| Fusarium graminearum, the casual agent of wheat Fusarium head blight (FHB), is an important filamentous fungi worldwidely, causing numerous yield losses of wheat. The epidemic of FHB in the middle and lower regions of the Yangtze River, thewinter wheat regions in the South China, and Heilongjiang province greatly threat the production of wheat and the food safety in China. Moreover, mycotoxins produced by Fusarium also pose great threats to the food security. Wheat graines contaminated with Fusarium mycotoxins are harmful for livestocks and human who have comsumed the contaminated grains and food made from them. Due to the inadequate of wheat FHB resistance resources, the progress of wheat breeding against FHB is quite limited. Chimcal compounds are still the major approch to control wheat FHB and the antidrug effects cannot be ignored. It is significance to investigate the mechanismes of infection and mycotoxin productions as well as the development processes, searching for the new target of fungicide. These works will provide information, based on which new broad-spectrum fungicide can be developed.In this study, we successfully constructed the mutant library of F. graminearum wild type strain 5035, the prominent strain in the middle and lower regions of the Yangtze River in China, by restriction enzyme mediated integration. By screening the REMI library, we obtained several important mutants showing obvious phenotypes. Major results are listed as following:1. The analysis of distribution pattern of REMI insertion sites in the library indicated that foreign DNA were integrated into the chromosomes of F. graminearum. In the genome, REMI events were biased towards the genomic region, and this proverty contributes to the high effeciency of gene tagging.2. By screening the library, we obtained 47 important mutants in total, among which 36 showed remarkable mutant in hypha growth and development while the other 11 mutants showed decreased pathogenicity when they were inoculated onto the spiletes of wheat.3. In these 47 muants, M594 were chose for further research. By TAIL-PCR, the flanking sequences of insertion site in M594 were isolated and blasted to NCBI. Blast results indicated that FG09719 was interupted in M594. FG07919 encodes a deduced protein homology to the type II myosin in yeast and animal, thus, and was designated as Myo2.4. We deleted Myo2 gene in the wild type strain 5035 to confirm the mutant effect in M594 and generated the null mutant△myo2. △myo2 showed abnormal colony and septa, decreased growth rate of hyphae and production ability of conidial spores. Conidial spores of△myo2 were tandemly attached head to tail and exhibited an abnormal morphology. In addition, the ascope developemnt was blocked by the deletion of Myo2 gene. What’s more important, the pathogenicity of△dmyo2 was totally lost and the mycotoxin level was also significantly decreased.5. In△myo2, the septa were distributed unevenly along the hyphae and the total number was decreased compared to the wild type strain 5035. In addition, the septa appeard in△myo2 were incomplete and could not span the hyhpae, which was also confirmed by the transmission electronic microscopy (TEM) observation.6. In order to examine the cellular localization of Myo2 protein, we fused green fluorescent protein (GFP) to the C terminal of Myo2 protein in wild type strain 5035 by homologous recombination. Under the epifluorescece microscopy (Nikon 90i) with B-2A filter, the fusion protein Myo2-GFP was observed and its dynamics was lively recorded. Myo2-GFP protein was recruited to the division site before the septum was formed, and subsequently assembled into a ring structure. The Myo2 protein ring constricts at the onset of cytokinesis and pull the invagination of plasma membrane to form septum. Myo2 protein ring constricts to a bright dot at the final stage and the spetum was concomitantly filled. When the septum was formed, the Myo2 protein was dispersed into the cytoplasma. The dynamics of Myo2 protein in the conidiation progress was similar to that in the hyphae.7. Gene expression file of△myo2 was analyzed to find the regulated genes. Based on the results, we noticed that the secondary metabolism was disturbed by the deletion of Myo2 gene. Several important genes involved in the biosynthesi pathway of aurofusarin and DON were significantly down regulated in△myo2, and this explained the decreased level of DON. Moreover, among these down regulated genes, FGSG00051 attracted our interests. FGSG00051 was involved into the process of response to plant defense reaction, and this gene was about 40000 fold down regulated in△myo2 and could hardly be detected.8. The cytokinesis process of fungi is totally different from that of plant cells and type II myosins are exculsively exsited in fungi and animal cells, but not in plant. Therefore, the Myo2 gene could be used as novel promising target of fungicide. The structures and functions are highly conserved in filamentous fungi, thus, the fungicde targeted to Myo2 or other essential proteins involved in cytokinesis would be used in a very wide range of plant diseas control.In this study, we successfully constructed a REMI mutant library of F. graminearum strain 5035, and a number of valuable mutants were obtained from this library. With our work, we provided materials for the future research on the meachism of the infecting and the development process of F. graminarum, and this will lead to more comprehensive undertanding of the whole precess of F. graminearum. Deep investigation on mutant M594 revealed that type II myosin played crucial roles in almost all the development states of F. graminearum. It is important that Myo2 protein is conserved in fungi but not exsited in plant and this proverty make it to be an ideal target for broad-spectrum fungicide. Therefore, our work is meaningful to the development of novel approaches to the plant disease control. |
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