Genetic Transformation And Molecular Mechanisms Of Fungicide Imazalil Of Penicillium Digitatum

Penicillium digitatum is the most important pathogen, causing the green decay of postharvest citrus. The method of a stable genetic transformation via Agrobacterium tumefaciens-mediated transformation (ATMT) was established in this study. The transformation efficiency could be reached to 1 per 105conidia, and the insertion event was most likely random and single copy, suggested that this method could be used to create a T-DNA inserted-mutant reservoir for study of gene function in P. digitatum.An unknown molecular machnism of imazalil-resistance was found in this study. To study if the mutation was account for the machnism of imazalil-resistance, the sequences of CYP51 gene and their promoter sequences were cloned from 7 imazalil-sensitive and 14 imazalil-resistant strains. Aligment of both deduced amino acid sequences of CYP51 genes and the CYP51 promoter sequences demonstrated no significatly related mutations were responsed to the resistance. Three CYP51 genes (PdCYP51, PdCYP51B and PdCYP51C) were identified from EST database of P. digitatum. The expressions of three CYP51s from 2 imazalil-sensitive and 7 imazalil-resistant strains were analyzed using real-time RT-PCR, and indicated no significantly different expression profile were found among these strains. These results suggested that neither the point mutation in PdCYP51 coding and promoter regions and nor the expression profile of these CYP51 genes could be count for the molecular machnism of imazlil-resistance found in this study.PdMfsl, a novel major facilitator superfamily gene from P. digitatum, was cloned and characterized in this study. PdMfsl contains an open reading frame (ORF) of 1,876 bp, with 3 introns of 55,49 and 71 bp. It encodes a protein of 566 amino acids shares high identity to members of the drug:H+antiporter efflux family of major facilitator surperfamily (MFS) transporters with 14 predicted transmembrane spanners (DHA14). Heterologous expression of PdMfsl in Saccharomyces cerevisiae resulted in the transformants displaying reduced sensitivity to DMIs and other unrelated compounds. The imazalil-resistant levels of P. digitatum field strains were positively correlated with the expression levels of PdMfsl. The expression of PdMfsl was elavated by the treatments of DMIs and other compounds in both imazalil-resistant and imazalil-sensitive strains. Disruption of PdMfsl did decrease the resistance of P. digitatum to DMIs fungicides and some other compounds, and the dysfunctioned resistance could be complemented by reintroducing PdMfsl to the disruptant (ΔPdMfsl), whereas PdMfsl overexpression mutants displayed increased resistance to these compounds. These results indicate that PdMfsl is a multidrug transporter of P. digitatum that could paritially function in resistance to DMIs and other toxic compounds. Pathogenicity test found that the lesion development in APdMfsl inoculated citrus fruits was slower than those of parental strain PdW03, PdMfsl null mutants, overexpresion and complementary mutants inoculated fruits, suggesting that PdMfsl also plays role in pathogenicity.