Abstract
BackgroundPenicillium digitatum is one of the most destructive postharvest pathogen of citrus fruits, causing fruit decay and economic loss. The emergence of fungicide-resistant strains made the control of P. digitatum more difficult. While the genome of P. digitatum is available, there has been few reports about its resistant mechanism from the transcriptome perspective and there has been no large-scale functional annotation of the genome using expressed genes derived from transcriptomes.MethodsTotal RNA of P. digitatum strain HS-F6 (prochloraz-resistant strain) and HS-E3 (prochloraz-susceptible strain) before and after prochloraz-treatment were extracted and sequenced on an Illumina Hiseq 2000 platform. The transcriptome data of four samples were compared and analyzed using differential expression analysis, novel transcripts prediction and alternative splicing analysis, SNP analysis and quantitative real-time PCR. ResultsWe present a large scale analysis about the transcriptome data of P. digitatum. The whole RNA was extracted from a prochloraz-resistant strain (HS-F6) and a prochloraz-susceptible strain (HS-E3) before and after prochloraz-treatment and sequenced by Illumina technology. A total of more than 100 million reads were generated and de novo assembled into 9760 transcripts that contained annotated genes after quality control and sequence assembling. 6625 single nucleotide variations (SNVs) were identified from the sequences aligned against the reference genome. 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 and 1100 differentially expressed genes in HS-F6 after prochloraz-treatment. 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 the two strains. 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) and 3 MATE (multidrug and toxic compound extrusion family) transporters. Meanwhile, the roles of typical MFS, ABC and MATE proteins in prochloraz resistance were investigated using real-time quantitative PCR.ConclusionsThe sequencing-based transcriptome data of P. digitatum demonstrate differences between prochloraz-resistant and prochloraz-susceptible strains with prochloraz-treatment. The differences existed in expressed transcripts, splice isoforms and GO categories, which would contribute to our knowledge on the molecular mechanisms involved in drug resistance of P. digitatum.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2043-x) contains supplementary material, which is available to authorized users.
Highlights
Penicillium digitatum is one of the most destructive postharvest pathogen of citrus fruits, causing fruit decay and economic loss
In 2015, we reported the identification and characterization of an Sterol regulatory element-binding proteins (SREBPs) protein, SreA, in P. digitatum and proved that it played an important role in the full virulence, prochloraz (PRC) resistance and expression of ergosterol biosynthetic genes in P. digitatum [10]
Identification of expressed transcripts in the P. digitatum transcriptome The whole RNA was extracted from a prochloraz-resistant strain HS-F6 and a prochloraz-susceptible strain HS-E3 before and after prochloraz-treatment and sequenced by Illumina technology
Summary
Penicillium digitatum is one of the most destructive postharvest pathogen of citrus fruits, causing fruit decay and economic loss. Control of P. digitatum is becoming more difficult because of the emergence of drug-resistant strains due to excessive use of demethylation inhibitor (DMI) fungicides [2,3,4]. Evidence on the transcriptional regulation of these target genes has emerged to explain the drug-resistant mechanisms of P. digitatum [7]. Hamamoto et al [11] reported that duplication of a 126-bp DNA element in the cyp promoter region led to the increased resistance of P. digitatum strains to the antifungal drug imazalil. Another case of imazalil-resistance is associated with up-regulated CYP51 expression caused by the insertion of a 199-bp miniature inverted-repeat transposable element (MITE) in the promoter region [12]. Comparative genomics analysis demonstrated that the difference in genome scale was not expectedly distinct between the resistant and susceptible strains, indicating the importance of transcriptional or post-transcriptional regulation in the fungicide resistance [16, 17]
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