| Curvularia leaf spot, caused by Curvularia lunata (Wakker) Boed., significantly reduces the yields of maize in some countries. The disease has been widely spread from northeast to southwest of China. Since the end of nineteen century, it had become one of the most serious maize diseases on restricting the maize yields. There are some researches on occurrence regulation, identification of resistance of variety resource and epidemiology of Curvularia lunata, but little has been known about the virulence genes and effects of selection of this disease.Reinoculation and two-dimensional electrophoresis were performed to analyze the virulence differentiation of Curvularia lunata. Resistant host inbred lines Shen135, Mo17, and 78599-1 were reinoculated (six generations) with low virulent isolate WS18 and C107, and dynamic changes were analyzed. Results showed that the disease index has no significant change for the first 2 generations of inoculation. At the third generation, the number and the expression of differential proteins increased on the protein profiles with the disease index significant increase. A number of of differential expressed proteins were found in the fifth generation mycelia when compared to the original one. In the experiment, 10 differentially expressed proteins were identified by MALDI-TOP/MS. Two proteins, iron/manganese superoxide dismutases and phosphoglycerate mutase 1 were induced in both isolates by all 3 inbred lines. Another protein, Ran protein Serine/threonine protein kinase, glycerol-3-phosphate and scytalone dehydratase were found to be up-regulated during reinoculation. On the other hand, triosephosphate isomerase, RNB domain and Brn1 were down-regulated. One unknown protein was also identified. Three proteins were related directly to the differentiation of virulence, including 2 proteins (Brn1, Scytalone dehydratase) were related to melanin biosynthesis, and 1 protein (NAD-dependent glycerol-3-phosphate) was related to the glycerol biosynthesis; 2 proteins (Serine/threonine protein kinase, Iron/manganese superoxide dismutases) were related to allergen, 4 proteins (Triosephosphate isomerase, Phosphoglycerate mutase, RNB domain, Ran/TC4 subfamily) were related to the metabolism of carbon or signaling pathway, and 1 protein (hydrolases or acyltransferases) was unkonwn to Curvularia lunata. Those induced and up-regulated proteins had close relationship with pathogen virulence differentiation, not only indicated the directional selection of resistance host to pathogen, but also can be virulence differentiation markers. It provided important clues and methods for studying on the mechanism of virulence differentiation.To analize the genes related to virulence, we have used the restriction enzyme-mediated DNA integration (REMI) method to establish a transformation system in Curvularia lunata CX-3 using the linearized plasmid PV2, which contains a hygromycin B phosphotransferase and a ampicillin (amp) resistance genes as selective markers. Out of 200 REMI transformants, 10 genetic stable albino transformants were screened by selective media for 5 generations. After inoculation of maize with the albino transformants and strain CX3, we found that those all albino transformants have low virulence and sporulation. After a plasmid rescue, the partial integrated vector and 430 bp of flanking fungal genomic DNA sequences were isolated from transformant A-2 and designated the fatty acid desaturase (FAD3), which is a major enzyme in synthesizing poly-unsaturated fatty acids (PUFAs), and maybe related to the conidial production and mycelial growth.
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