Proteomic And Expression Profiling Of Cottons (Gossypium Hirsutum) During Early Infection By Verticillium Dahliae

Cotton (Gossypium hirsutum L.) is one of the most important fiber resources in the textile industry. However, Verticillium wilt results in substantial reduction in cotton production every year. The causal fungus, Verticillium dahliae Kleb. (V. dahliae), is a soilborne filamentous fungi and leads to an economically significant vascular wilt disease which affects a wide range of crops and woody plants in temperate climates. Despite the economic importances, not much is known about the molecular mechanisms of Verticillium wilt. Historically, there are two seemingly contradictory hypotheses concerning the mechanism of infection for Verticillium wilt. One states that Verticillium wilt results from toxin activity, whereas the other states that it is predominantly the result of vessel occlusion in the plant.In recent years, the so-called'omic'technologies (e.g. transcriptomics, proteomics) have emerged as powerful tools in the fields of biology to profile "life complexity". In the present study, we established efficient and reliable pathogen-inoculation and proteomic methods, allowing the identification of proteins during the initial interaction between cotton and V. dahliae. The pathogen could be divided into three categories: strong, intermediate and weak pathogenicity respectively based on the disease severity. In the present study, V. dahliae with strong pathogenicity was used as test fungus for proteomics research. In further expression profiling study, cottons were infected by the type of weak pathogenicity V. dahliae for gaining more information about no pathogenity symptom after infection of V. dahliae, and then the different expression genes of cotton roots were analyzed using Solexa sequencing technology.1. Establish efficient protein extraction method compatible with proteomic analysis for the cotton seedlingBy comparing four previously proteins extraction protocols, we set up a suitable protein extraction method from cotton (G. hirsutum L.) seedlings, a recalcitrant plant tissue for protein extraction. The results suggested that trichloroacetic acid-acetone-phenol extraction method (Method D) produced the best sample for two dimensional electrophoresis (2-DE) and the most proteins spots (321), particularly basic proteins in the 2-DE image. The extraction method combining acetone precipitation-phenol (Method B) displayed 216 spots in the 2-DE image. The trichloroacetic acid-acetone precipitation method (Method A) is not suitable for the cotton seedlings proteins extraction because only small molecular weight proteins were visualized in the 2-DE image. The phenol extraction method (Method C) showed 240 spots in the 2-DE gel but with higher gel background. We subsequently modified the Method D to extract protein from cotton seedlings. After optimization, more than 900 protein spots were detected on the 2-DE gel with pH 3-10 nonlinear gradient strip and 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel using Coomassie brilliant blue G-250 staining. The result indicated that the optimized Method. D is more suitable for proteomic studies of cotton seedlings.Using optimized Method D, different proteome of cotton leaf was studied after cotton was infected with V. dahliae. A protein, flavanone 3-hydroxylase (F3H), showed a significantly up-regulation in cotton leaf after V. dahliae infection. Further research revealed F3H and the downstream genes of F3H in proanthocyanidins (PAs) biosynthesis were also significantly induced and showed coordinate expression patterns during wounding. The results suggest that PAs in cotton act an important role in response to infection by V. dahliae and wounding. Over all, cotton leaf show a few different protein, and cotton roots should be a good sample to study V. dahliae-host interactions because the infection initiated from roots.2. Proteomic analysis of cotton roots during early infection by V. dahliaeAccording to optimized Method D with modification, a protein extraction for cotton roots was established. To isolate early-interaction proteins of cotton, we harvested seedling root tissue at 0 h,24 h, and 72 h after inoculation. The proteins were analyzed on two-dimensional (2-D) gels by using pH 3-10 nonlinear gradient strips and 10.5% SDS-PAGE gels. Averages of 830±11 protein spots were detected on the gels stained by Blue-silver staining. In total, we identified 34 differentially expressed proteins of cotton roots by peptide-mass fingerprinting (PMF). Because a complete cotton protein database is not available, the searches were performed on all available higher plant proteins database.Of the 34 differential protein spots, one protein was significantly upregulated, whereas 10 were suppressed. Four proteins showed a temporary increase in expression at 24 h. They include pathogenesis-related proteins (PR); the proteins related to the production of reactive oxygen species (ROS); the proteins related to protein folding and modification; or the proteins related to glycolysis and energy generation; and other proteins.In conclusion, it has been postulated that cotton can establish early defense reactions (such as PR protein and ROS) after infection with V. dahliae. However, it is not sufficient to efficiently protect the cotton plant, and V. dahliae is able to successfully invade cotton by suppressing or down-regulating the expression of defense-related genes. The normal physiological function of cotton is quickly impaired by V. dahliae, and some related proteins (such as some proteins are related to glycolysis and energy metabolism, protein folding and modifying, cell cytoskeleton) show significant downregulation.3. Toxin inhibition experiments relate to ROS accumulation and cell deathPlant tissues staining assay confirmed that a sudden release of ROS and cell death accompany V. dahliae infection in the cotton vasculature. Further Research indicated that culture supernatant of V. dahliae alone can cause leaf symptoms of chlorosis and wilt symptoms.To determine whether V. dahliae uses toxin-induced cell death to manipulate its host, tobacco leaves were infiltrated with cycloheximide (CHX), an inhibitor of protein synthesis. CHX efficiently delayed the development of leaf lesions. However, ROS inhibitor diphenyleneiodonium chloride (DPI) and proteasome inhibitor MG132 did not prevent V. dahliae toxin to cause leaf lesions. These results suggest that V. dahliae toxin actively induces plant cell death by modifying protein synthesis in the host. This modification, however, does not involve the ROS burst or the ubiquitin/26S proteasome system.Moreover, lesions formed more quickly on older leaves rather that young leaves. This indicates that V. dahliae toxin-triggered cell death is correlated with leaf age, and this finding is consistent with the observation that lesions and defoliation symptoms start at the base and move to the apex of the infected plant.4. Expression profiling of cotton roots after, infection by weak pathogenicity V. dahliaeAlthough the cotton showed no significantly wilt syptom after infected by V. dahliae with weak pathogenicity, it was confirmed by PCR that V. dahliae has invaded into roots and stems of cotton. In order to meet the Solexa sequencing for demanding high quality RNA sample, an efficient and economic RNA ectraction method from cotton roots was established by consulting several methods. Different expression genes were analyzed using Solexa sequencing method from roots tissue at 72 h after inoculation. Totally, we identified 2681 different expression genes, which include 1017 up-regulation genes and 1664 down-regulation genes.To identify the functions of all different genes in the metabolic pathways, the genes were analyzed using BLAST similarity search in known pathways in the KEGG database. Four pathways were significantly upregulated and three pathways were significantly downregulated. Four up-regulated pathways include flavone and flavonol biosynthesis, flavonoid biosynthesis, stilbenoid, diarylheptanoid and gingerol biosynthesis and metabolism of xenobiotics by cytochrome P450. Antioxidation and antimicrobe of the metabolic products in these pathways have been reported except for metabolism of xenobiotics by cytochrome P450. The cytochromes P450 enzymes take part in metabolism of chemicals, and they could detoxify chemicals from V. dahliae. Three downregulation pathways were identified, two of three pathways relate to ribosome and spliceosome pathways, which take part in gene expression and translation. Their downregulation suggested that normal physiological function of cotton was impaired, which is consistent with serious tissue destruction of cotton roots. Another downregulation pathway is steroid biosynthesis. A few reporters were found about interaction of steroid and microbe. Then genes expression in the two pathways was analyzed using realtime-PCR. The results showed that most of genes in steroid biosynthesis prsents temporary downregulation, and then significant upregulation. The expression pattern corresponds to pathogenity symptom of cotton roots after infection of weak pathogenity V. dahliae (necrosis of cotton lateral roots in early stage of infection were found, and some new cotton lateral roots were formed as times go on). A plausible explanation is that metabolic products of steroid biosynthesis promote root formation and growth, which is of paramount importance for cotton living.