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Functional analysis of a novel ABC transporter ABC4 from Magnaporthe grisea

Archna Gupta, Bharat B. Chattoo
DOI: http://dx.doi.org/10.1111/j.1574-6968.2007.00937.x 22-28 First published online: 1 January 2008

Abstract

The ATP-binding cassette (ABC) superfamily of membrane transporters has been implicated to play a role in pathogenesis in various phytopathogenic fungi. In an insertional mutagenesis screen for pathogenicity mutants of Magnaporthe grisea obtained via Agrobacterium tumefaciens-mediated transformation (ATMT), a novel gene belonging to the ABC transporter family was identified. The gene ABC4 was predicted to be 5045 bp in length coding for a protein of 1654 amino acids. The mutant did not form functional appressoria and was nonpathogenic. When compared with wild type, the mutant showed increased sensitivity to certain antifungal compounds and phytoalexins, implying the role of ABC4 in multidrug resistance (MDR) as well as establishment in the host. Reverse transcriptase PCR showed the expression of ABC4 in wild-type strain while it was absent in the mutant abc4. In real-time PCR, the expression of ABC4 was seen to be enhanced in the presence of various drugs tested. The data suggests that ABC4 is required for the pathogenicity of M. grisea, helping the fungus to cope with the cytotoxic environment during infection.

Keywords
  • ABC transporter
  • ABC4
  • Magnaporthe grisea
  • appressorium
  • multidrug resistance

Introduction

ATP-binding cassette (ABC) transporters are transmembrane proteins that couple the energy of ATP hydrolysis to the selective transfer of substrates across biological membranes (Higgins, 1992). The ABC superfamily comprises an extremely diverse class of membrane-transport proteins responsible for the controlled efflux and influx of substances across cellular membranes. The ABC transporters have been identified in a wide variety of organisms, including mammals, yeast, fungi, bacteria and insects (van Veen & Konings, 1998), and play a major role in multidrug resistance (MDR). They are minimally composed of four domains, with two transmembrane domains (TMDs) responsible for allocrite binding and transport and two nucleotide-binding domains (NBDs) responsible for coupling the energy of ATP hydrolysis to conformational changes in the TMDs. These proteins act as virulence factors and provide protection against plant defense compounds during pathogenesis (Stergiopoulos et al., 2002, 2003).

Worldwide attempts are being made to study host–pathogen interactions using the Magnaporthe grisea–rice pathosystem. Magnaporthe grisea is the first plant pathogenic fungus to be fully sequenced (Dean et al., 2005), providing a detailed insight into its genome. Magnaporthe grisea undergoes a series of defined morphogenetic developmental steps, leading to the production of a specialized infection structure called the appressorium (Hamer et al., 1988; Talbot, 2003), which is essential for pathogenicity. Although the major steps in infection process have been documented, the molecular details including the number and nature of genes involved remains largely unknown. Insertional mutagenesis is an effective way to study the genes involved in infection and pathogenicity. Agrobacterium tumefaciens can transfer DNA to a broad group of organisms and has been used as an effective tool for insertional mutagenesis (Krysan et al., 1999; Rho et al., 2001). Agrobacterium tumefaciens-mediated transformation (ATMT) of M. grisea was used, in an attempt to identify the genes involved in appressorium formation. Analysis of regions flanking the T-DNA integration site in one of the appressorium mutants showed insertion in a gene belonging to the ABC family of transporters. The gene was found to be essential for appressorium development as well as pathogenicity of M. grisea.

Materials and methods

Fungal culture

Magnaporthe grisea B157, corresponding to the international race IC9, was previously isolated in the author's laboratory from infected rice leaves (Kachroo et al., 1994). The fungus was grown and maintained on Oatmeal agar (Hi-Media, Mumbai, India). Fungal conidia were harvested by scraping the biomass grown on Oatmeal agar plates with a sterile surgical blade, resuspended in sterile water and purified by passing through a glass wool column.

ATMT of the fungus

The vector used for transformation was pABC (Gupta & Chattoo, 2007), where hygromycin phosphotransferase gene is cloned under a gpdA promoter (glyceraldehyde-3-phosphate-dehydrogenase). Magnaporthe grisea was transformed with pABC via ATMT as described (Mullins et al., 2001). Magnaporthe grisea transformants were selected on complete medium, CM (Talbot et al., 1997), supplemented with Hygromycin B (Sigma Chemical, St Louis, MO) to a final concentration of 200 μg mL−1. Fungal genomic DNA was extracted as described by Dellaporta (1983), and inverse PCR (Ochman et al., 1988) was carried out to amplify the T-DNA flanking sequence. The PCR products were cloned at the EcoRV site of pBluescript KS+ for sequencing using a CEQ Beckman Coulter 8000 sequence analyzer.

Appressorial assay

Approximately 50 μL of 104 spores mL−1 were placed on the hydrophobic side of a gel bond film (Amersham Biosciences, Uppsala, Sweden) and incubated under moist conditions for 12–16 h. The spores were checked under a microscope for appressorium formation.

Infection assay

Barley explant infection was carried out as described (Clergeot et al., 2001), where droplets of spore suspension (c. 105 spores mL−1) were placed on leaf segments placed on water agar plus kinetin (2 mg L−1) plates. Disease symptoms were recorded after 4–5 days. For wound infection, the leaves were abraded with a needle before inoculating the spore suspension. Fungal cell staining was carried out using lactophenol cotton blue solution (Merck, Darmstadt, Germany) and observed under an epifluorescence microscope (Nikon, Eclipse 80i, Kanagawa, Japan) with a CCD camera attached.

Drug assays

The drugs cycloheximide, camptothecin, methotrexate, miconazole and resveratrol were purchased from Sigma Chemical. Stock solutions were made in dimethyl sulfoxide. Cells were grown in minimal agar plates supplemented with the required drug concentrations for 7 days at 28 °C for drug sensitivity assays.

Reverse transcriptase PCR (RT-PCR)

Fungus was grown in CM for 48 h and further grown for 5 h in the presence and absence of various drugs. Fungal biomass was harvested and frozen in liquid nitrogen. Total RNA was isolated using Trizol reagent (Invitrogen Life Technologies, CA). Five micrograms of total RNA was used to synthesize the first-strand cDNA using MuMLV reverse transcriptase (Fermentas GmBH, St Leon-Rot, Germany) and oligo (dT)12 in a 20 μL reaction system. Five microliters of this RT product was used to carry out the RT-PCR using ABC4-specific primers: Forward primer 5′-ATGGCGTTCATCCGGCAAA-3′; Reverse primer 5′-CTACTTGCGCCGACTAAACAA-3′.

Real-time PCR

Quantitative PCR was performed by monitoring in real time the increase in fluorescence of the SYBR Green dye on a LightCycler system for real-time PCR (Roche Applied Science, Mannheim, Germany), according to the manufacturer's instructions. The primers used for the amplifications were TubL1 (GAG TCC AAC ATG AAC GAT CT); TubR1 (GTA CTC CTC TTC CTC CTC GT); ABC4L1 (GTC ATG CTC GAG GAG AAC AA); and ABCR1 (TAT CCC TCC TCC TTG ACG TT). Thermal cycling conditions consisted of 10 min at 95 °C, followed by 40 cycles of 10 s at 95 °C, 10 s at 55 °C and 15 s at 72 °C. Each RT-PCR quantification was carried out in triplicate. Values for each gene were normalized to the expression level of the respective control condition and used further to calculate the ratio of the expression levels of the requisite transcripts.

Results

Identification of ABC4

In an ATMT screen for pathogenicity mutants, a mutant abc4 defective in appressoria formation was identified. Integration of T-DNA at a single site was confirmed by Southern hybridization, and the gene disrupted was identified by inverse PCR. Homology searches were carried out using the M. grisea genome database (http://www.broad.mit.edu/annotation/fungi/magnaporthe/), and the sequence showed identity to gene coding for the ABC family of proteins (GenBank accession no. XM_368307).

Features of ABC4 gene

When compared with the M. grisea database, the ABC4 comprised the locus MGG_00937.5. The ABC4 gene was found to be 5045 bp in length consisting of two exons. The gene was predicted to code for a 1654 amino acid (aa) protein, consisting of two halves, where each half is composed of the ABC family signature sequence, a hydrophobic domain and a hydrophilic domain. An ATP–GTP-binding site motif A or a P-loop motif was present, which is a characteristic feature of ABC transporters. Hydropathy analysis using TCDB (transport classification database) showed that each half is composed of six transmembrane spanning regions.

ABC4 showed the highest similarity to Neurospora crassa (59%) ABC transporter (accession no. CAD79694), whereas it did not show any significant similarity to the transporters reported earlier from M. grisea (ABC1, ABC2, ABC3). When compared with Saccharomyces cereviseae transporters, ABC4 showed maximum similarity (29%) to a hypothetical transporter (YOLO75C), and to STE6 (22%) among the characterized transporters. A phylogenetic tree was made by comparing the ABC4 protein sequence with other sequences using blast pairwise alignment (Fig. 1). The phylogram showed that ABC4 falls into a separate family of fungal MDR transporters.

Figure 1

Phylogram of ABC transporters related to ABC4. clustalx (Thompson et al., 1997) dendrogram depicting the phylogenetic relatedness of Magnaporthe ABC4 with the ABC proteins from the indicated eukaryotes. Bootstrapping (500 trials) was utilized in generating the phylogenetic tree using the Neighbor-joining algorithm.

Importance of ABC4 in M. grisea pathogenesis

The spores from the mutant abc4 when inoculated on a hydrophobic surface (gel bond film) gave rise to a germ tube ending in abnormal (seen as a swelling at the tip of the germ tube) appressoria (Fig. 2a). When inoculated on onion epidermis, the mutant was unable to elaborate the infection hyphae as compared with the wild type (Fig. 2b).

Figure 2

Appressorium formation and infection assay. (a) Mutant abc4 is unable to form appressoria on a gel bond film. Conidia were allowed to germinate on the hydrophobic side of the gel bond film for 16 h and visualized under a microscope. The scale bar corresponds to 10 μm. (b) Mutant abc4 unable to form infection hyphae on onion epidermis. Conidia were allowed to germinate onion epidermis for 48 h and visualized under a microscope after staining the fungus with lactophenol cotton blue. A, B157; C, abc4; B, D, Corresponding fluorescent images. Scale bar corresponds to 10 μm. (c) Barley infection assay where droplets of spore suspension (c. 105 spores mL−1) were placed on leaf segments placed on water agar plus kinetin (2 mg L−1) plates, and disease symptoms were recorded after 4–5 days. A, infection before wounding; B, infection after wounding.

In the barley infection assay, disease lesions developed in the leaves inoculated with spores from the wild-type fungus, but in the case of the mutant, no lesions were observed (Fig. 2c). Disease symptoms were seen on the leaves inoculated with spores of abc4 when infection was carried out after wounding the leaves, but the degree of infection was less as compared with the wild type.

Multidrug resistance

Putative conserved domains for a multidrug transport system and a sodium transport system were predicted for ABC4 in the NCBI conserved domain database. There was no difference observed in mycelial growth in the presence of NaCl, but the length of the germ tube was greatly reduced in the mutant abc4, as compared with the wild-type strain (Fig. 3). This might be due to the involvement of ABC4 in sodium transport.

Figure 3

Effect of NaCl on abc4. Spore suspension was inoculated on the hydrophobic side of a gel bond film in the presence of NaCl, and germ tube growth was observed after 16 h. Bar corresponds to 10 μm. The effect on the growth of germ tube and appresorium formation was seen at 0, 0.2, 0.5 and 1.0 M NaCl concentrations for both B157 and abc4.

Many ABC transporters are reported to mediate the efflux of various compounds (Stergiopoulos et al., 2002), thus imparting resistance to such antifungal compounds, drugs, antimetabolites or ions. The growth of the mutant abc4 was compared with that of wild type in the presence of various antifungal agents such as methotrexate (antimetabolite inhibiting the synthesis of folic acid), miconazole (sterol biosynthesis inhibitor), camptothecin (alkaloid and a plant metabolite), cycloheximide (protein synthesis inhibitor) and resveratrol (phytoalexin from grapevine). The mutant abc4 showed increased sensitivity to resveratrol, miconazole and cycloheximide, whereas the difference in growth was not significant in case of camptothecin and methotrexate (Table 1).

View this table:
Table 1

Drug sensitivity of wild-type strain B157 and mutant abc4

CompoundB157 EC50 (μg mL−1)B157 MIC (μg mL−1)abc4 EC50 (μg mL−1)abc4 MIC (μg mL−1)
Resveratrol10030050100
Miconazole3.0102.06
Cycloheximide0.31.50.31.0
Camptothecin0.11.00.11.0
Methotrexate>500>500>500>500
  • * Effective concentration of the tested drug inhibiting radial growth by 50% on minimal agar plates.

  • Minimum inhibitory concentration (MIC) of the tested drug sufficient to inhibit the growth on minimal agar plates.

  • The growth experiments were done in triplicate; with three plates for every drug concentration each time.

Expression of ABC4 mRNA

RT-PCR was carried to monitor mRNA expression of the ABC4 gene. ABC4 expression was absent in the mutant, while the expression was seen in the wild-type strain when grown in CM with or without drugs. ABC4 expression was absent in the mutant, while the expression was seen in the wild-type strain when grown in CM with or without drugs and expression levels were seen to be more in treated samples as compared with the untreated one (Fig. 4).

Figure 4

Expression of mRNA for ABC4. RT-PCR was carried out using total RNA from treated and untreated samples. RT-PCR was carried out to amplify the 5-kb ABC4 gene. Equal loading of lanes with rRNA was checked with ethidium bromide staining. Lanes 1, abc4 (CM); 2, B157 (CM); 3, B157 (100 μg mL−1 resveratrol); 4, B157 (80 μg mL−1 miconazole); 5, B157 (100 μg mL−1 cycloheximide); 6, B157 (10 μg mL−1 camptothecin); 7, B157 (200 μg mL−1 methotrexate).

Quantitative analysis of the relative transcript levels of ABC4 was performed using real-time PCR. The same cDNA was used for real-time PCR to monitor the fold induction of ABC4 in the presence of various drugs. The calibrator and target used were TUB (β-tubulin) and ABC4 genes, where the primers were designed to amplify 78 and 110-bp fragments, respectively. The unknown samples were the ones treated with the drug and the reference samples were the untreated ones both for tubulin and the ABC4 gene. Control samples contained no template DNA. The relative quantification results of ABC4 gene are shown in Table 2. The ABC4 was induced about 10.50-, 10.24-, 5.26-, 4.28- and 4.08-fold in the presence of resveratrol, miconazole, cyclohexamide, camptothecin and methotrexate, respectively.

View this table:
Table 2

Relative transcript levels of ABC4 in presence of drugs by real-time PCR

CompoundC t target (ABC4)C t reference (TUB)Normalized ratio (relative transcript level)
None (control)22.8316.881.0
Resveratrol19.1416.7310.50
Miconazole19.3516.7110.24
Cycloheximide20.2916.605.26
Camptothecin21.1916.744.28
Methotrexate21.4216.704.08
  • The relative levels of ABC4 mRNA in each of the sample was normalized using the Ct obtained for the tubulin gene. The values represent the number of times the gene is expressed in presence of the drug compared with the untreated sample grown in absence of any drug (set at 1.00). The results are averages of three repetitions.

Discussion

The identification of a novel ABC transporter, essential for virulence in the blast fungus M. grisea, is reported. Mutant abc4 was impaired in appressorium formation, implying the role of ABC4 in the pathogenic development of M. grisea. As a consequence of the defective appressorium formation, the mutant was also not capable of infection. Nevertheless, lesser intensity disease lesions were observed when infection was carried out on wounded leaves indicating that ABC4 is indispensable for appressorium-mediated penetration but may cause infection once inside the host cells.

ABC4 showed the highest similarity to N. crassa (59%) ABC transporter but not to the transporters reported earlier from M. grisea (Urban et al., 1999; Lee et al., 2005; Sun et al., 2006). ABC4 is related to the Ste6 protein from S. cereviseae (22%), which is an ABC transporter required for the export of a-factor during mating in MAT a cells (McGrath & Varshavsky, 1989). The locus (MGG_00937.5) that harbors the ABC4 gene in M. grisea has been reported to be one of the expressed sequence tags from M. grisea mated culture library (Unisequence ID: Mag30405205; Cogeme database). Related structure homology shows that the conserved domain in the second half of ABC4 is homologous to Treponema pallidum (natA) ABC transporter, which is an ATP-binding protein involved in drug resistance, nodulation, lipid transport, and lantibiotic immunity. blast search suggests that apart from conferring an MDR function, M. grisea abc4 may be involved in lipid as well as Na+ transport. The reduction in the growth of the germ tube in the mutant might be related to Na+ transport. Domain spanning from 460 to 684 and 1289 to 1503 residues show homology to ABC subfamily A, which is known to mediate the transport of a variety of lipid compounds. The ABCA protein in humans is suggested to be involved in the removal of cholesterol and phospholipids from cells onto high-density lipoprotein particles (Young & Fielding, 1999). Cdr1p and Cdr2p confer azole resistance and act as phospholipid translocases in Candida albicans (Smriti et al., 2002). Regions from 460 to 761 and 1048 to 1507 residues are predicted to be similar to CcmA and Mdl B, respectively, which are ATPase components of the ABC-type multidrug transport system. The region over 1289–1507 aa shows homology to ‘NatA,’ which is an ABC-type Na+ transport system. Similar predictions are made for ABC homologs in N. crassa (CAD 79694), Aspergillus fumigatus (XP_753691), Ustilago maydis (XP_759601) and Yarrowia lipolytica (XP_504037). The increase in sensitivity of the mutant to miconazole, a sterol biosynthesis inhibitor, suggests a similar role of ABC4 in lipid transport function.

Mutant abc4 showed sensitivity to the drugs resveratrol and miconazole, and also to cycloheximide to some extent. The mRNA expression of the ABC4 gene was increased in the presence of various drugs while it was absent in the mutant, confirming the expression and role of ABC4 in multidrug resistance. In real-time PCR, the mRNA expression of ABC4 was induced in the presence of drugs. The expression levels correlated with the growth of the mutant in the presence of the drug. Maximum induction of ABC4 was found in the presence of resveratrol, a phytoalexin from grapevine, where ABC4 was 10.50-fold induced as compared with the untreated sample. The ABC transporter from Botrytis cinerea (accession no. AJ006217), showing 29% identity to ABC4, has been reported to affect the sensitivity towards resveratrol and the mutant was reduced in virulence on grapevine (Schoonbeek et al., 2001). Phytopathogenic fungi encounter toxic environments during plant invasion as a result of plant defense response. The interaction between rice and M. grisea is accompanied by production of many antifungal proteins and phytoalexins in the host (Kodama et al., 1992). Increased sensitivity of the mutant to grapevine phytoalexin resveratrol may be the cause of loss of virulence of the mutant.

In conclusion, ABC4 is essential for the pathogenic development of M. grisea on the host. It is required to cope with the cytotoxic environment and provides protection against plant defense mechanisms.

Acknowledgements

This study was funded by Council of Scientific and Industrial Research (CSIR), New Delhi, India and Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India.

Footnotes

  • Editor: Anthony George

References

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