Cloning And Characterization Of A Novel MFS Transporter Gene Pdmfs2 In Penicillium Digitatum

Penicillium digitatum is the most destructive postharvest pathogen of citrus fruits, causing about 90%of the losses during postharvest citrus packing, storage, transporting, and marketing. Additionally, control of this disease is further complicated by the emergence of drug-resistant strains due to the extensive use of demethylation inhibitor fungicides (DMIs). Under this circumstance, an understanding of the potential molecular mechanisms involved in DMIs resistance is of great significance because it will provide a basis for the designing of novel antifungal chemicals with greater efficacy. Identifying new target genes and the designing of new drugs is of great significance to control the spread of green mold caused by P. digitatum. The major facilitator superfamily (MFS) is a large protein family with diverse physiological functions in all kingdoms of life. Many of these transporters have low substrate specificity and play a role in sensitivity to drugs and fungicides. Furthermore, over-expression of such transporters can lead to multidrug resistance (MDR) of microorganisms.In order to understand the molecular mechanisms involved in DMIs resistance of P. digitatum, a novel MFS transporter gene has been found and researched in this article. Major results of this study are as follows:1. In this work, a novel MFS transporter gene, Pdmfs2, was isolated from P. digitatum. Sequence analysis revealed that the gene Pdmfs2 had a length of 2115 bp, with 5 introns of 51,56,51,62 and 305 bp, located between positions 211-261 bp,333-388 bp, 681-731 bp,804-865 bp and 1,571-1,875 bp, respectively. The full-length DNA of Pdmfs2 had a 1590 bp open reading frame encoding a full-size MFS transporter with 529 amino acids. PdMFS2 shares a high degree of similarity with members of the MFS transporters from other fungi, and the MFS1 domain has been found. We designated this cloned gene as the Pdmfs2, being predicted to encode a major facilitator superfamily transporter 2 in P. digitatum.2. qRT-PCR was performed to compare the transcription levels of Pdmfs2 in the prochloraz-resistant strain (PdHS-F6) and prochloraz-sensitive strain (PdHS-E3). The result showed that the background transcription level of Pdmfs2 in PdHS-F6 was about 1.73-fold as high as that in PdHS-E3. Additionally, the Pdmfs2 transcription level in PdHS-F6 was up-regulated after treating with 7.0 μg/mL prochloraz for 6 h, and the normalized expression value was 2.37. This demonstrated that the expression of Pdmfs2 in PdHS-F6 can be induced by prochloraz.3. The Pdmfs2 disruption (APdmfs2) and complementation (COPdmfs2) strains were obtained by A. tumefaciens-mediated transformation (ATMT). The deletion of Pdmfs2 in PdHS-F6 led to increased susceptibility to prochloraz and lower EC50 value compared with the PdHS-F6 wild-type or complementation strain. PdHS-F6 is highly resistant to triazole drug prochloraz with an EC50 value of 7.896 mg/L. The EC50 value of prochloraz for the ΔPdmfs2 strain was 6.782 mg/L, which was less than the value of PdHS-F6. However, complementation of Pdmfs2 restored the EC50 value of the ΔPdmfs2 strain (EC50 value of prochloraz for the COPdmfs2 strain was 7.813 mg/L). Additionally, the diameters of colonies of APdmfs2 strains on PDA plates supplemented with prochloraz (5.0 μg/mL,10.0μg/mL) were smaller than those of PdHS-F6 and COPdmfs2 strains. These results demonstrated that Pdmfs2 is required for prochloraz resistance, and PdMFS2 could pump prochloraz out of cells.4. Conidiation assays showed that the Pdmfs2 disruption strain was defective in conidia yield, while the complementation of Pdmfs2 could restore this phenotypic feature to a large extent. Disruption of Pdmfs2 (ΔPdmfs2) in PdHS-F6 caused a decrease of-29% conidia compared with the PdHS-F6 wild-type and COPdmfs2 strains, indicating that Pdmfs2 is involved in conidiation.5. A virulence assay was performed directly on citrus fruits. The mean diameter of the macerated lesions of the fruits incubated with the ΔPdmfs2 conidial suspension was approximately 4.48 cm at 3 days post inoculation, whereas the mean macerated lesion diameter of the fruits incubated with the PdHS-F6 conidial suspension was about 5.48 cm. A virulence assay for the COPdmfs2 strain showed that the average diameter of the macerated lesions induced by COPdmfs2 was 5.44 cm. The deletion of Pdmfs2 rendered the ΔPdmfs2 strain less virulent compared with the wild-type PdHS-F6, and the virulence of COPdmfs2 was comparable to that of PdHS-F6. These results revealed that Pdmfs2 plays a role in the virulence of P. digitatum toward citrus fruits.