Molecular Mechanisms Of Inducing Resistance To Cucumber Fusarium Wilt By Nonpathogenic Fusarium Oxysporum CS-20

Fungal fusarium wilt is a devastating soil-borne disease caused by pathogenic Fusarium oxysporum, and leads to severe yield reduction or no harvest. There are no effective methods to control the disease currently. The reasons are short of resistant varieties and chemical control agents. Thus the biological technology research of inducing resistance in plant has become an important pathway to control the fusarium wilt. The strain of Fusarium oxysporum CS-20 was isolated in Florida from a soil suppressive to fusarium wilt of watermelon, and it had good effects to control the crop of Fusarium wilt. However, the molecular mechanism of inducing defence resistance by the strain CS-20 in plant has not been clearly demonstrated. Moreover, it still remains unknown about which factors of the strain CS-20 can active defence resistance and how their molecular mechanisms work. So the lack of research has limited the application of the strain CS-20. Therefore, the susceptible cucumber cultivar （Cucumis sativus L.9930） and the strains CS-20 were the objects of this study. There were two aims. One was to make the defense analysis of related mechanism in cucumber seedlings after root inoculating the strain CS-20, and the other was to excavate and elucidate the function and synthetic product of key effector genes of polyketide biosynthase （PKS） and nonribosomal peptide synthetase （NRPS） in process of inducing resistance. The results of research were as follows:1. The mechanism of defence in cucumber seedlings in relation to root colonization by nonpathogenic Fusarium oxysporum CS-20 was analyzed. Biological research results showed that the disease severity index （DSI） of Fusarium wilt decreased significantly after 3 days post inoculation （dpi） of the inoculum of strain CS-20 in seedlings roots, compared with the FOC-BN treatment. The observation of colonization in roots with the use of green fluorescent protein （GFP）-tagged strains CS-20 and FOC-BN proved that the infection of FOC-BN progressed more quickly than that of CS-20. Moreover, the strain FOC-BN had infected the central cylinder of the lateral roots and led the xylem vessels of the taproot to rot seriously after 5 dpi. Molecular mechanism results showed that the CS-20-mediated induced systemic resistance （ISR） was activated by the signaling pathways of salicylic acid （SA）, jasmonic acid （JA） and JA/ethylene, which were involved in both systemic acquired resistance （SAR） and ISR, and the genes of Ca2+/CaM signal transduction pathway were upregulated specifically after CS-20 inoculation in cucumber plants.2. Based on the genome of the srain CS-20, the prediction of bioinformatics showed that there were 10 PKS genes,14 NRPS genes,10 PKS-NRPS hybrid genes and 3 prenyleransfersase （DAMT） genes in the genome of the strain CS-20, and 30 homologous genes in the strain FOC-BN identified by the analysis of BLAST. The quantitative reverse-transcriptase polymerase chain reaction （qRT-PCR） analysis of PKS-NRPS genes showed that the value expression of genes were significantly different between CS-20 and FOC-BN in comparison with cultured hyphae in vitro before and after 72 dpi. There were 9 genes of the strain CS-20 expressed specifically in relation to the induce resistance to cucumber fusarium wilt, with 4 genes （FOWE₁0925, FOWE 10935, FOWE₀1643 and FOWE₁09588） being upregulated and the other 5 genes （FOWE₀0999, FOWE₁2767, FOWE₀3871, FOWE₀6548 and FOWE₁1163） downregulated. But the data of comparison also showed that there were 15 genes upregulated distinctively in the strain FOC-BN in relation to its pathogenicity.3. The split marker gene deletion method was used to delete the 9 genes related to the induced resistance in the strain CS-20, together with PEG mediated genetic transformation of Protoplasts, screening of resistance to hygromycin and verification by PCR and Southern bolt. The assay obtained 9 gene deletion mutants （⊿FOWE₀0999 （T87）,⊿FOWE₁2767 （T28）, ⊿FOWE₁3871 （T19）,⊿FOWE₁6548 （T5）,⊿FOWE₁0925 （T17）,⊿FOWE₁1163 （T16）, ⊿FOWE₁0935 （T13）,⊿FOWE₀1643 （T9）,⊿FOWE₁9588 （T20）） successfully. The wild type （WT） strain CS-20 was found to be significantly different with the other 8 mutants in morphology, although no difference was traced wihen compared with the mutant ⊿FOWE₁1163 （T16）; There was also significant difference between the WT CS-20 and the other of 9 mutants in their everyday colony growth diameter （p<0.05）. The growth rate of the two mutants ⊿FOWE₁0925 （T17） and ⊿FOWE₁1163 （T16） corresponded with the WT strain, but that of the other 7 mutants were lower than the WT strain CS-20.4. Using challenge inoculation and split-root inoculation methods to analyze the difference levels of inducing resistance of 9 candidate genes, the results of experiments showed that the DSI of 6 gene mutants（⊿FOWE₀0999,⊿FOWE₁2767,⊿FOWE₀3871,⊿FOWE₀6548, ⊿FOWE₁0925 and ⊿FOWE₁1163） were higher than that of the WT strain CS-20. So these 6 genes were closely related with the induce resistance to cucumber fusarium wilt, and they were regarded as the key effector genes. The qRT-PCR analysis revealed that when compared with the WT strain CS-20, the level expression of NPR1 in the roots and leaves were downregulated after inoculating the 6 key effector gene mutants, and corresponded with the water control check （CK）. After root inoculation with the 6 key effector gene mutants, the expression levels of LOX1 and PAL1 were lower than that of WT CS-20 significantly, but the expression level of PR3 was only downregulated significantly after inoculation with ⊿FOWE₁2767. In contrast to the signal genes expression level in root, there was difference only in the expression level of PAL1 in the leaves, with another signal genes expression level keeping around the horizontal curve.5. The structure analysis of function domains of the 6 key effector genes showed that all of them consisted of ketosynthase （KS）, acyl transferase （AT）, β-ketoreductase （KR）, enoyl reductase （ER）, dehydrogenase （DH）. The six genes belonged to the highly reducing （HR）-PKSs and produced a large diversity of linear and cyclic nonaromatic compounds. Based on the protein sequence of all domains of PKS enzymes, the phylogram showed that each function domain of the 6 key effector genes was in an independent small branch with distant relationship between each other.6. There was no report about the synthetic product of 6 key effector genes. Using liquid chromatography-mass spectrometry （LC-MS） to detect the components of fermentation crude extract of the WT CS-20 and 6 key effector gene mutants, together with the analysis of difference comparison and the metabolic spectrum database, the result of speculation were as follows. The key effector gene FOWE₁0999 might synthetize the product of 11,12,13-trihydroxy-9-octadecenoic acid, and the molecular structural formula was C18H34O5; The key effector gene FOWE₁2767 might synthetize the product of 9,10,13-trihydroxy-11-octadecenoic acid, and the molecular structural formula was C18H34O5; The key effector gene FOWE₀3871 might synthetize the product of 5-hydroxy-2-octadecenoic acid, and the molecular structural formula was C18H34O3; The differential and linear compound was not found between the mutant ⊿FOWE₀6548 and the WT CS-20; The key effector gene FOWE₁0925 might synthetize the product of 8-methoxy-13-hydroxy-9,11-octadecadienoic acid, and the molecular structural formula was C19H34O4; the key effector gene FOWE 11163 might synthetize the product of 10-hydroxy-undecanoic acid, and the molecular structural formula was C11H22O3. The 5 conjectural compounds were unknown and new compounds, and whether they were as elicitors to induce resistance need to certify in the next research.To sum up, the study explained the defence mechanism in relation to root inoculation by the strain CS-20 in the cucumber seedlings, and by focusing on the strain CS-20, it elucidated the key effector genes and their function originated from the genes of synthetizing secondary metabolite innovatively. Therefore, the study illustrated the molecular mechanisms of inducing resistance to cucumber fusarium wilt by nonpathogenic Fusarium oxysporum CS-20 systemically, and laid a theoretical foundation for controlling the fusarium wilt caused by pathogenic Fusarium oxysporum.