Cloning and primary functional analysis of Pdmfs1, a major facilitator superfamily transporter from the fungal pathogen Penicillium digitatum
Wang Jy and Li HY
Program and Abstracts, 11th International Citrus Congress (ISC Congress), 26-20 October 2008, Wuhan, China. 333 pages.
2008
บทคัดย่อ
Cloning and primary functional analysis of Pdmfs1, a major facilitator superfamily transporter from the fungal pathogen Penicillium digitatum
Recent studies indicate that transporters in the major facilitator superfamily (MFS) in plant pathogens function in the secretion of endogenous fungal pathogenicity factors (e.g. toxins) and in protection against exogenous toxic compounds, such as plant defense compounds (e.g. phytoalexins) and fungicides. Therefore, transporters can act as virulent and fungicide-resistant factors. This study elucidates the function of MFS in the fungicide-resistance of Penicillium digitatum (Pers.:Fr) Sacc., the most important causal agent of postharvest decay of citrus. A gene (Pdmfs1) in the MFS was cloned by thermal asymmetric interlaced PCR (TAIL-PCR) following the an amplification of a conserved fragment using degenerated primers…Comparison of the sequence of genomic DNA and cDNA revealed that Pdmfs1 contains a 1698-bp open reading frame (ORF) interrupted by 3 introns, encoding a protein with 566 amino acids. The nucleotide sequence of Pdmfs1 is available at GenBank (Accesion No. AM412556). The Blast datable (http://www.ncbi.nlm.nih.gov/BLAST/) demonstrated that Pdmfs1 is highly homologous with other fungal MFS transporters from Neosartorya fischeri (60%), Aspergillus clavatus(60%), and Botrytis cinerea (56%). A prediction of transmembrane (TMS) domains of Pdmfs1 using a program of TMHMM (http://www.cbs.dtu.dk/services/TMHMM) suggested the presence of 14 TMS domains. The full length Pdmfs1 ORF was colned into yeast expression vector pYES2, and then transformed to a hypersensitive Saccharomyces cerevisiae strain yorl (ΔLc64ΔLc65). The sensitivity comparison between the transformants with pYES2+Pdmfs1and with pYES2 (empty vector) indicated that the expression of Pdmfs1 in yor1 resulted in an increase of its resistance against the 14-α-demethylation inhibitor fungicides imazalil, prochloraz, and myclobutanil. Expression analysis using real time PCR (RT-PCR) indicated that the expression of Pdmfs1 in PDw03, a naturally imazalil-resistant isolate of P. digitatum, is about 10 times higher than that of the naturally imazalil-sensitive isolate Pd23. The expression level of PDw03 was activated by the treatment of 0.1 ppm imazalil for 30 min. Therefore, the data suggests that Pdmfs1 might be involved in protection of P. digitatum against fungitoxic compounds.