GhHb1: a nonsymbiotic hemoglobin gene of cotton responsive to infection by Verticillium dahliae

Verticillium wilt of cotton is a widespread and destructive disease that is caused by the fungus pathogen Verticillium dahliae. Although no cotton cultivar is immune to the disease, some genotypes exhibit superior wilt tolerance. To gain an insight into the molecular mechanisms responsible for wilt tolerance, we employed the method of suppression subtractive hybridization (SSH) to isolate genes whose expression is up-regulated after inoculation of the pathogen in a wilt-tolerant cotton cultivar (Gossypium hirsutum cv. BD18). Among the identified candidate ESTs, a cDNA representing a nonsymbiotic hemoglobin gene (designated GhHb1) was further characterized in this study. Northern blot hybridization demonstrated that GhHb1 shares similar characteristics to some other nonsymbiotic hemoglobin genes including the hypoxic stress-induced expression. Sub-cellular localization analysis indicated that GhHb1 proteins were predominantly present in the nucleus with a minor amount appearing in the cytoplasm. Two novel features of GhHb1 were also identified, indicating that GhHb1 expression is activated in the cotton roots after inoculation with V. dahliae and that exogenous hydrogen peroxide induces GhHb1 expression. These results suggest that the GhHb1 may play a role in the defense response of G. hirsutum against V. dahliae invasion.     

海岛棉GbHyPRP1克隆及其转基因拟南芥抗黄萎病验证

在对黄萎病菌胁迫处理的海岛棉Pima 90-53根组织全长c DNA文库分析中,筛选到一个与黄萎病胁迫相关的杂合富含脯氨酸蛋白(hybrid proline-rich protein)基因,将其命名为Gb Hy PRP1。该基因c DNA序列全长1747 bp,开放阅读框945 bp,编码一个由314个氨基酸残基组成的蛋白,包含信号肽、N端富含脯氨酸域及C端Pollen Ole e I域。同源序列分析显示,Gb Hy PRP1与来自雷蒙德氏棉、陆地棉和亚洲棉的Hy PRP1蛋白序列相似性最高,分别为95.95%、93.87%和91.34%。q RT-PCR分析结果显示,受黄萎病菌胁迫后海岛棉根部Gb Hy PRP1表达显著下调。将Gb Hy PRP1基因克隆至植物超表达载体,农杆菌介导转化拟南芥获得转基因植株。病指统计分析表明Gb Hy PRP1过量表达显著降低了拟南芥对黄萎病的抗性。据此推测Gb Hy PRP1参与棉花抗黄萎病,可能是一个重要的负调控因子。     

Ectopic expression of the cotton non-symbiotic hemoglobin gene GhHbd1 triggers defense responses and increases disease tolerance in Arabidopsis

Plant non-symbiotic hemoglobins (nsHbs) play important roles in a variety of cellular processes. Previous evidence from this laboratory indicates that the expression of a class 1 nsHb gene (GhHb1) from cotton is induced in cotton roots challenged with the Verticillium wilt fungus. The present study examined further the expression patterns of the GhHb1 gene in cotton plants and characterized its in vivo function through ectopic overexpression of the gene in Arabidopsis thaliana. Expression of GhHb1 in cotton plants was induced by exogenously applied salicylic acid, methyl jasmonic acid, ethylene, hydrogen peroxide (H(2)O(2)) and nitric oxide (NO). Ectopic overproduction of GhHb1 in Arabidopsis led to constitutive expression of the defense genes PR-1 and PDF1.2, and conferred enhanced disease resistance to Pseudomonas syringae and tolerance to V. dahliae. GhHb1-transgenic Arabidopsis seedlings were more tolerant to exogenous NO and contained lower levels of cellular NO than the wild-type control. Moreover, transgenic plants with relatively high levels of expression of the GhHb1 gene developed spontaneous hypersensitive lesions on the leaves in the absence of pathogen inoculation. Our results indicate that GhHb1 proteins play a role in the defense responses against pathogen invasions, possibly by modulating the NO level and the ratio of H(2)O(2)/NO in the defense process.     

Functional divergence of GLP genes between G. barbadense and G. hirsutum in response to Verticillium dahliae infection

Germin-like proteins (GLPs) play important roles in plant disease resistance but are rarely reported in cotton. We compared the expression of GLPs in Verticillium dahliae inoculate G. hirsutum (susceptible) and G. barbadense (resistant) and enriched 11 differentially expressed GLPs. 2741 GLP proteins identified from 53 species determined that GLP probably originated from algae and could be classified into 7 clades according to phylogenetic analysis, among which Clade I is likely the most ancient. Cotton GLP (two allopolyploids and two diploids) genes within a shared clade were highly conserved. Intriguingly, clade VII genes were mainly located in gene clusters that derived from the expansion of LTR transposons. Clade VII members expressed mainly in root which is the first battle against Verticillium dahlia and could be induced more intensely in G. barbadense than G. hirsutum. The GLP genes are resistant to Verticillium dahliae, which can be further investigated against Verticillium wilt.     

陆地棉品种和骨干品系黄萎病抗性鉴定

选育和推广抗病品种是防治陆地棉黄萎病的主要措施,为了早日实现多类型、多区域大面积抗病品种的应用,本研究选取107份遗传背景差异较大的种质,利用河北省农林科学院棉花研究所小安舍试验站黄萎病病圃进行了3年黄萎病抗性重复鉴定。鉴定得到抗病品系8个,占7.5%;耐病品种(系)20个,占18.7%。本研究表明,当前被作为育种亲本的抗病品系还太少,需要深入开展抗病遗传机制,以及与其他经济性状协同改良的关系,为陆地棉抗病育种提供理论指导;达到抗病或接近抗病水平的大部分品种(系)来自于海陆野远缘后代,具有外源基因血统,证明了远缘杂交是陆地棉黄萎病抗性改良的有效手段。     

Identification and characterization of differentially expressed ESTs of Gossypium barbadense infected by Verticillium dahliae with suppression subtractive hybridization

Cotton wilt defense reaction is a complicated continuous process and involves a battery of genes. In this study, we adopted suppression subtractive hybridization (SSH) technique to isolate differentially expressed ESTs from Gossypium barbadense variety 7124 during Verticillium wilt defense process. An array of 1165 clones from the subtractive library has been screened with reverse northern blotting, of which 131 ESTs were considered as over-expressed and 16 ESTs were down-regulated. Sequence analysis and blast search showed that 83 ESTs were homologous to 45 unique sequences in the databases. Among all these differentially expressed ESTs, at least three kinds of genes were characterized. The majority of ESTs with deduced identity to aerobic metabolism enzymes strongly expressed in the infection process. Likewise, ESTs similar to those reported for pathogen-related protein genes were also picked out in this study. These ESTs in combination with other kinase-like genes and a defensin-like EST constituted an assembly of genes responded during pathogens' infection. These results imply that sea-island cotton undergoes strong oxidative stress and results in a series of defense responses when attacked by V. dahliae. To our knowledge, this is the first report on the isolation of global ESTs during sea-island cotton defense reaction.     

海岛棉几丁质酶基因GbCHI的克隆与功能分析

几丁质酶是植物主要的病程相关(PR)蛋白之一。前期工作中利用比较蛋白质组学方法, 从海岛棉7124根部蛋白中分离到一个IV型几丁质酶(GbCHI)。文章通过同源克隆获得了海岛棉GbCHI基因的cDNA序列, 并对该基因的表达特征及其蛋白的抑菌功能进行了分析鉴定。qRT-PCR实验结果表明GbCHI基因在棉花根、茎、叶、花和胚珠中均有表达, 其表达受大丽轮枝菌、水杨酸(SA)、乙烯(ACC)和茉莉酸(JA)诱导; 亚细胞定位分析显示GbCHI蛋白主要分布在细胞膜上; 体外抑菌实验证明GbCHI蛋白能显著抑制大丽轮枝菌孢子的萌发和菌丝的生长。这些研究结果为了解GbCHI的功能及其在抗黄萎病棉花分子育种中的应用提供了实验依据和思路。     

海岛棉几丁质酶基因GbCHI的克隆与功能分析

几丁质酶是植物主要的病程相关(PR)蛋白之一。前期工作中利用比较蛋白质组学方法,从海岛棉7124根部蛋白中分离到一个IV型几丁质酶(GbCHI)。文章通过同源克隆获得了海岛棉GbCHI基因的cDNA序列,并对该基因的表达特征及其蛋白的抑菌功能进行了分析鉴定。qRT-PCR实验结果表明GbCHI基因在棉花根、茎、叶、花和胚珠中均有表达,其表达受大丽轮枝菌、水杨酸(SA)、乙烯(ACC)和茉莉酸(JA)诱导;亚细胞定位分析显示GbCHI蛋白主要分布在细胞膜上;体外抑菌实验证明GbCHI蛋白能显著抑制大丽轮枝菌孢子的萌发和菌丝的生长。这些研究结果为了解GbCHI的功能及其在抗黄萎病棉花分子育种中的应用提供了实验依据和思路。     

Overexpression of GhPFN2 enhances protection against Verticillium dahliae invasion in cotton

Growing evidence indicates that actin cytoskeleton is involved in plant innate immune responses,but the functional mechanism remains largely unknown.Here,we investigated the behavior of a cotton profilin gene(GhPFN2) in response to Verticillium dahliae invasion,and evaluated its contribution to plant defense against this soil-borne fungal pathogen.GhPFN2 expression was up-regulated when cotton root was inoculated with V.dahliae,and the actin architecture was reorganized in the infected root cells,with a clear increase in the density of filamentous actin and the extent of actin bundling.Compared to the wild type,GhPFN2-overexpressing cotton plants showed enhanced protection against V.dahliae infection and the actin cytoskeleton organization in root epidermal cells was clearly altered,which phenocopied that of the wild-type(WT) root cells challenged with V.dahliae.These results provide a solid line of evidence showing that actin cytoskeleton reorganization involving GhPFN2 is important for defense against V.dahliae infection.     

Dynamic infection of Verticillium dahliae in upland cotton

• Verticillium wilt, an infection caused by the soilborne fungus Verticillium dahliae, is one of the most serious diseases in cotton. No effective control method against V. dahliae has been established, and the infection mechanism of V. dahliae in upland cotton remains unknown.
• GFP‐tagged V. dahliae isolates with different pathogenic abilities were used to analyse the colonisation and infection of V. dahliae in the roots and leaves of different upland cotton cultivars, the relationships among infection processes, the immune responses and the resistance ability of different cultivars against V. dahliae.
• Here, we report a new infection model for V. dahliae in upland cotton plants. V. dahliae can colonise and infect any organ of upland cotton plants and then spread to the entire plant from the infected organ through the surface and interior of the organ.
• Vascular tissue was found to not be the sole transmission route of V. dahliae in cotton plants. In addition, the rate of infection of a V. dahliae isolate with strong pathogenicity was notably faster than that of an isolate with weak pathogenicity. The resistance of upland cotton to Verticillium wilt was related to the degree of the immune response induced in plants infected with V. dahliae. These results provide a theoretical basis for studying the mechanism underlying the interaction between V. dahliae and upland cotton. These results provide a theoretical basis for studying the mechanism underlying the interaction between V. dahliae and upland cotton.

     

Isolation of Glycoproteins from Verticillium dahliae and Their Phytotoxicity

Verticillium dahliae Kleb. is a phytopathogenic fungus that causes cotton wilt-disease. Glycoproteins secreted by V. dahliae have been found to play an important role in wilting syndrome. In this study the glycoproteins were purified consecutively by ConA-Sepharose 4B affinity chromatography, Sephadex G-150 gel filtration, two-dimensional gel electrophoresis and SDS gradient gel electrophoresis. The N-terminal residual sequence of a 26 kD glycoprotein was analyzed. Plant-wilting tests were carried out by injection of glycoproteins, and those treated by heat, ConA and zeatin, into cotton leaves, respectively. Results showed that heat and ConA treatment abolished the wilt-causing activity of the glycoproteins, and zeatin alleviated the wilt syndrome of cotton. Furthermore, the glycoproteins were found to be effective elicitors in inducing the biosynthesis of sesquiterpene aldehyde phytoalexins in suspension cell cultures of Gossypium barbadense L., and heat-treatment lowered, but not abolished the elicitor activity. However, application of native glycoproteins at the concentration higher than 5 mg/L resulted in cell death.     

大丽轮枝菌分泌糖蛋白的分离及其致萎性研究

利用伴刀豆球蛋白（ＣｏｎＡ） 亲和层析、Ｓｅｐｈａｄｅｘ Ｇ＿１５０ 凝胶层析、双向电泳、ＳＤＳ梯度电泳等手段对大丽轮枝菌（Ｖｅｒｔｉｃｉｌｌｉｕｍ ｄａｈｌｉａｅ Ｋｌｅｂ．）分泌的糖蛋白复合物进行分离,对其中一约２６ ｋＤ 的组分进行了Ｎ 端氨基酸序列分析。以棉花（ Ｇｏｓｓｙｐｉｕｍ ｂａｒｂａｄｅｎｓｅ Ｌ．）叶片为材料,进行了糖蛋白致萎性实验。结果表明,沸水浴或ＣｏｎＡ 处理的蛋白致萎性消失,Ｚｅａｔｉｎ 使糖蛋白致萎性减弱。该糖蛋白能够诱导海岛棉培养细胞中棉酚等倍半萜的合成,棉酚的积累随着糖蛋白浓度的增加而增加,但到一定程度后下降,此时较高浓度的大丽轮枝菌分泌糖蛋白引起植物细胞死亡     

基因沉默技术在抗真菌病害中的应用和展望

真菌病害严重威胁作物的产量和品质,给国家和人民造成巨大的经济损失。尤其是引起维管束病害的土传真菌,化学农药的作用效果很不理想。利用抗性基因进行遗传育种是目前生物防治的重要手段之一,但对于缺乏抗性资源的物种,面对强大的土壤真菌病害,研究者也时常束手无策。近年来,利用RNA干扰技术发展而来的宿主诱导的基因沉默(Host induced gene silencing,HIGS)策略,在抗病虫害领域逐渐崭露头角,但由于真菌侵染的复杂多样性及土壤传播的特性,HIGS在土壤真菌病害中的应用充满神秘和挑战。本研究室近期揭示了棉花黄萎病(一种严重的土壤真菌病害)的"罪魁祸首"——大丽轮枝菌的侵染结构和侵染过程;并首次证明了宿主植物内源小RNA能够跨界进入病原菌细胞中降解致病基因表达的抗病作用;在此基础上,本研究室利用HIGS在棉花上获得了对黄萎病抗性较高的品系,成功地开辟了抗土壤黄萎真菌病害的新天地,研究结果显示出基因沉默技术在这一领域强大的应用潜力和前景。