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Keywords:

  • pathogenic fungi;
  • genome sequencing;
  • fungicide resistance

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Penicillium digitatum, causing citrus green mold, is one of the most devastating pathogenic fungi for postharvest fruits. The disease control is becoming less efficient because of the dispersal of fungicide-resistant strains. However, genome-scale analyses of its resistance mechanism are scarce. In this work, we sequenced the whole genome of the R1 genotype strain Pd01-ZJU and investigated the genes and DNA elements highly associated with drug resistance. Variation in DNA elements related to drug resistance between P. digitatum strains was revealed in both copy number and chromosomal location, indicating that their recent and frequent translocation might have contributed to environmental adaptation. In addition, ABC transporter proteins in Pd01-ZJU were characterized, and the roles of typical subfamilies (ABCG, ABCC, and ABCB) in imazalil resistance were explored using real-time PCR. Seven ABC proteins, including the previously characterized PMR1 and PMR5, were induced by imazalil, which suggests a role in drug resistance. In summary, this work presents genome information of the R1 genotype P. digitatum and systematically investigates DNA elements and ABC proteins associated with imazalil resistance for the first time, which would be indicative for studying resistant mechanisms in other pathogenic fungi.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Green mold decay, caused by Penicillium digitatum (Pers.: Fr.) Sacc., is a worldwide problem not only in citrus-growing regions, but also anywhere citrus fruits are enjoyed. Penicillium digitatum is responsible for about 90% of the total loss of postharvest citrus fruits during packing, storage, transportation, marketing, and consumption (Kanetis et al., 2007; Macarisin et al., 2007). Chemical control of citrus green mold using sterol demethylation inhibitor (DMI) fungicides, such as imazalil, is widely adopted around the world, but extensive use of imazalil has caused the emergence of fungicide-resistant populations since the 1980s (Eckert, 1987).

The mechanism of DMI fungicide resistance in P. digitatum was initially reported by Hamamoto et al. (2000). They found a group of imazalil-resistant isolates (designated as the R1 group), of which the drug target gene CYP51 was overexpressed in the presence of a 126-bp DNA element duplicated at the CYP51 promoter region (Hamamoto et al., 2000). Another type of imazalil-resistant P. digitatum was found in 2007. These isolates (the R2 group) also had upregulated CYP51 gene expression caused by the insertion of PdMLE1, a 199-bp miniature inverted-repeat transposable element (MITE), into the promoter region (Ghosoph et al., 2007). Interestingly, our recent work revealed that when PdMLE1 is inserted upstream of CYP51B, a gene homologous to CYP51, CYP51B would be overexpressed and consequently confer DMI fungicide resistance (R3 group; Sun et al., 2011ab). Apart from the CYP51 gene family, many other transporter genes are also associated with fungicide resistance. In P. digitatum, two ATP-binding cassette (ABC) transporter family genes (PMR1 and PMR5) and one major facilitator superfamily (MFS) gene (PdMFS1) have been revealed to mediate DMI fungicide efflux (Hamamoto et al., 2001; Nakaune et al., 2002; Wang et al., 2012). However, both the PdMLE1 element and the transporters are abundant in P. digitatum, which indicates that more of them may be involved in the drug resistance.

Recently, the genome sequences of two P. digitatum strains have become available: the DMI-resistant strain Pd1, which has the similar genotype to the R3-resistant P. digitatum, and the sensitive strain PHI26 (Marcet-Houben et al., 2012). Comparative genomics revealed a weak relationship between the resistant phenotype and genome variations, which suggested that insertion of PdMLE1 should be the major, if not the only reason for DMI fungicide resistance in the Pd1 strain. In this work, we sequenced the genome of another P. digitatum strain, Pd01-ZJU, which belongs to the R1 resistance group. DMI-fungicide-resistance-associated DNA elements and ABC transporters were then investigated at the genomewide level.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Strain, culture, and DNA preparation

The strain Pd01 (assigned as Pd01-ZJU in this paper) of P. digitatum used in this study was single-spore-isolated from infected citrus in the Zhejiang province and has been previously characterized (Zhu et al., 2006) and deposited in the CBS fungal collection bank with accession number CBS130525. Fungal cultures were maintained on potato dextrose agar (PDA) or potato dextrose broth (PDB) at 25 °C unless otherwise indicated. Conidial suspensions in 20% glycerol were stored at −80 °C. Conidia were produced by culturing fungal strains on PDA at 25 °C. For DNA preparation, 5 μL (106 spores mL−1) of conidia suspension was added into PDB media for 72 h (160 r.p.m., 25 °C). Then, mycelia were harvested and homogenized in liquid nitrogen for DNA extraction using DNeasy Plant Mini Kit (Qiagen, Mississauga, ON). The extracted DNA was quantified and qualified with 1% agarose gel before sequencing.

Genome sequencing and annotation

The draft genome sequence of Pd01-ZJU was generated using a hybrid strategy combining Illumina GAII (Illumina, San Diego, CA) and Roche GS FLX Titanium (Roche Diagnostics, Basel, Switzerland) sequencing technologies integrated by Shanghai Biochip Ltd (Shanghai, China). A fragment library was prepared for GS FLX sequencing. A 300-bp insertion paired-end library (PE) and a 3-kb and another 5-kb mate-pair library (MP) were constructed, and they were sequenced with the GAII platform. Raw data generated by GS FLX were assembled by Newbler (www.454.com). These contigs were then extended and scaffolded by adding the short reads of GA II PE and MP libraries using SSPACE (Boetzer et al., 2011).

Gene prediction for the Pd01-ZJU genome was performed by both ab initio and cDNA based methods with GeneMark (Besemer & Borodovsky, 2005), Genscan (Burge & Karlin, 1997), and AUGUSTUS (Stanke et al., 2004). Primary gene annotation was carried out by homolog searching against NCBI nonredundant protein database (nr) using blastp (E-value < 1e-3, identity > 30% and query coverage > 50%). The whole-genome transposable elements analysis was implemented using both de novo and homology-based methods via RepeatModeler and RepeatMasker, respectively (setting default parameters; Bergman & Quesneville, 2007).

Classification of ABC transporter subfamily

To identify the putative ABC transporters in Pd01-ZJU, we adopt the method as described previously (Kovalchuk & Driessen, 2010) with slight modifications. Briefly, the ABC subfamily data set downloaded from NCBI database was used as query sequences for blast searching against predicted Pd01-ZJU proteome. Genes showing the highest similarity with known ABC genes were selected for further analysis. Cutoff e-values of < 1e-4 were applied for protein similarity in all hits.

Multiple sequence alignment of ABC proteins was performed using ClustalW (Thompson et al., 2002). The phylogenetic tree was then constructed using MEGA5 (Tamura et al., 2011) with the maximum-likelihood method and 1000 bootstrap replicates. Protein domains of ABC subfamilies were obtained from the InterPro database (http://www.ebi.ac.uk/interpro/).

Quantitative PCR analysis

The expression levels of genes responding to fungicide imazalil were measured by qPCR on an ABI 7300 Real-Time PCR system (ABI). Five microliter conidia suspension (106/mL) was added to PDB medium and cultured at 28 °C for 2 days. The mycelia were then treated with 100 μg mL−1 imazalil for 15 min before filtered for total RNA isolation as described previously. The first-strand cDNA synthesis using EasyScriptTM First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) was followed by the standard procedure of the manual. qPCR was carried out using the SYBR Premix Ex TaqTM (Perfect Real-Time) kit (TaKaRa Biotech. Co., Dalian, China) according to the manufactures' instructions. Experiments were conducted in triplicates. The expression of the target gene related to the reference β-tubulin encoding gene was calculated using the inline image method (Livak & Schmittgen, 2001). anova was applied to determine significant differences of the test genes with respect to controls.

Data deposition

This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number ANGJ00000000. The version described in this paper is the first version, ANGJ01000000.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Genome features and comparison with other strains

The genome assembly of P. digitatum Pd01-ZJU is c. 26 Mb in length, including 76 unordered scaffolds that are larger than 200 bp (average coverage > 300×; Supporting Information, Table S1), which excludes the mitochondrial genome of 28 978 bp (Sun et al., 2011ab). The 35 super scaffolds (> 100 kb) account for c. 98% of the genome. The average G+C content of P. digitatum is c. 48.4%. A total of 9006 protein-coding genes and 162 tRNAs were identified in Pd01-ZJU, showing a similar coding capacity to other Penicillium species. Approximately 8200 (91% of its gene set) P. digitatum genes showed > 35% protein identity to their homologs in GenBank database (Coordinators, 2013). 87% of the predicted genes had their best hits in Penicillium and another 9% in Aspergillus. Transposon-like elements comprised about 1% of Pd01-ZJU genome.

The Pd01-ZJU genome shared high similarity with recently published genomes of another two P. digitatum strains Pd1 and PHI26 (Marcet-Houben et al., 2012), both in genome size and organization (99.89% and 99.8% average identity, respectively, at nucleotide level). Rather than PHI26, Pd1 showed better genomic synteny with Pd01-ZJU (Fig. 1), with a total of 1775 high-confidence single nucleotide polymorphisms (SNPs) identified between them. One hundred and three SNPs in Pd01-ZJU were located at coding regions, which led to synonymous mutation (27%) or nonsynonymous mutation (73%) on nucleotide sequences (Table S2).

image

Figure 1. Genomic synteny between three Penicillium digitatum strains: Pd1, PHI26, and Pd01-ZJU.

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Fungicide-resistance-related DNA elements in P. digitatum

Our previous study has demonstrated that the PdMLE1 plays a very important role in DMI fungicide resistance for P. digitatum (Sun et al., 2011ab). A blast search and Southern blot against the Pd01-ZJU genome revealed that there were at least 17 copies of intact PdMLE1 elements in P. digitatum (Sun et al., 2013). Herein, we estimate the variation in copy number and chromosomal location of PdMLE1 in three P. digitatum strains. Seven, eleven, and thirteen intact PdMLE1 elements were identified in Pd01-ZJU, Pd1, and PHI26 assemblies, respectively, with each containing several imperfect elements. It is interesting that most of the imperfect PdMLE1 elements are located at the distal end of scaffolds in all the three genome assemblies, indicating the growing genome complexity caused by PdMLE1 mobility. Locations of PdMLE1 elements were compared between P. digitatum strains, which revealed that most of them were not conserved in genome arrangement (Fig. 2), indicating the recent translocation events occurred in P. digitatum (Fig. 2).

image

Figure 2. Genome locations of PdMLE1 among threes strains. A: Pd01-ZJU; B: PHI26; C: Pd1.

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Due to the powerful transcriptional activity, an insertion of PdMLE1 to the proper location might lead to the undersigned phenotype. Therefore, we checked through the annotated genomes and collected the information of genes downstream of the PdMLE1 elements. The result showed that most of these genes have unknown function (Table S3). Only a few genes, such as serine/threonine protein kinase, phosphotransferase, and amino acid permease, were found in different strains. Currently, there is no evidence for these genes being involved in fungicide resistance or being overexpressed. But it is possible that the translocation of PdMLE1 would eventually increase the adaptation to special niches.

The other DNA element that assisted in the emergence of the R1 resistance group in P. digitatum consisted of 126-bp nucleotides. We checked its copy number in three strains. The tandem repeat of 126-bp sequence was only found in Pd01-ZJU, which was in accord with our hypothesis that this 126-bp sequence was duplicated incidentally rather than that acquired from elsewhere. However, so far, it is still unclear whether this sequence could act as a promoter or functioned only as a transcriptional enhancer.

ABC transporter family in P. digitatum

Taking advantage of available ABC transporter family classification in human (Dean et al., 2001), yeast (Paumi et al., 2009), and filamentous fungi (Kim et al., 2013), we conducted an exhaustive search of putative ABC genes in P. digitatum. A total of 46 chromosome-encoded ABC family transporters were identified in the Pd01-ZJU genome, dividing into eight subfamilies, that is, ABCB, ABCC, ABCD, ABCE, ABCF, ABCG, ABCI, and Ydr061w. Genes in subfamilies ABCB (10 genes, 22%), ABCD (12 genes, 26%), and ABCG (13 genes, 28%) took a higher proportion than that of other subfamilies. These genes were scattered around different scaffolds in P. digitatum. blast searching against another two strains and nearby species, Penicillium chrysogenum, Penicillium marneffei, and Talaromyces stipitatus, indicated that most genes were conserved across genus Penicillium, and only three genes (Pd001g10790, Pd009g18793, and Pd003g15407) were lost in either of Pmarneffei and Tstipitatus (Fig. 3).

image

Figure 3. Classification of ABC subfamilies in Penicillium digitatum. The maximum-likelihood method with the JTT model was used to construct the unrooted phylogenetic tree of ABC proteins. The bootstrap consisted of 1000 replicates. PC, Penicillium chrysogenum; PM, Penicillium marneffei; TS, Talaromyces stipitatus.

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Phylogenetic analysis was carried out to determine the evolutionary relationship among these 46 genes. Results showed that they were classified into nine subgroups (Fig. 3). Subfamilies ABCB and ABCC were much closer in phylogenetic distance: both contained ABC transporter domain (IPR001140) and ATPase domain (IPR003593). ABCG subfamily genes had an additional domain, ABC-2 type transporter (IPR013525), which was consistent with phylogenetic distance with other ABC subfamilies. Interestingly, two genes, Pd005g17512 (ABCF subfamily) and Pd001g10745 (ABCG subfamily), were distinct from their respective family members in the phylogenetic tree. Of these, Pd005g17512 was only half the length in predicted protein size compared with other ABCF subfamily members, so it was probably misannotated.

ABC subfamily proteins involved in imazalil resistance

To explore whether more transporters may be involved in drug resistance, the ABC proteins, subgroups ABCC and ABCG (all members), as well as part of subgroup ABCB (Pd004g16828, Pd008g18475, and Pd002g13917), for which proteins basically contained the two typical ABC family domains (transmembrane domain and ATPase domain), were selected for imazalil-inducing expression analysis. Totally, 7 of 29 tested ABC genes were upregulated (expression fold change > 1.5), including previously characterized PMR1 and PMR5. Of these, two genes belonged to the subgroups ABCB and ABCC (Pd008g18475 and Pd003g15518, respectively), and the other five genes were the members of ABCG subfamily (Fig. 4). Of note, Pd001g10258, PMR1, and Pd009g18793 were highly expressed, and the expression change ratio for Pd009g18793 even reached 200. Although this was the first time for most of these transporters being characterized in P. digitatum, when blast searching against the GenBank database, we found that these upregulated genes were highly similar to the multidrug transporters in Aspergillus spp. (62–79% peptide sequence identity), suggesting conserved evolution of these genes. Moreover, the expression magnitude also indicated that the uncharacterized transporters with higher expression changes might be more important for DMI fungicides exportation in P. digitatum.

image

Figure 4. Upregulated transporters induced by imazalil.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Traditional chemical control for green mold has led to several types of resistant groups in P. digitatum that evolved under selective pressure due to fungicide use (Hamamoto et al., 2000; Ghosoph et al., 2007; Sun et al., 2011ab). Two P. digitatum genome sequences have been available thanks to the effort from colleagues in Spain, enabling us to now release the genome of another resistant strain, Pd01-ZJU. These strains were high similar in genome structure and composition. A few genomic variations around PdMLE1 locations were found, and some of these were probably caused by translocation. The ABC transporter family was systematically classified, and the novel function of drug transportation for several genes was also indicated. The genome information we offered will provide the opportunity to better understand the genome evolution of P. digitatum under fungicide pressure. Likewise, those ABC transporters involved in imazalil transportation could be the putative target for future drug development.

Mobile element mediating drug target gene overexpression is the common mechanism of fungicide resistance in phytopathogens [e.g. a 2.1- to 5.6-Kb truncated retrotransposon in Blumeriella jaapii (Ma et al., 2006), a 65-bp DNA element in Monilinia fructicola (Luo & Schnabel, 2008), and a 553-bp DNA sequence in Venturia inaequalis (Schnabel & Jones, 2001)]. Such insertions increased expression of the downstream gene and resulted in fungicide resistance. However, little is known about how these DNA elements functioned in detail. In our previous work, we found a 199-bp DNA element (PdMLE1) insertion in the promoter region of the gene CYP51B and demonstrated that this element was a transposon belonging to the MITE family. PdMLE1 contained promoter sequences, and we had located the 30-bp core promoter sequences (5′-CCGAGAC AGATAAACTATATGTGATGTTTA-3′; Sun et al., 2013). In the current study, we confirmed that PdMLE1 was an active transposon in P. digitatum with several unique insertions in different P. digitatum strains, which made the fungus more resilient to environmental pressure. The mechanism in which PdMLE1 acted in P. digitatum could be a general model for explaining fungicide resistance resulting from transposons in other fungal pathogens. Also, as PdMLE1 is a species-specific transposon, it is still very interesting to understand where PdMLE1 originated and how it evolved.

Transporters mediate almost all life activities in the cell, including nutrient assimilation, protein exchange, and toxic compound exportation. The ABC transporters were highly conserved in the genus Penicillium. The roles of typical ABC transporters involved in imazalil exportation were investigated, and in addition to PMR5 and PMR1, several other ABC proteins were indicative of conferring drug resistance. This study demonstrates that more genes than previously known may participate in the drug resistance. The MFS family genes are another type of transporters that are essential for all organisms. There are more than one hundred MFS genes in P. digitatum, but only one had been reported to be involved in DMI fungicide resistance. As indicated by our expression analysis of the ABC genes, we believe that more MFS genes will be revealed to have a role in drug transport.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

This work was supported by the National Foundation of Natural Science of China (31071649), China Agriculture Research System (CARS-27), and the Special Fund for Agro-scientific Research in the Public Interest (201203034).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
fml12235-sup-0001-TableS1-S3.docxWord document30K

Table S1. Genome features of Pd01-ZJU.

Table S2. Single nucleotide polymorphisms in the coding region of Pd01-ZJU compared with Pd1 and PHI26.

Table S3. Genome locations of PdMLE1 among three P. digitatum strains.

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