A novel ABC transporter gene ABC2 involved in multidrug susceptibility but not pathogenicity in rice blast fungus, Magnaporthe grisea☆

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Abstract

We cloned a novel ATP-binding cassette (ABC) transporter gene ABC2 from the rice blast fungus, Magnaporthe grisea. ABC2 protein had nucleotide-binding folds (NBF) and predicted transmembrane domains (TMD6) arranged in a duplicate [NBF–TMD6]2 configuration and showed the highest amino acid homology with BMR1 of Botrytis cinerea. Transcription of the gene was up-regulated by treatment with many toxicants, including several blasticides, demethylation inhibitors (DMI), and antibiotics. ABC2 disruptants displayed an increased sensitivity to bitertanol, myclobutanil, and tebuconazole (DMIs), camptothecin (alkaloid) and cycloheximide (antibiotic). This result demonstrated that ABC2 is a member of multidrug transporters. We performed infection assay of ABC2 disruptants towards rice. However, no obvious difference of disease severities between ABC2 disruptants and wild-type strain was observed, demonstrating that ABC2 is not involved in pathogenicity.

Introduction

Fungicide treatment is the most important method for the control of plant diseases caused by phytopathogenic fungi. Fungicide resistant strains have appeared in many phytopathogenic fungi, such as Botrytis cinerea, Penicillium digitatum, and Venturia inaequalis, which have led to difficulties in the control of these diseases [1].

Rice blast fungus, Magnaporthe grisea (Hebert) Barr [anamorph, Pyricularia grisea Sacc.], is the most devastating rice pathogen. Strains of this fungus which are resistant to phosphorothiolates, kasugamycin, and blasticidin S first appeared in Japan in the 1970s [1], and carpropamid resistant strains also appeared in 2001 in Japan [2]. To develop the efficient control strategy towards such strains, it is important to clarify the fungicide resistance mechanism.

Until now, molecular mechanisms of fungicide resistance such as mutation of target protein [1], [3], overproduction of target enzyme [4], and detoxification of fungicide [1], [3] have been designated. Recently, it was demonstrated that active efflux of fungicides mediated by ATP-binding cassette (ABC) transporters also contributes to fungicide resistance in several filamentous fungi, such as Aspergillus nidulans [5], [6], [7], P. digitatum [8], [9], and B. cinerea [10], [11], [12], [13].

ABC transporters form a large protein superfamily found in almost all living organisms [14]. In the genome of Saccharomyces cerevisiae, for example, a total 30 such proteins are found [15]. ABC transporters actively transport chemically or functionally diverse compounds from the inside of the cell to the outside using the energy from ATP hydrolysis [14]. PDR5 of S. cerevisiae extrudes more than 100 compounds [16]. For this reason, ABC proteins are often called ‘multidrug transporter’. All ABC proteins have a similar molecular architecture with a hallmark domain organization that includes the presence of evolutionarily conserved nucleotide-binding folds (NBF), containing the Walker A and Walker B motifs and the ABC signature [17], [18], as well as several transmembrane domains (TMD). In most ABC transporters, the TMDs and NBFs are arranged in a duplicate configuration—[NBF–TMD6]2 or [TMD6–NBF]2 [15].

In addition, the natural functions of some ABC transporters concerning pathogenicity and microorganism competition have been demonstrated. For example, ABC1 of M. grisea [19], BcatrB of B. cinerea [12], and Gpabc1 of Gibberella pulicaris [20] act as pathogenicity factors. It is thought that these ABC proteins are involved in efflux of phytoalexins or phytoanticipins. BcatrB is involved in competition with phenazine-producing Pseudomonas spp. by extruding this antibiotic [21].

We previously cloned a novel ABC transporter gene (ABC2) fragment from M. grisea by degenerate polymerase chain reaction (PCR) [22]. In this paper, we present the corresponding full-length gene structure of ABC2. The contribution of ABC2 to fungicide susceptibility and pathogenicity towards rice was examined by creating ABC2 disruptants. Furthermore, comparison of ABC2 with ABC1 was carried out.

Section snippets

Fungal strains and culture conditions

Magnaporthe grisea strain P-2b (MAFF235001, race: 303) and all the transformants were maintained on potato dextrose agar (PDA, Difco laboratories, Sparks, MD) at 25 °C in the dark. For nucleic acid extraction, M. grisea strains used were cultured in potato dextrose broth (PDB, Difco) for 3–4 days at 25 °C with agitation at 120 strokes/min. Conidia were produced on oatmeal plates during incubation at 25 °C. After 2 weeks of culture, the aerial mycelia were brushed off, and the plates were incubated

Characterization of ABC2 gene

A novel ABC transporter gene ABC2 of M. grisea was cloned by PCR-based strategy. The genomic sequence of ABC2 has submitted to DDBJ under accession number AB091269. Sequence analysis revealed an ORF of 4455 nucleotides encoding a deduced protein of 1484 amino acids (Fig. 1). This ORF was interrupted by two introns of 84 and 80 bp in length; they were verified by RT-PCR. The upstream of ABC2 -coding region contains a 6/8 conserved Kozak sequence (TCACGATG) spanning the putative translation start

Discussion

In this study, we cloned and characterized a novel ABC transporter gene ABC2 from M. grisea to investigate the contribution of ABC transporters to toxicant susceptibility of this fungus. We found that ABC2 is a determinant of susceptibility to chemically or functionally unrelated toxicants. However, decreases in EC50 value of ABC2 disruptants towards DMIs were not so drastic as towards camptothecin and cycloheximide. Therefore, it is possible that ABC2 together with the other ABC transporter

Acknowledgment

We thank Dr. Francois Kraus (University of Tokyo) for advice in the preparation of the manuscript.

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    ☆

    This study was supported by the program for promotion of basic research activities for innovative biosciences of the Bio-oriented Technology Research Advancement Institution (BRAIN).

    1

    Present address: Laboratory of Experimental Therapeutics, Korea Cancer Center Hospital, Konglung-Dong, Nowon-Ku, Seoul 139-706, South Korea.

    2

    Present address: Research and Development Center, Unitika Ltd., Uji-kozakura, Uji, Kyoto 611-0021, Japan.

    3

    Present address: Laboratory for Adaptation and Resistance, RIKEN Plant Science Center, Hirosawa, Wako, Saitama 351-0198, Japan.

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