| Penicillium digitatum is the most destructive postharvest pathogen of citrus fruits, causing fruit decay and economic loss. Triazole antifungal drugs have been used to prevent this disease for many years. However, control of the disease is further complicated by the emergence of drug-resistant strains due to the extensive use of triazole antifungal drugs. This paper aims to research the resistant mechanisms of P. digitatum towards prochloraz and its virulent mechanisms towards citrus fruits and providind theoretical basis to the designing of new antifungal drugs with more efficacy. Using methods of molecular biology and bioinformatics, the function of a putative sterol regulatory element-binding protein in P. digitatum was researched and its relation with the resistance of P. digitatum has been revealed. Using the technology of RNA-seq, the trancsriptome of P. digitatum before and after prochloraz-treatment have been sequenced and a large scale analysis of the transcriptome was presented. The resistant mechanisms of P. digitatum were studied from the transcriptome prospect for the first time. Major results of this study are as following:1. An orthologus gene encoding a putative sterol regulatory element-binding protein (SREBP) was identified in the genome of P. digitatum. Using the technology of PCR, Genome walking and RACE (rapid-amplification of cDNA ends), the whole sequence of this SREBP-encoding gene was obtained and named sreA. The putative SreA protein contains a conserved domain of unknown function (DUF2014) at its carboxyl terminus and a helix-loop-helix (HLH) leucine zipper DNA binding domain at its amino terminus, domains that are functionally associated with SREBP transcription factors.2. The deletion of sreA (AsreA) in a prochloraz-resistant strain (PdHS-F6) by Agrobacterium tumefaciens-mediated transformation led to increased susceptibility to prochloraz and a significantly lower EC50 value compared with the HS-F6 wild-type or complementation strain (COsreA). A virulence assay showed that the AsreA strain was defective in virulence towards citrus fruits, while the complementation of sreA could restore the virulence to a large extent.3. Results of quantitative real-time PCR demonstrate that transcriptional abundances of cyp51 genes were significantly decreased in the AsreA strain, especially with regard to cyp51A. The normalized expression values of cyp51A. cyp51B, and cyp51C in the AsreA strain compared with the wild-type were 0.02,0.29 and 0.47, respectively, which reflect that sreA is an important regulator of cyp51 genes. Further analysis revealed that prochloraz-induced expression of cyp51A and cyp51B in PdHS-F6 was completely abolished in the AsreA strain. These results demonstrate that sreA is a critical transcription factor gene required for prochloraz resistance and full virulence in P. digitatum and is involved in the regulation of c51 expression.4. Resistant mechanisms of P. digitatum strain HS-F6 (highly-resistant) were studied from the transcriptome prospect. Total RNA was extracted from prochloraz-resistant strain HS-F6 and prochloraz-susceptible strain HS-E3 before and after prochloraz-treatment, and sequenced by Illumina technology. Gene expression profiling analysis was performed upon prochloraz treatment in HS-F6 and HS-E3, and differential expression analysis was used to identify genes related to prochloraz-response and drug-resistance:there are 224 differentially expressed genes in HS-E3 after prochloraz-treatment; 154 of them are down-regualted and 70 of them are up-regulated. In HS-F6, there are 1100 differentially expressed genes; among them 698 genes are down-regualted and 402 are up-regulated. Moreover, gene expression profile in prochloraz-resistant strain HS-F6 is quite different from that in HS-E3 before prochloraz-treatment,1520 differential expression genes were identified between two strains.5. Gene ontology (GO) term enrichment and KEGG enrichment were then performed to classify the differential expression genes. Among these genes, there are a lot of transporter encoding genes; including 14 MFS (Major Facilitator Superfamily) transporters,8 ABC (ATP-binding cassette transporter) transporters, and 3 MATE (multidrug and toxic compound extrusion family) transporters. Then the roles of typical MFS, ABC and MATE proteins in prochloraz resistance were explored using real-time PCR. Among them,6 MFS proteins,3 ABC proteins and 3 MATE proteins were induced by prochloraz in HS-F6 or express differently in prochloraz-susceptible and-resistant strains. The results confirmed the credibility of the transcriptome data and demonstrate that these transporters are related with the response of P. digitatum to prochloraz. |
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