genomewide investigation into dna elements and abc transporters involved in imazalil resistance in ...

R E S EA RCH L E T T E R

Genomewide investigation into DNA elements and ABCtransporters involved in imazalil resistance in Penicillium

digitatum

Xuepeng Sun1, Ruoxin Ruan1, Lingyun Lin2, Congyi Zhu1, Tianyuan Zhang1, Mingshuang Wang1,Hongye Li1 & Dongliang Yu3

1Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Institute of Biotechnology, Zhejiang University, Hangzhou, Zhejiang, China;2Zhejiang Institute of Freshwater Fisheries, Huzhou, Zhejiang, China; and 3College of Life and Environmental Sciences, Hangzhou Normal

University, Hangzhou, Zhejiang, China

Correspondence:

Hongye Li, Key Laboratory of Molecular

Biology of Crop Pathogens and Insects,

Institute of Biotechnology, Zhejiang

University, Hangzhou, Zhejiang 310058,

China. Tel./fax: (+86)057188982328;

e-mail: [email protected]

Received 9 July 2013; revised 4 August 2013;

accepted 8 August 2013.

DOI: 10.1111/1574-6968.12235

Editor: David Studholme

Keywords

pathogenic fungi; genome sequencing;

fungicide resistance.

Abstract

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 investi-

gated the genes and DNA elements highly associated with drug resistance. Vari-

ation 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 environmen-

tal adaptation. In addition, ABC transporter proteins in Pd01-ZJU were charac-

terized, 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 pre-

sents 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

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 con-

sumption (Kanetis et al., 2007; Macarisin et al., 2007).

Chemical control of citrus green mold using sterol deme-

thylation 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. digit-

atum was initially reported by Hamamoto et al. (2000).

They found a group of imazalil-resistant isolates (desig-

nated 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-resis-

tant 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., 2011a, b).

Apart from the CYP51 gene family, many other trans-

porter genes are also associated with fungicide resistance.

FEMS Microbiol Lett && (2013) 1–8 ª 2013 Federation of European Microbiological SocietiesPublished by John Wiley & Sons Ltd. All rights reserved

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In P. digitatum, two ATP-binding cassette (ABC) trans-

porter 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 genome-

wide level.

Materials and methods

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 acces-

sion number CBS130525. Fungal cultures were main-

tained on potato dextrose agar (PDA) or potato dextrose

broth (PDB) at 25 °C unless otherwise indicated. Conid-

ial 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 lL(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 (Illu-

mina, San Diego, CA) and Roche GS FLX Titanium

(Roche Diagnostics, Basel, Switzerland) sequencing tech-

nologies 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 plat-

form. 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 per-

formed 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 Repeat-

Modeler 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 fur-

ther analysis. Cutoff e-values of < 1e-4 were applied for

protein similarity in all hits.

Multiple sequence alignment of ABC proteins was per-

formed 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 suspen-

sion (106/mL) was added to PDB medium and cultured

at 28 °C for 2 days. The mycelia were then treated with

100 lg 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

FEMS Microbiol Lett && (2013) 1–8ª 2013 Federation of European Microbiological SocietiesPublished by John Wiley & Sons Ltd. All rights reserved

2 X. Sun et al.

(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 b-tubulinencoding gene was calculated using the 2�DDCt method

(Livak & Schmittgen, 2001). ANOVA was applied to deter-

mine 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

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 > 3009; Sup-

porting Information, Table S1), which excludes the

mitochondrial genome of 28 978 bp (Sun et al.,

2011a, b). 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. Transpo-

son-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-confi-

dence single nucleotide polymorphisms (SNPs) identified

between them. One hundred and three SNPs in Pd01-

ZJU were located at coding regions, which led to synony-

mous mutation (27%) or nonsynonymous mutation

(73%) on nucleotide sequences (Table S2).

Fig. 1. Genomic synteny between three

Penicillium digitatum strains: Pd1, PHI26, and

Pd01-ZJU.


Genome dissection of imazalil resistance in P. digitatum 3

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., 2011a, b). 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 ele-

ments 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).

Due to the powerful transcriptional activity, an inser-

tion 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

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


4 X. Sun et al.

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 inci-

dentally rather than that acquired from elsewhere. How-

ever, 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 sub-

families. 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, indi-

cated that most genes were conserved across genus

Penicillium, and only three genes (Pd001g10790,

Pd009g18793, and Pd003g15407) were lost in either of

P. marneffei and T. stipitatus (Fig. 3).

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. Inter-

estingly, 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 mem-

bers, 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 upregulat-

ed (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 con-

served evolution of these genes. Moreover, the expression

magnitude also indicated that the uncharacterized trans-

porters with higher expression changes might be more

important for DMI fungicides exportation in P. digitatum.

Discussion

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.,

2011a, b). 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 resis-

tant strain, Pd01-ZJU. These strains were high similar in

genome structure and composition. A few genomic varia-

tions 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 pres-

sure. Likewise, those ABC transporters involved in imaza-

lil transportation could be the putative target for future

drug development.

Mobile element mediating drug target gene overexpres-

sion is the common mechanism of fungicide resistance in



phytopathogens [e.g. a 2.1- to 5.6-Kb truncated retrotrans-

poson 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′-CCGAGACAGATAAACTATATGTGATGTTTA-3′; Sun et al., 2013).

In the current study, we confirmed that PdMLE1 was an

active transposon in P. digitatum with several unique inser-

tions 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

Fig. 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.

Fig. 4. Upregulated transporters induced by imazalil.


6 X. Sun et al.

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 exporta-

tion were investigated, and in addition to PMR5 and

PMR1, several other ABC proteins were indicative of con-

ferring 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 analy-

sis of the ABC genes, we believe that more MFS genes

will be revealed to have a role in drug transport.

Acknowledgements

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).

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Supporting Information

Additional Supporting Information may be found in the

online version of this article:

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.


8 X. Sun et al.

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