Study On The Function Of Blue Light Receptors BcWCL2 And BcVVD1 In Botrytis Cinerea

Botrytis cinerea belongs to necrotrophic plant pathogen and infects more than1000 plant species worldwide to cause serious damage in diseased plants,leading to huge economic losses of agricultural yields.As one of the important environmental signals,light can affect the growth and development,secondary metabolism and pathogenicity expression by the fungi.The perception of light signals with different wavelength ranges depends on different photoreceptors that the fungi possess.In fungi,blue light receptor is an important class of photoreceptors,which includes WC-1,WC-2 and VVD.Previous studies reported that Bc WCL1 in B.cinerea regulate the growth and pathogenicity of this fungus,but the function of other two blue light receptors has remained unclear.From this viewpoint,the present work was designed to further clarify specific functions of these three blue light receptors that regulate growth and pathogenicity of B.cinerea and the interaction among them;namely,the author mutated two genes bcwcl2 and bcvvd1 of B.cinerea to obtain reliable comparative data between the wild and mutant types.The results obtained are summarized as follows:1.The author knocked out and complemented bcwcl2 gene.To demonstrate that bcwcl2 is involved in the photoresponse of Botrytis cinerea,we examined the expression of the photoresponse genes in?bcwcl2 in dark and light,and found that these genes can be specifically induced by light in the wild type,but not in?bcwcl2,indicating that bcwcl2 have function in the photoresponse of Botrytis cinerea.2.The author explored the regulation of growth and development by the bcwcl2gene.The growth rate and conidiation of the mutant(?bcwcl2)under light was opposed to each other the former was significantly reduced and the latter increased than that of the wild type.The?bcwcl2 mutant produce conidia in the dark without forming sclerotium,which is distinct from the wild type.Furthermore,the author examined the germination rate of?bcwcl2(at 4h)and germ tube length(at 8h after incubation)and found that it was lower than that of the wild type.The above results indicate that the bcwcl2 gene plays a role in the growth and conidium production by B.cinerea.3.In order to clarify the regulation of pathogenicity expression by bcwcl2,the author selected tomato leaves and apple fruits as hosts to conduct inoculation experiments under different lighting conditions after inoculation;continuous lighting,continuous non-lighting(darkness)and alternated lighting and non-lighting treatment at the same time length(12 h).The results showed that the pathogenicity of?bcwcl2was significantly lower than that of the wild type.From these results,the author concluded that the regulation of the pathogenicity by bcwcl2 is not dependent on light.In addition,the author examined the ability of?bcwcl2 to secrete ROS(Reactive oxygen species),some cell wall degrading enzymes(polygalacturonase,cellulose and protease)and organic acid and found that the ability of?bcwcl2 to produce ROS,polygalacturonase and organic acid was significantly reduced.The expression of some genes related to the toxin production are also decreased.The above results indicate that bcwcl2 can positively regulate the pathogenicity of B.cinerea.4.In order to explore the function of the bcvvd1 gene,the author knocked out and complemented this gene.Eventually,the?bcvvd1 mutant lost the ability to produce conidia under light,while sclerotium was still produced in the dark.The growth rate and pathogenicity expression by the?bcvvd1 mutant are not different from the wild type.The above results indicate that the bcvvd1 gene can promote the production of conidia by B.cinerea,but has no regulation on the growth,sclerotium production and pathogenicity of this fungus.5.In order to further clarify the function of the bcvvd1 gene,we examined whether this gene is involved in the light adaptation of B.cinerea.The wild and?bcvvd1 types were lighted for different periods of time(0-5h),and the light-regulated genes bcltf1 and bcltf2 were examined for their expression by a fluorescence quantitative analysis method.The results indicate that short-time illumination(0-1h)cause significant induction of the expression of the genes in both the wild and mutant types,whereas long-term illumination(2-5h)reduce the expression level of these genes in both.Obviously,it appeared that there is light adaptation in both wild type and?bcvvd1,indicating that bcvvd1 is not involved in the light adaptation of B.cinerea.6.To reveal the interaction among the three blue light receptors,the author examined the interaction between Bc WCL1,Bc WCL2 and Bc VVD1.Bc WCL1 was shown to interact with Bc WCL2 in previous works,but the interaction between other proteins are not clear.The present works indicate that Bc VVD1 can interact with Bc WCL1 and Bc WCL2 by a yeast two-hybrid expression system.In addition,the author knocked out the PAS domain and the Zn F domain of Bc WCL2 and found that the interaction between Bc WCL2^{?Zn F} and Bc VVD1 was reduced.These results suggest that Zn F domain takes part in the interaction between Bc WCL2 and Bc VVD1.7.In order to prove that Bc WCL2 can act as a transcription factor to regulate the pathogenicity of Botrytis cinerea,the subcellular localization analysis of Bc WCL2was firstly carried out.It was found that Bc WCL2 was localized in the cytoplasm and nucleus,and then the Zn F domain of Bc WCL2 was knocked out.The result showed that the pathogenicity of bcwcl2^{?Zn F} was significantly lower than that of wild type.These results preliminarily demonstrate that Bc WCL2 can act as a transcription factor to regulate the pathogenicity of Botrytis cinerea.In summary,the present work successfully verified the functions of bcwcl2 and bcvvd1 in several aspects including the growth and pathogenicity expression by B.cinerea.In addition,the interactive relationship among three blue light receptors Bc WCL1,Bc WCL2 and Bc VVD1 of this fungus was clarified extensively.In general,the research in this paper enriched the understanding of the photoreceptor and laid a solid foundation for the comprehensive analysis of the light signal pathway of B.cinerea.