| Fusarium head blight (FHB) of wheat is an important fungal disease. It mainly occurrs in the middle and lower regions of the Yangtze River valley, the winter wheat regions in the South China, and the Heilongjiang province in the northeastern region of China. Fusarium graminearum is the main causal agent. Fusarium asiaticum is the predominant species in F. graminearum clade in China. FHB not only causes yield loss and quality reduction but also can contaminate grains with a variety of potent mycotoxins that are detrimental to human and domesticated animal health. Up to now, the control of FHB has mainly depended on the frequent use of chemical fungicides. Consequently, the incidence of fungicide-resistant FHB pathogens has increased dramatically, resulting in marked decreases of FHB control. Effective control of FHB relies on the development of new strategies. Thus, it is urgent to investigate new targets that can be used for the development of antifungal compounds for FHB control in wheat.The fungal cell wall is very important for the cell morphogenesis and pathogenicity of the pathogens. Chitin is the main structurally important component of the fungal cell wall but absent in plant cells. Chitin synthases that catalyze chitin biosynthesis would be an ideal target for developing new antifungal compounds for controlling the disease. Molecular and functional characterization of the chitin synthase genes could provide academic evidence for developing new antifungal strategies and new antifungal compounds.In this dissertation a set of chitin synthase gene disruption mutant strains of F. asiaticum were created via Agrobacterium tumefaciens-mediated transformation and homologous recombination techniques based on the Molecular Biology, Microbiology, Plant Pathology and Cell Biology methods. The differences between the mutant strains and the wild-type strain on chitin synthase activity, chitin content, pathogenicity, cell wall ultrastructure, growth and development were systematically characterized. The main results were as following:1. F. asiaticum genome contains eight chitin synthase genes which can be assigned to seven classes based on their derived amino acid sequences. The seven classes are CHS1 (class I), CHS2 (class II), CHS3a (class III), CHS3b (class III), CHS4 (class IV), CHS5 (class V), CHS6 (class VI), and CHS7 (class VII). In this study, the chitin synthase genes CHS1, CHS2, CHS3a, CHS3b, CHS4, CHS5, and CHS6, besides the CHS7, from F. asiaticum were functionally characterized. 2. Seven homologous recombination vectors for the chitin synthase gene disruption were constructed. Six (besides the CHS3b) individual chitin synthase gene disruption mutant strains of F. asiaticum were generated and identified by PCR, Southern, and RT-PCR analyses. The attempt to obtain a CHS3b gene-deficient mutant was unsuccessful, possibly due to a lethal effect on the mutant.3. The?chs1,?chs2, and?chs5 mutant strains have a significant reduction in chitin synthase activity, chitin content, and conidiation compared to the wild-type strain 5035:1) The?chs1,?chs2, and Achs5 mutant strains have a 58%,53%, and 78% reduction, respectively, in chitin synthase activity compared to the wild-type strain; 2) The?chs1,?chs2, and?chs5 mutant strains have a 35%,19%, and 42% reduction, respectively, in chitin content of the mycelia compared to the wild-type strain under the PDB culture condition, a 23%,16%, and 37% reduction, respectively, under the PDB containing 1.2 mol/L sorbitol culture condition, and a 50%,30%, and 52% reduction under the PDB containing 1.2 mol/L NaCl culture condition; 3) The?chs1,?chs2, and Achs5 mutant strains also have a 25%,41%, and 67% reduction, respectively, in chitin content of the conidiospores compared to the wild-type strain; 4) The?chs1 and?chs2 mutant strains have a 22%, and 37% reduction, respectively, in conidiation compared to the wild-type strain in the CMC broth while the Achs5 mutant strain lost the ability of conidia formation; 5) Under the CMC supplemented with 20% PEG4000 culture condition, the?chs1 and?chs2 mutant strains have a 30%, and 44% reduction, respectively, in conidiation compared to the wild-type strain while the Achs5 mutant has a 33% conidiation compared to the wild-type strain; 6) The?chs3a,?chs4, and?chs6 mutant strains have no apparent effect on the chitin synthase activity, chitin content, and conidiation.4. The?chs1,?chs2, and Achs5 mutant strains are more sensitive to various stress stimuli such as sorbitol, NaCl, SDS, H2O2, and HCl than the wild-type strain. The germination of the conidia and mycelial growth were clearly slower in the three mutant strains than the wild-type strain 5035. The?chs1,?chs2 and Achs5 mutants had an increase in pigmentation that appeared to secrete more red pigments into the medium compared with the wild-type strain 5035. The Achsl and Achs5 mutant strains had more nuclei per cellula than did the wild-type strain. The Achs5 mutant strain had the balloon-like hyphae that were frequently observed in some parts of the mycelium and the hyphal tips. The transmission electron microscopy (TEM) assays revealed that the macroconidia, ascospores and mycelia of Achsl, Achs2, and Achs5 mutant strains had an altered cell wall ultrastructure compared to the wild-type strain 5035. The Achs3a, Achs4, and Achs6 mutant strains had a cell wall structure similar to the wild-type in different development stages.5. The Achsl, Achs2, and Achs5 mutant strains have a dramatic reduction in pathogenicity. On 21 days post inoculation, the infected spikelets inoculated with the Achsl, Achs2, and Achs5 mutant strains were reduced by 40%,35%, and 57%, respectively, compared to those from strain 5035 on wheat cultivar Sumai3 and 60%, 50%,86%, respectively, on cultivar Annong8455. Pathogenicity of the Achsl, Achs2, and Achs5 mutants on wheat seedlings was also reduced. On 7 days post inoculation, the lesion length on Annong8455 was reduced by 30%,42%, and 48%, respectively, with the?chsl,?chs2, and?chs5 mutant strains compared to that on strain 5035.6. The CHS1-complemented strain, CHS2-complemented strain, and CHS5-complemented strain have a 73%,94%, and 25% increase, respectively, in chitin synthase activity compared to their corresponding strains carrying a single deficient mutant gene. The three CHS-complemented strains also displayed a partially functional complementation in chitin content, conidiation, pathogenicity, and the cell wall ultrastructure compared to the wild-type strain 5035.7. A highly efficient A. tumefaciens-mediated transformation system was established. The frequency of homologous recombination between the T-DNA and the target gene was up to 100%. There are no multi-copy or ectopic integration of transforming DNA in the transformants identified.These results indicated that CHS1, CHS2, and CHS5 genes were the pivotal chitin synthase genes in F. asiaticum. The Chs1, Chs2, and Chs5 proteins play an essential role in the morphogenesis and pathogenicity of F. asiaticum. Therefore chitin synthases, and the chitin thereof could be the ideal targets for controlling the FHB. These results may serve as basis and provide materials for developing new strategies and new antifungal compounds for controlling FHB disease in agriculture. |
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