Construction Of A High Efficiency Of Gene-targeting System And Functional Analysis Of PdErg3in Penicillium Digitatum

Penicillium digitatum, causing citrus green mold, is the most important pathogen of postharvest citrus. The post-harvest loss caused by this disease usually accounts for about90%of the total loss. We know little about the molecular mechanism of pathogenicity and drug resistance of P. digitatum in spite of the importance. Disruption or replacement of the targeted genes is efficient and absolutely essential for determining the functions of a interested gene. Although we had established Agrobacterium tumefaciens-mediated transformation (ATMT) for this pathogen before, the gene disruption frequency was very low amongst transformants recovered through ATMT in many cases, and for some genes, we were even unable to disrupt. This shortage greatly hampers the functional characterization of interested genes in P. digitatum. In this study, a high efficient system for gene targeting in P. digitatum was established. In addition, the function of PdErg3B was explored. The results are as follows:1. Establishment of a high efficient system for gene-targeting in P. digitatumThe low efficiency of gene targeting in filamentous fungi is related to the presence of the active repairing mechanism of DNA double-strand breaks (DSBs) named non-homologous end joining (NHEJ) pathway. While the heterodimer consisting of Ku70and Ku80is a key factor for NHEJ pathway. Ku80ortholog, PdKu80, in P. digitatum was identified by the blast search of the genomic sequence of P. digitatum (unpublished) using Ku80homolog of Aspergillus oryzae (AoKu80). The putative PdKu80gene was2673bp in length, which contained10introns and encoded712amino acids. PdKu80disruption plasmid pTFCM-△PdKu80was constructed by replacing the open reading frame of PdKu80with hygromycin resistance gene cassete through two-step PCR and gene cloning technology. The vector pTFCM-△PdKu80was transformed into wild-type strain PdKH8of P. digitatum via ATMT,82putative transformants were picked up from the hygromycin B-containing medium. PCR followed by Southern blot identification indicated3transformants were PdKu80-targeted mutants, named△PdKu80. The vegetative growth, conidiation and pathogenicity for△PdKu80had not been affected.Using the similar strategy, the PdBrlA and PdMpkA disruption plasmids pNEO1300-APdBrlA and pNEO1300-△PdMpkA were constructed. They were transformed into the wild-type and△PdKu80of P. digitatum, respectively. The PdBrlA-disrupted mutants and PdMpkA-disrupted mutants were selected on the neomycin-containing medium, followed by diagnostic PCR identification. Calculation of the gene-targeting efficiency indicated that when the wild-type PdKH8was used as targeting strain, the efficiencies PdBrlA and PdMpkA targeting were2.2%and4.0%, respectively. However, when APdKu80was used as targeting strain, the efficiencies of PdBrlA and PdMpkA targeting were33.3%and13.0%respectively. These results indicated that deletion of the PdKu80gene in P. digitatum clearly increased gene targeting frequencies.2.The founctions of PdErg3BThe research indicated that two genes, PdErg3A and PdErg3B, encoding C-5sterol desaturase are present in P. digitatum. PdErg3A had939bp nucleotides, without intron, encoding312amino acids. PdErg3B was1130bp in length, and encoded350amino acids, with an intron located at nt271to nt347bp. The disruption of PdErg3B ddi not affect the growth, sporulation, and the biosynthesis of ergosterol, but increased the resistance of P. digitatum to DMI fungicides such as imazalil, difenoconazole and tebuconazole. This results indicated that PdErg3B was not related to the growth, sporulation and the ergosterol biosynthesis, but related to azole resistance.