| Light signal transduction pathway is an important research topic of photoreaction studies. Functional research of red light receptor phytochrome is one of the foundations to understand the light signal transduction pathway. Phytochromes are widely distributed in plants, algae, bacteria, filamentous fungi and other organisms. Our laboratory found that the growth and pathogenicicty of Botrytis cinerea are regulated by light. To explore the molecular mechanisms of light reaction in Botrytis cinerea, the function of phytochrome Bcphy3 were studied by the methods of molecular genetics. The results were as follows:1. To predict phytochrome gene, the bioinformatics were used. At first, searching the phytochrome in the Botrytis cinerea genome database; then BLAST on the NCBI, we found that there were three phytochrome in the Botrytis cinerea:Bcphyl, Bcphy2 and Bcphy3. The results show that Bcphy3 belongs to the Botrytis cinerea phytochrome through further analysis conserved functional domains and transmembrane regions of BcPHY3, and the homologous evolutionary relationships with other fungi phytochrome.2. Bcphy3 was knocked out by means of homologous recombination mutation strategy with PEG-mediated protoplast transformation. One mutant strain was obtained from 126 transformants,which was identified by the PCR analysis of genomicDNA, RNA transcript and Southern blot. The mutant strain was named △Bcphy3-P21.3. Compared with the wild-type strain Bc05.10, △Bcphy3-P21showed slower growth ratereduced biomass accumulation, and less pigment in mycelium. These results indicate that Bcphy3 plays an important role in regulating vegetative hyphae development and pigment synthesis. Observed by transmission electron microscopy, the cell wall structure of ABcphy3-P21 was damaged. Besides, the expression of chitin synthesis gene were reduced in △Bcphy3-P21. At the same time, ABcphy3-P21 chitin content decreased. These results show that Bcphy3 is important for the integrity of the cell wall. In addition, Observed by Optical microscope, △Bcphy3-P21 was dificient in conidiation and spore germination, but produced more microspores, indicating that Bcphy3 can regulate the reproductive development.4. ABcphy3-P21 usually form initial lesions or had no lesions on tomatoes, peppers, onions, tobacco leaves and so on. In addition, △Bcphy3-P21 didn’t form appressorium on onion epidermal and its mycelia can not invade into onion epidermal as shown by trypan blue dyeing. Further analysis of pathogenic factors showed that:there were no ROS in the process of ABcphy3-P21 infection on tobacco leaves. Analysis of cell wall degrading enzymes and oxalic acid secretion in vitro culture demonstated that △Bcphy3-P21 does not secrete proteases, but can secrete polygalacturonase, cellulase and oxalic acid. Therefore, Bcphy3 is crucial to the early stages of infection.5. Red light can promote the colony growth of △Bcphy3-P21 but not affect its conidiation; However, red light can promote conidiation of wild-type strain Bc05.10 but does not affect its colony growth. Then it can be concluded that Bcphy3 is responsive to red light and promote asexual reproduction of Botrytis cinerea.6. When grown on the CM medium with osmotic stress, oxidative stress, cell membrane stress and cell wall stress, the relative growth rates of △Bcphy3-P21 were lower than the wild-type Strain Bc05.10. That is to say mutant are more sensitive to stress. This illustrates that Bcphy3 plays an important role in stress tolerance.Based on the results above, we consider that Bcphy3 plays a vital role in development of vegetative hyphae, pigments synthesis, the cell wall integrity, sexual and asexual reproduction, the early stages of infection and stress resistance. What is more, Bcphy3 can respond to red light and promote conidiospore formation. This study broadens the scope of the study of fungal phytochrome and provide important theoretical basis to further study the optical signal transduction pathway in fungi. |
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