One Pathogenicity-related Gene And It’s Functional Characterization Of Fusarium Oxysporum F. Sp. Cubense Race4

To date, there are4races of Fusarium oxysporum f. sp. cubense oxysporum and race4(Focr4) is considered as one of the most destructive pathogens of banana by causing serious wilting disease. However, its pathogenicity mechanism is still not clearly understood yet. Since the Focr4genome has been sequenced and the mutant library of Focr4has been built. The full length cDNA of the pathogenicity-related gene in Focr4-1562was cloned. An analysis of the inactivated gene and a study of the phenotype of the gene-knockout mutant would serve as the basis for studying the specific mutation and pathogenesis of Foc.In this study, DNA Southern blots analysis of Focr4-1562mutant created in our laboratory showed that this mutant was single-copy insertions. The full length cDNA of the inactivated pathogenic gene in Focr4-1562was cloned and the inactivated gene of knockout/complementary from the wild type Focr4-193-6was analysed. By comparing the phenotypes of the T-DNA insertion mutant Focr4-1562, gene-knockout transformant ΔFocr4-1562and the wild type Focr4-193-6, speculated functions of mpfol gene through observing the phenotypes of the mutant. the results can be summarized as follows:(1)The full cDNA sequence of Focr4-1562gene was cloned and submitted to GenBank as mpfo1(accession No. JQ669955). The ORF of the gene is composed of five exons separated by four introns. Based on online information, It has been suggested that the gene may be the channel protein of a regulating gene located on the endoplasmic reticulum.(2) Construction of the knock-out vector of the mpfol gene by labeling it with green fluorescent protein (GPF), using genomic DNA of strain Focr4-193-6as template and based on pathogenic tests to screen for changes in pathogenic phenotypes, the knock-out transformant ΔFocr4-1562was obtained. ΔFocr4-1562and ΔFocr4-1562were compared with the wild type Focr4-193-6. The pathogenicity was significantly diminished. The pathogenicity of the reverse mutation transformant, ΔFocR4-1562cp was as same as the wild type Focr4-193-6. The study revealed that the mpfol of F. oxysporum was probably the pathogenicity-related gene.(3) The phenotypes of the T-DNA insertion mutant Focr4-1562, gene-knockout transformant ΔFocr4-1562and the wild type Focr4-193-6. The results showed that the growth rate of ΔFocr4-1562was slower on complete medium with reduced conidia production and germination while the colony color was also different. ΔFocr4-1562was more resistant to cell wall degrading enzyme, and the utilization rate of carbon source was reduced. The results suggested that the mpfol gene was related to the structural changes in the cell wall.