Whole transcriptome analysis of Penicillium digitatum strains treatmented with prochloraz reveals their drug-resistant mechanisms

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, whi...

Transcriptome analysis of fungicide-responsive gene expression profiles in two Penicillium italicum strains with different response to the sterol demethylation inhibitor (DMI) fungicide prochloraz

“…In the past decades, conventional synthetic DMIfungicides, such as prochloraz and imazalil, were widely applied to control Penicillium decay, but undesirably, a considerable number of resistant isolates including P. digitatum and P. italicum strains have developed [5][6][7]. The mechanisms underlying DMI-fungicide resistance have been elucidated for P. digitatum species by transcriptomic analysis [11]. However, how to develop DMIresistance in P. italicum species is still not clear, might due to rare opportunity to find highly DMI-resistant P. italicum strain(s).…”

“…Fungal resistance to azole-fungicides including a number of DMI-fungicides has been usually ascribed to overexpression of specific drug-efflux pumps such as ABC and MFS transporters [8, 42-50, 53, 54, 57, 58, 88]. Specially, ABC and MFS transporter-encoding genes, each containing multiple isoforms, were reported to be simultaneously up-regulated in the prochloraz-resistant P. digitatum [11]. The similar up-regulation of multiple ABC and MFS gene members (i.e., ABC1-2 and MFS1-4) was also observed in the prochloraz-treated Pi-R rather than Pi-S (Table 4 and Fig.…”

“…However, resistance to these DMI fungicides has frequently occurred for the Penicillium molds in the past decade, especially for P. digitatum isolates with high DMIresistance [5,7], considerably reducing the efficacy of the fungicides. Up to date, we have got some understanding on the mechanism of azole fungicide resistance in P. digitatum [8][9][10][11][12][13]. However, little information is available to explain P. italicum resistance induced by the DMI fungicides.…”

“…Unlike ABC and MFS transporters, MATE proteins function predominantly in bacterial drug-resistance [59][60][61]. To date, the MATE contribution to fungal drug-resistance was only reported in the ectomycorrhizal fungus Tricholoma vaccinum [62] and the citrus pathogenic fungus P. digitatum [11].…”

“…RNA sequencing (RNA-seq) technology has become a powerful tool to profile transcriptomic response to reveal azole-resistance mechanism for some pathogenic fungi including prochloraz-resistant P. digitatum [11], voriconazole-resistant A. fumigatus [81], tetraconazoleresistant Cercospora beticola [82], tebuconazole-resistant Fusarium culmorum [83], and fluconazole-resistant Candida glabrata [84]. Our earlier report has elucidated the mechanism of P. digitatum resistance to DMIfungicide prochloraz through RNA-seq analysis [11]. Nevertheless, the molecular mechanism(s) of P. italicum resistance to such fungicides are poorly understood.…”