulation of chitinase and β -1,3-glucanase, which resulted in the resistance of callus cells to the VD. toxin. Western blot using a polyclonal antibody against β -1,3-glucanase identified 28 kD protein that was induced by VD-toxin, SA, or VD-toxin plus SA.

棉花黄萎病真菌Verticillium dahliae木聚糖酶基因的克隆、表达和酶学性质分析

【目的】从棉花黄萎病真菌Verticillium dahliae中克隆木聚糖酶基因,并在毕赤酵母中进行异源表达,研究酶学性质。【方法】通过多序列比对设计简并引物,扩增出真菌V.dahliae木聚糖酶基因片段,再采用基因组步行PCR技术获得全长木聚糖酶基因序列。经BLAST比对并结合GT-AG原则分析,该基因含有一个大小为63bp的内含子,利用DpnI介导的缺失方法对含内含子的全长木聚糖酶基因进行剪接,获得该基因的全长cDNA。将克隆到的cDNA在毕赤酵母GS115进行了表达,重组酶经纯化后进行酶学性质分析。【结果】BLAST比对显示,该cDNA推测的氨基酸序列和已知木聚糖酶的最高一致性为72%。测得该酶最适反应温度为45℃,最适反应pH值为6,在pH5-9维持50%以上的活性,对山毛榉材木聚糖具有最好的水解效果。Mg2+和Ca2+对酶有激活作用,分别提高了33.7%和16.6%,EDTA,β-巯基乙醇和NaN3对酶的活性基本没有影响,Tween-80和DMSO使酶活性提高了28.4%和12.8%。【结论】本文从引起棉花黄萎病的真菌V.dahliae中克隆到的木聚糖酶基因是在GenBank上登录的第一个来自棉花黄萎病真菌的木聚糖酶基因序列。本文所用的克隆方法可以高效的从植物病原真菌和白腐真菌克隆只含一个内含子的11家族的新木聚糖酶基因,避免了摸索原始菌株酶表达诱导条件,检测酶的活性等繁琐的操作。酶学性质分析显示该酶在低聚木糖的制备,面包改良上有潜在的应用价值。

Fusion Expression of Raphanus sativus-Antifungal Protein 1 (Rs-AFP1) in Escherichia coli and Its Antifungal Activity on Verticillium dahliae

The Raphanus sativus L. antifungal protein 1 (Rs_AFP1) gene was isolated by polymerase chain reaction (PCR). The complete open reading frame and the fragment encoding the putative mature protein were inserted into the prokaryotic expression vector pET_32b(+), respectively. Subsequent expression showed that the Rs_AFP1 was produced in E. coli as a 27 kD fusion protein only when the N_terminal signal peptide was removed. After treatment with thrombin to remove part of the N_terminal His.tag sequence, the bacterially expressed Rs_AFP1 was used for fungal growth inhibition assay which was conducted on Verticillium dahliae Kleb., a soil_born fungus causing the cotton wilt disease. Results showed that, in the liquid medium, the Rs_AFP1 fusion protein at a concentration of 0.3 g/L clearly inhibited the growth of V. dahliae and the germination of spores. Thus the bacterially expressed fusion protein had the antifungal activity against V. dahliae.

The cotton WRKY transcription factor GhWRKY70 negatively regulates the defense response against Verticillium dahliae

WRKY transcription factors (TFs) play important roles in the regulation of biotic and abiotic stresses. However, the functions of most WRKY TFs in upland cotton (Gossypium hirsutum) are still unknown. In this study, we functionally identified a group Ⅲ WRKY transcription factor, GhWRKY70, in upland cotton. Reverse transcription-quantitative PCR analysis showed that GhWRKY70 expression was induced by Verticillium dahliae, salicylic acid (SA) and methyl jasmonate. Virus-induced gene silencing of GhWRKY70 increased the resistance of cotton to V. dahliae. Specifically, jasmonic acid (JA) response-associated genes were upregulated and SA-related genes were downregulated in GhWRKY70-silenced cotton plants. Overexpression of GhWRKY70 reduced tolerance to V. dahliae in Arabidopsis thaliana. Transgenic Arabidopsis plants showed increased expression of SA-associated genes and reduced expression of JA response-associated genes. These results suggest that GhWRKY70 negatively regulates tolerance to V. dahliae in at least two ways: (ⅰ) by upregulating the expression of SA-associated genes and (ⅱ) by reducing the expression of JA-associated genes.

The Influence of the Verticillium dahliae Kleb Infection on the Anti-Enzyme Inside the Body of the Cotton with Different Root Injured Degree

This study was to explore the influence of the Verticillium dahliae Kleb inflection on the anti-enzyme inside the body of the cotton with a different root injured degree. When the cotton seedling was long, with four leaves, it was flushed with water carefully, and then the following were obtained： （1） complete root seedling; （2） cut root seedling - by cutting off the lower part, 3-5 cm of the root, with a disinfected knife; （3） injured root seedling - by cutting off most of the side roots, but keeping the main root. Three kinds of cotton seedlings with different roots were immersed separately in different concentrations of the germ liquid （V. dahliae） of 20 mL each. Through 0- 48 h, the wilt degree of the seedling was recorded, and the related anti-enzyme of the variety was measured. After being immersed in the germ liquid, there was a significant difference in the wilt degree of the three kinds of injured root. When the germ liquid was in the ratio of 1：10, the complete root seedling was the lightest with no wilt; the injured root seedling was the second with a 2-degree wilt; but the cut root seedling was the most serious with a 3- degree wilt. At the same time, the changes in the peroxidase and malondialdehvde activities were determined. Peroxidase （POD） activities in the cut root seedling were 38.2 U mg^-1 min^-1, in the injured root seedling were 42.96 U mg^-1 min^-1, and in the complete root seedling were the highest at 49.2 U mg^-1 min^-1. The malondialdenvde （MDA） content in cut root seedling was 39.483 mmol g^-1, injured root seedling was 27.12 mmol g^-1, and the complete root seedling was only 3.845 mmol g^-1 The activity of the related anti-enzymes, such as POD was high or low, the quantity of the MDA was more or less, which they met the order of the harm of the seedlings. The change of SOD activities in cut root seedling was the most obvious as well. After injuring and inflecting the young roots, the exterior pathological reaction of the seedling and the dynamic state biochemical reaction of the related enzymes inside the plant body were studied. It showed that the plant exterior pathology responded to the test, with the internal biochemical reaction fitting together mutually.

A review of the pathogenicity mechanism of Verticillium dahliae in cotton

Verticillium wilt,caused by the notorious fungal pathogen Verticillium dahliae,is one of the main limiting factors for cotton production.Due to the stable dormant structure microsclerotia,long-term variability and co-evolution with host plant,its pathogenicity mechanism is very complicated,and the interaction mechanism between pathogen and host plant is also unclear.So identification and functional analysis of the genes involved in the pathogenicity or virulence of this fungus will benefit to uncover the molecular pathogenic mechanism of V.dahliae.In this review,many multifunction genes covering microsclerotia development,pathogen infection,effector proteins,transcription factors,horizontal gene transfer and trans-kingdom RNA silencing have been summarized to provide a theoretical basis to deep understand the molecular pathogenicity mechanism of V.dahliae and promote to effectively control Verticillium wilt.Furtherly,these pathogenicity-related genes may be considered as targets for effective control of Verticillium wilt in cotton.

Extracellular superoxide dismutase VdSOD5 is required for virulence in Verticillium dahliae

Plants produce reactive oxygen species(ROS) to defend pathogens. To counteract this attack, certain pathogens express superoxide dismutases(SODs) to scavenge host-derived ROS. However, the roles of SODs in Verticillium dahliae, an important vascular pathogen, are not clear. Our previous study has shown that a putative extracellular SOD(VdSOD5) of V. dahliae is significantly induced by culturing in cotton tissues, suggesting that VdSOD5 may play an important role in host–pathogen interactions and virulence. Here, we showed that VdSOD5 encoded a superoxide dismutase with a cofactor copper-binding site and a functional signal peptide that can conduct protein secretion in an invertase-mutated yeast strain. The mutations in VdSOD5(ΔVdSOD5) did not change the normal vegetative growth and conidial production but reduced the virulence of V. dahliae on susceptible host cotton. Further studies showed that the transcription of Vd SOD5 was significantly up-regulated during the early stage of infection, and the loss-of-function of VdSOD5 decreased culture filtrate and fungal tissue SOD activities of V. dahliae by 74 and 28%, respectively. Compared to the wild-type strain Vd991, the ΔVdSOD5 showed the same sensitivity to the intracellular ROS generator menadione. Furthermore, nitroblue tetrazolium(NBT) staining demonstrated that VdSOD5 functioned in the detoxification of superoxides generated by host roots during infection. These results suggest that VdSOD5 of V. dahliae is an important virulence factor, secreted out of cells to combat host-derived ROS.

RAPD Patterns Characteristic for VCGs of Cotton Verticillium Wilt Pathogen,Verticillium dahliae

Nine primers were employed to detect molecular polymorphisms in 103 Verticillium dahliae isolates that represent diverse groups of Vegetative Compatibility (VC). Our results showed that these isolates confer two distinctive RAPD groups (RPGs). RPG1, consists of isolates belonging to vegetative compatibility group I (VCG I ), while RPG2 encompasses VCGⅢ and VCGⅣ . The genetic diversity associated with VCGⅢ was greater than that associated with VCG1. Five RAPD fragments, with frequencies more than 0.96 in VCG I but less than 0.1 in VCGⅢ, produced characteristic fragments for VCG I (defoliating type), suggesting strong correlation between RPGs and VCGs.

Tolerance of New Introgressive Hybrid and Backcross Forms Pathogenic Micromitisms (<i>Verticillium dahliae</i>Kleb and Fusarium oxysporum f.sp. vasinfectum)

The article is based on the use of experimental polyploidy method, with the introduction of new introgressive hybrid forms combining several species genotype with pathogenic Verticillium dahliae Kleb. and the effects of mycotoxins separated from the Fusarium oxysporum f.sp. vasinfectum micromicette on the yield of plant seeds. New artificial complex hypertension forms based on experimental polyploidy Verticillium dahliae Kleb. and Fusarium oxysporum f.sp. vasinfectum combine the potential of resistance to mycotoxins separated from microcrystals, making a tremendous contribution to the selection of new varieties and to the effectiveness of selection as a result of the use of genetic selective research as genetic-selective genetic-selector studies.

Down regulation of cotton GbTRP1 leads to accumulation of anthranilates and confers resistance to Verticillium dahliae

Background:Verticillium wilt,caused by Verticillium dahliae,is called a "cancer" disease of cotton.The discovery and identification of defense-related genes is essential for the breeding of Verticillium wilt-resistant varieties.In previous research we identified some possible broad-spectrum resistance genes.Here,we report a tryptophan synthesis-related gene GbTRP1 and its functional analysis in relation to the resistance of cotton to V.dahliae.Results:Expression analysis shows that GbTRP1 is suppressed at 1 h and 6 h post V.dahliae infection,but activated at 12 h and 24 h,and the expression of GbTRP1 is highly induced by treatment with salicylic acid and jasmonic acid.Sub-cellular localization studies show that GbTRP1 is localized in the chloroplast.Suppression of GbTRP1 expression leads to lesion-mimic phenotypes and activates the immune response in cotton by showing enhanced resistance to V.dahliae and B.cinerea.Metabolomic analysis shows that anthranilic compounds significantly accumulated in GbTRP1-silenced plants,and these metabolites can inhibit the growth of V.dahliae and B.cinerea in vitro.Conclusions:Our results show that suppression of GbTRPI expression dramatically activates the immune response and increases resistance of cotton to V.dahliae and B.cinerea,possibly due to the accumulation of anthranilate compounds.This study not only provides genetic resources for disease resistance breeding,but also may provide a basis for new chemical control methods for combatting of fungal disease in cotton.

miR398b negatively regulates cotton immune responses to Verticillium dahliae via multiple targets

MicroRNAs(miRNAs)play essential roles in plant defense responses,although such roles have not been identified in cotton in response to the plant pathogenic fungus Verticillium dahliae.In this study,the functions of miR398b and its target genes in cotton-V.dahliae interaction were investigated.The transcript levels of miR398b were down-regulated by V.dahliae infection and miR398b overexpression in cotton made the plants more susceptible to V.dahliae.The results suggest that miR398b negatively regulates cotton resistance to V.dahliae.This may occur by miR398b repression of some CC-NBS-LRR genes via translational inhibition,interfering with defense responses and leading to cotton susceptibility to V.dahliae.Alternatively,miR398b may guide the cleavage of the mRNAs of GhCSD1,GhCSD2 and GhCCS,each of which functions in reactive oxygen species(ROS)regulation and homeostasis,thereby causing excessive ROS accumulation in miR398b-overexpressing plants in response to V.dahliae infection.This study suggests conserved and novel roles of miR398b in the cotton–V.dahliae interaction.These discoveries may be coupled with new strategies in cotton breeding programs to improve resistance to V.dahliae.

Studies on the Pathogenicity Differentiation of Verticillium dahliae on Cotton Varieties

Verticillium dahliae may be classified into the two basic groups, defoliate and non-defoliate, in terms of the pathogenicity reaction of 19 isolates of Verticillium dahliae on 16 cotton varieties. According to the comprehensive resistant or susceptible expression of 8 defoliate isolates on 8 upland cotton varieties R02,R04, R05, R06, R08, R09, R11 and R14, the defoliate isolates were for the first time divided into three categories: strong pathogenic (XS4 as the representative), mediun with inclination to strong pathogenic (T9 as the representative) and medium pathogenic (VD8 as the representative). Through the resistance identification of varieties such as R05, R06, R08, R09, R11 and R14, the defoliate isolate VD8 originated from Nantong of Jiangsu Province may be efficiently distinguished from the defoliate isolate 19 originated from USA. In nondefoliate category, utilizing 4 upland varieties R15, R14, R13 and R10, it might be possible to identify the different pathogenic genes in 7 isolates XJ4, XJ1, AY, VD404, VD326, LY, BP2 and may be nominated as No. 1 to No. 7 biological races respectively. The upland variety R01 is capable of resisting to all the isolates used in the experiments. While upland variety R12, may be used to identify strongly pathogenic isolate, which displayed a specific function in identifying strong pathogenic isolate strains.

Molecular Cloning and Characterization of Genes Involved in Cotton(Gossypium barbadense L.) Response to Verticillium dahliae

Verticillium dahliae Kleb.is a necrotrophic plant pathogen which causes serious soil borne vascular disease in cotton.The molecular basis the defense response of cotton to this pathogen is

Comparison of methods of purification and identification of Verticillium dahliae toxins

The complexes excreted by VerticiUium dahliae are phytotoxins, which are responsible for most of the symptoms associated with Verticillium wilt disease. Verticillium dahliae toxins （VD-toxins） can be purified by different methods. In the present study, we reported a simpler, more effective method to purify VD-toxins. The supematant of V. dahliae culture was frozen, lyophilized and dialyzed by 1 kDa Dialysis Membranes （MWCO）. We also partially identified the characteristics of the purified VD-toxins. The results showed that the components of VD-toxins include glycoprotein within 35.8-83.2 kDa. The phytotoxic activity of VD-toxins was remained after VD-toxins were pretreated by high temperature, Concanavalin-A, and proteinase E, respectively. These data suggest that VD-toxins are heat-stable, and the protein fraction and glycosyl are both important contributors to the phytotoxic activity. VD-toxins purified effectively from the culture filtrates of V. dahliae may help in further understanding the mechanisms of interactions between V. dahliae and plants.

Genotypic Resistance of F_1 Cotton Hybrids by Inoculation with Different Virulent Isolates of the Fungus Verticillium Dahliae Klebahn

The plant pathogen Verticillium dahliae causes severe cotton losses in Uzbekistan. To create cotton varieties that are resistant to the more virulent races of V.dahliae we wanted to determine

DNA methylation-dependent epigenetic regulationof Verticillium dahliae virulence in p

As a conserved epigenetic mark, DNA cytosine methylation, at the 5’ position (5-mC), plays importantroles in multiple biological processes, including plant immunity. However, the involvement of DNAmethylation in the determinants of virulence of phytopathogenic fungi remains elusive. In this study,we profiled the DNA methylation patterns of the phytopathogenic fungus Verticillium dahliae, one of themajor causal pathogens of Verticillium wilt disease that causes great losses in many crops, and exploredits contribution in fungal pathogenicity. We reveal that DNA methylation modification is present in V.dahliae and is required for its full virulence in host plants. The major enzymes responsible for theestablishment of DNA methylation in V. dahliae were identified. We provided evidence that DNAmethyltransferase-mediated establishment of DNA methylation pattern positively regulates fungalvirulence, mainly through repressing a conserved protein kinase VdRim15-mediated Ca2? signaling andROS production, which is essential for the penetration activity of V. dahliae. In addition, we furtherdemonstrated that histone H3 lysine 9 trimethylation (H3K9me3), another heterochromatin markerthat is closely associated with 5-mC in eukaryotes, also participates in the regulation of V. dahliaepathogenicity, through a similar mechanism. More importantly, DNA methyltransferase genes VdRid,VdDnmt5, as well as H3K9me3 methyltransferase genes, were greatly induced during the early infection phase, implying that a dynamic regulation of 5-mC and H3K9me3 homeostasis is required for anefficient infection. Collectively, our findings uncover an epigenetic mechanism in the regulation ofphytopathogenic fungal virulence.

VdPKS1 is required for melanin formation and virulence in a cotton wilt pathogen Verticillium dahliae

Verticillium dahliae is a soil-borne phytopathogenic fungus that causes vascular wilt disease in a broad range of hosts. This pathogen survives for many years in soil in the form ofmelanized microsclerotia. To investigate the melanin synthesis in V.. dahliae, we identified a polyketide synthase gene in V. dahliae, namely VdPKS1. PKS1 is known to involve in the dihydroxynaphthalene melanin synthesis pathway in many fungi. We found that VdPKS1 was required for melanin formation but not for microsclerotial production in E dahliae. The VdPKS1 gene-disruption mutant （vdpksl） formed melanin-deficient albino microsclerotia, which did not affect the fungal colonization in host tissues but significantly reduced the disease severity. Gene transcription analysis in the wild-type and the vdpks1 strains suggested that VdPKS1 gene-disruption influenced the expression of a series of genes involved in ethylene biosynthesis, microsclerotial formation and pathogenesis. Our results suggest that the VdPKS1-mediated melanin synthesis is important for virulence and developmental traits of E dahliae.