| Fusarium asiaticum is a necrotrophic fungal pathogen for important cereal crops,causing sever rotting of their roots,stems,spikes and leaves as a result of infection.A kind of mycotoxin,deoxynivalenol(DON),was accumulated in these infected plants.This compound is toxic to human and animal livestock,and the exposure risk is directly through ingestion of mycotoxin-containing cereals.One of the basic approaches to solve this problem was to clarify mycological characteristics of this fungal pathogen and to supply basic information for establishing an efficient method to control this pathogen.The focal point of the present research project was to analyze external stimuli to affect its physical and developmental activities of this fungus.Light is one of the most important signals to make profound influences on growth,development,secondary metabolism and pathogenicity/virulence expression by the fungi.Fungi can perceive optical signal through a variety of photoreceptors,such as blue light receptors WC1,WC2 and VVD.In order to clarify roles and functions of blue light receptors in F.asiaticum,two mutants(AFawcl and ?Favvd.)for the Fawcl and Favvd genes had been obtained in our laboratory.In the present study for the master's thesis,the author placed the main focus on another blue light receptor gene Fawc2 of F.asiaticum to fully understand its own function and communication with other blue light photoreceptors.The main results obtained are listed below:1.In the first experiment,the author conducted the bioinformatics analysis for the Fawc2 gene and indicated that DNA sequence of Fawc2 was basically homologous to the wc2 genes in other fungal species.However,the presence of different sequences in Fawc2 was suggestive of diverse functions of the genes.From the amino acid sequence analysis of FaWC2(transcript of the Fawc2 gene),the author showed that it contains two domains;1)PAS domain for protein-protein interactions and 2)GATA zinc finger domain for transcriptional regulation.In addition,the amino acid sequence alignment analysis showed that WC2 proteins are highly conserved among different fungal species.2.In the subsequent experiment,the author constructed a knock-out mutant for the Fawc2 gene of the wild type strain EXAP-08,according to the homologous recombination strategy based on PEG-mediated protoplast transformation.The mutant strain obtained exhibited different phonotypic characters from the wild type;that is,1)attenuated pigmentation,2)increased aerial hypha formation,3)accelerated growth rate and 4)defective sexual reproduction.These results indicate that the Fawc2 gene has regulatory functions in colony pigmentation,sexual reproduction and vegetative growth.3.Subsequently,the author examined the ?Fawc2 mutant for its photoreactivation of carotenoid synthesis and UV tolerance.The results showed that the accumulation of carotenoid in AFawc2 was obviously lower than that of the wide type under light exposure conditions.In fact,the mutant strain showed the loss of the photoinductivity for carotenoid synthesis and the significant reduction of the expression of two genes CarA and CarB,which were related to carotenoid synthesis.In addition,the UV tolerance became weaker,and the gene Phrl for photoreactivation was less expressed in the ?Fawc2 mutant than the wild type strain.These results indicated that the Fawc2 gene promote the photoreactive ability for carotenoid biosynthesis and UV tolerance,suggesting that this gene plays an important role in light responsive process of F.asiaticum.4.In the following experiment,the author conducted an inoculation assay for the?Fawc2 mutant strain to examine its infection capability.Eventually,the mutant caused yellow and soft lesion at the inoculation site of asparagus,followed by rapid decay.Additionally,the author inoculated the mutant into wheat spikes in the field and detected the occurrence of abnormal spikes with increased spikelets.These results indicate that the deletion of Fawc2 became more infectious or virulent to host plants.As a matter of fact,these results were contrary to the function of the Fawc1 gene reported previously.From these results,it was clear that Fawc1 and Fawc2 regulate their pathogenicity in a different mode.Finally,the author examined the ability of the mutant to penetrate and successfully indicated its enhanced hyphal penetration.5.In the following experiment,the author constructed the double mutant?Fawc1/wc2 by the method mentioned above and examined whether Fawc2 has any effects on the mutant AFawcl.Eventually,the present double mutant was similar to the original mutant(?Fawc1)in some phenotypic characteristics;1)hyphal growth and development,2)light responsibility and 3)pathogenicity or virulence expression.These results strongly suggest that Fawc2 has no influence on the phenotype expression by the ?Fawcl mutant,and that FaWC2 cannot affect functions of FaWC 1 in the blue light signal pathway.6.In the final experiment,the author constructed an additional double mutant(?Favvd/wc2)and examined whether Fawc2 has any effect on the mutant ?Favvd.The results showed that there was no difference in growth and development,light response and pathogenicity between ?Favvd/wc2 and ?Fawc2.Judging from these results,the author speculated that Fawc2 affects ?Favvd and FaWC2 has influences on the function of FaVVD in the blue light signal pathway.FaWC2 may be located in the upstream of FaVVD.In the present study,the author analyzed the blue light photoreceptor gene Fawc2 from several aspects and found that the function of Fawc2 in F.asiaticum was different from Fawcl in this fungus and wc2 in other fungi.Moreover,the author clarified the relationship between Fawc2 and Fawcl/Favvd.In conclusion,all results and findings obtained in this study could be contributed to provide an experimental basis for understanding the blue light signal pathway of F.asiaticum and more importantly for laying a foundation for further study of fungal photobiology. |
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