| Cotton is an important cash crop in the world. Verticillium dahliae is a destructive, soil-borne fungal pathogen that causes severe losses in cotton yield and fiber quality. A more thorough mining and understanding of the functional mechanisms of genes related to resistance to Verticillium wilt will benefit efforts to genetically improve crop plants. By screening suppression subtractive hybridisation(SSH) and c DNA libraries of tolerant cotton induced by V. dahliae, we have previously isolated several genes related to resistance to Verticillium wilt. Polyamine metabolism pathway was selected by further analysis of the enrichment of these genes, cotton polyamine oxidase(Gh PAO), S-adenosylmethionine decarboxylase(Gh SAMDC), spermine synthase(Gh SPMS) and thermospermine synthase(Gh ACL5) genes stand out by analysis of the expression pattern for genes of polyamine pathway under V. dahliae stress through real-time RT-PCR method. Thus, it is very important to research the function and the underlying mechanism of cotton PAO/SAMDC/SPMS/ACL5 with V. dahliae infection, which would provide great significance for parsing of the mechanism of cotton plant defence response. In the present study, the main concepts were shown as fllows:(1) Assessment of the effect on responsiveness to V. dahliae infection and the change of related substrates and products by virus-induced gene silencing of Gh PAO/Gh SAMDC/Gh SPMS/Gh ACL5 in cotton.(2) By constitutive expression of Gh PAO/Gh SAMDC/Gh SPMS/Gh ACL5 in Arabidopsis thaliana, evaluation of the effect on responsiveness to V. dahliae infection, and dissection the mechanism at the level of molecular biology, cytology and physiological biochemistry.(3) By quantification of polyamine levels in tolerant and susceptible cotton cultivars after inoculation with V. dahliae, and the effect of exogenous polyamine solutions on the growth of V. dahliae in vitro, uncover the relevance between polyamine levels and the defense reponse against V. dahliae infection.The main results were summarized as follows:1. Constitutive expression of Gh PAO in Arabidopsis thaliana produced improved resistance to V. dahliae, along with decreased biomass of V. dahliae conidia and the number of wilting leaves that appeared after inoculation. Substrates of putrescine, spermidine and spermine were maintained at high levels, and PAO-specific activity,hydrogen peroxide, salicylic acid and camalexin(a phytoalexin) levels were distinctly increased in Gh PAO-overexpressing Arabidopsis plants during V. dahliae infection when compared with wild-type plants. Moreover, polyamine metabolism genes responsible for polyamine levels were induced in Gh PAO-overexpressing plants under V. dahliae stress, and depend on spermine and salicylic acid signalling in Arabidopsis.2. The extracted and highly purified recombinant Gh PAO protein displayed FAD-specific absorption maxima. The optimal p H and temperature for maximum Gh PAO activity using spermine as the substrate occurred at p H 8.0 and 40°C. Spermine was found to be the most preferred substrate for Gh PAO, Gh PAO converts spermine to spermidine as a partial back-conversion-type PAO. The application of putrescine, spermidine, spermine and thermospermine significantly inhibited conidial proliferation, hyphal growth and colony growth in vitro, spermine displayed the highest antifungal activity. With mock treatment, polyamine levels displayed no consistent pattern between 10 susceptible and 10 tolerant cotton cultivars. After V. dahliae infection, the levels of putrescine, spermidine and spermine were higher in tolerant cotton cultivars than in susceptible ones.3. After treatment with exogenous spermine, the camalexin content was significantly increased in both Arabidopsis and cotton plants when compared with mock-treated plants. Moreover, spermine induced the expression of mitogen-activated protein kinases and cytochrome P450 proteins for promoting the accumulation of camalexin in Arabidopsis and cotton plants. In addition, camalexin efficiently inhibited growth of V. dahliae in vitro. Silencing of Gh PAO in cotton plants led to a highly susceptible response to V. dahliae infection, with the increased disease index reaching a susceptible level. Gh PAO silencing significantly compromised levels of putrescine, spermidine, spermine, hydrogen peroxide and phytoalexin camalexin during V. dahliae infection, Gh PAO depends on spermine and camalexin signaling.4. Spermine metabolism related genes, Gh SAMDC and Gh SPMS, were induced early and strongly by inoculation with V. dahliae and application of plant hormones in resistant cotton plants. Silencing of Gh SPMS or Gh SAMDC in cotton leaves enhanced plant susceptibility to Verticillium infection. Exogenous supplementation of Spm to the silenced cotton plants improved resistance. When compared with the wild-type, constitutive expression of Gh SAMDC in Arabidopsis thaliana was associated with greater Verticillium wilt resistance and higher accumulations of Spm, salicylic acid, and leucine during the infection period. By contrast, transgenic Arabidopsis plants that over-expressed Gh SPMS were unexpectedly more susceptible than the wild-type to V. dahliae and they also had impaired levels of putrescine and salicylic acid.5. In resistant plants, expression of GhACL5 was enhanced after inoculation with V. dahliae when compared with the control. In the susceptible cultivar, Gh ACL5expression was indistinguishable from that detected in the control samples, except for a marked increase in the former at 36 hpi. Expression of Gh ACL5 was also induced upon treatment with plant hormones salicylic acid, jasmonic acid and ethylene in resistant cotton plants, but gene silencing in cotton enhanced their susceptibility to V. dahliae infection, along with increased disease index. Silencing of the cotton ACAULIS5 gene(Gh ACL5) in cotton reduced the amount of thermospermine and led to a severe dwarf phenotype. Constitutive expression of Gh ACL5 in Arabidopsis thaliana significantly increased plant height and elevated the level of thermospermine. |
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