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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary material
  9. References
  10. Supporting Information

The filamentous fungus Alternaria alternata contains seven pathogenic variants (pathotypes), which produce different host-specific toxins and cause diseases on different plants. The strawberry pathotype produces host-specific AF-toxin and causes Alternaria black spot of strawberry. This pathotype is also pathogenic to Japanese pear cultivars susceptible to the Japanese pear pathotype that produces AK-toxin. The strawberry pathotype produces two related molecular species, AF-toxins I and II: toxin I is toxic to both strawberry and pear, and toxin II is toxic only to pear. Previously, we isolated a cosmid clone pcAFT-1 from the strawberry pathotype that contains three genes involved in AF-toxin biosynthesis. Here, we have identified a new gene, designated AFTS1, from pcAFT-1. AFTS1 encodes a protein with similarity to enzymes of the aldo-ketoreductase superfamily. Targeted mutation of AFTS1 diminished the host range of the strawberry pathotype: ΔaftS1 mutants were pathogenic to pear, but not to strawberry, as is the Japanese pear pathotype. These mutants were found to produce AF-toxin II, but not AF-toxin I. These data represent a novel example of how the host range of a plant pathogenic fungus can be restricted by modification of secondary metabolism.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary material
  9. References
  10. Supporting Information

Host-specific toxins produced by fungal plant pathogens are generally low-molecular-weight secondary metabolites and are critical determinants of host-specific pathogenicity or virulence in several plant–pathogen interactions (Kohmoto et al., 1995; Markham and Hille, 2001; Wolpert et al., 2002). The imperfect fungus Alternaria alternata contains seven variants, which produce host-specific toxins and cause necrotic diseases on different plants (Nishimura and Kohmoto, 1983; Kohmoto et al., 1995). As A. alternata is one of the most cosmopolitan fungal species and is generally saprophytic (Rotem, 1994), these host-specific forms have been designated as pathotypes of A. alternata (Nishimura and Kohmoto, 1983; Kohmoto et al., 1995).

Host-specific toxins from A. alternata pathotypes are diverse in structure (Supplementary material, Fig.S1) (Kohmoto et al., 1995). However, AF-toxin of the strawberry pathotype, AK-toxin of the Japanese pear pathotype and ACT-toxin of the tangerine pathotype have a common moiety, 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid (EDT), in their structures (Figs 1A and S1) (Nakashima et al., 1985; Nakatsuka et al., 1986; Kohmoto et al., 1993).

image

Figure 1. Host-specific toxins from two pathotypes of A . alternata. A. AF-toxins produced by the strawberry pathotype. EDT, 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid; 2-HV, 2-hydroxyvaleric acid; 2,3-DHIV, 2,3-dihydroxy-isovaleric acid. B. AM-toxins produced by the apple pathotype. L-AMV, l-α-amino-methoxyphenyl-valeric acid; L-APV, l-α-amino-phenyl-valeric acid; L-AHV, l-α-amino-hydroxyphenyl-valeric acid; 2-HIV, 2-hydroxy-isovaleric acid; L-Ala, l-alanine; DHA, dehydroalanine.

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We previously isolated the AKT gene cluster involved in AK-toxin biosynthesis from the Japanese pear pathotype that causes black spot on a narrow range of susceptible Japanese pear cultivars (Tanaka et al., 1999; Tanaka and Tsuge, 2000; 2001). In DNA gel blot analysis, the AKT homologues were also detected from the strawberry and tangerine pathotypes, but not from other pathotypes and non-pathogenic strains of A. alternata (Tanaka et al., 1999; Masunaka et al., 2000; Tanaka and Tsuge, 2000). This result reveals that these three pathotypes share the common genes required for EDT biosynthesis.

The strawberry pathotype causes Alternaria black spot of strawberry (Maekawa et al., 1984). This pathotype affects only one Japanese strawberry cultivar, Morioka-16 (Maekawa et al., 1984). Interestingly, this pathotype was also found to be pathogenic to Japanese pear cultivars susceptible to the Japanese pear pathotype (Maekawa et al., 1984). Such a host range can be explained by the toxicity of AF-toxins. Most strains produce two related molecules, AF-toxins I and II (Fig. 1A) (Maekawa et al., 1984; Nakatsuka et al., 1986; Hatta et al., 2002). AF-toxin I is toxic to both strawberry and pear; toxin II is toxic only to pear (Maekawa et al., 1984; Nakatsuka et al., 1986).

We screened a genomic cosmid library of strain NAF8 of the strawberry pathotype with the AKT gene probe and isolated a clone, pcAFT-1, which contains AFT1-1, AFT3-1 and AFTR-1 with strong similarity to AKT1, AKT3-1 and AKTR-1 of the Japanese pear pathotype respectively (Fig. 2A) (Hatta et al., 2002). The common genes of these two pathotypes have more than 90% nucleotide identity with one another. We also analysed chromosomal distribution of the AFT genes using pulsed-field gel electrophoresis (PFGE) and found that all the genes are present on a small chromosome of 1.05 Mb (Hatta et al., 2002). Targeted mutation of AFT1-1 and AFT3-1 in NAF8 produced strains that lacked the 1.05 Mb chromosome (Hatta et al., 2002). These mutants lost the ability to produce AF-toxins, resulting in loss of pathogenicity to strawberry and pear. However, they grew and sporulated normally in culture, indicating that the 1.05 Mb chromosome is dispensable for saprophytic growth. Thus, it appears that AF-toxin biosynthesis genes are clustered on a conditionally dispensable (CD) chromosome (Covert, 1998; Hatta et al., 2002).

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Figure 2. The AFT gene cluster of the strawberry pathotype. A. Partial structure of the cosmid clone pcAFT-1. The cosmid clone was isolated from a genomic library of strain NAF8 and identified to encode AFT1-1, AFTR-1 and AFT3-1 (Hatta et al., 2002). The arrowed bars indicate protein-coding regions with introns (white segments). The arrowheads denote the orientation and location of oligonucleotide primers S1-5′ and S1-3′ used in PCR and RT-PCR experiments. Plasmids pAFT1-1 and pORFS1 were used as probes for hybridization experiments. B, BamHI; C, ClaI; E, EcoRV; H, HindIII; P, PstI; X, XhoI; Xb, XbaI. B. Amino acid sequence alignment of the ORFS1-encoding protein with other proteins. Amino acid sequence encoded by ORFS1 (accession no. AB119280) was aligned with hypothetical ORF (EAA) of N. crassa (accession no. EAA29581), StcV of A. nidulans (accession no. U34740) and Aad of P. chrysosporium (accession no. L08964). Amino acids that are conserved between the ORFS1 product and any of the others are indicated as white letters on a black background. The putative active site residues Asp-75, Tyr-80, Lys-108 and His-172 are indicated by asterisks.

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Here, we report the characterization of a new gene, AFTS1, identified from the cosmid clone pcAFT-1. AFTS1 encodes a protein with similarity to members of the aldo-ketoreductase superfamily (Jez et al., 1997). This gene is involved in the biosynthesis of AF-toxin I, but not of AF-toxin II, and is specifically required for pathogenicity to strawberry. DNA gel blot analysis revealed that the AFTS1 homologue is also present in the apple pathotype, which produces cyclic depsipeptide AM-toxin (Fig. 1B) and causes Alternaria blotch on certain apple cultivars (Okuno et al., 1974; Ueno et al., 1977). Here, we also describe how the strawberry and apple pathotypes share the orthologous genes required for biosynthesis of AF-toxin I and AM-toxins, although these toxins are chemically of different classes.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary material
  9. References
  10. Supporting Information

ORFS1 encodes an enzyme of the aldo-ketoreductase superfamily

The intergenic region between AFT1-1 and AFTR-1 in pcAFT-1 was found to encode a transposon-like sequence, named TLS-S1, and an open reading frame (ORF), named ORFS1 (Fig. 2A). TLS-S1 has significant similarity to transposase genes of fungal transposons that are members of the hAT transposon family (Kempken and Kück, 1996; Okuda et al., 1998). TLS-S1, however, contains several termination codons and is probably a pseudogene (data not shown).

An ORFS1 cDNA was prepared from total RNA of NAF8 by reverse transcription polymerase chain reaction (RT-PCR) with primers S1-5′ and S1-3′ (Fig. 2A). RT-PCR amplification produced a 1.1 kb fragment of DNA. Comparison of the genomic sequence with that of the RT-PCR product indicated that the gene has three introns (54, 50 and 50 bp) and four exons (219, 211, 335 and 333 bp) and potentially encodes a 366-amino-acid protein (Fig. 2).

A blast database search revealed that the ORFS1-encoded protein has similarity to members of the aldo-ketoreductase superfamily over its entire length (Fig. 2B). The aldo-ketoreductases are monomeric proteins that bind nicotinamide cofactor and catalyse the reduction of aldehydes and ketones in a diverse range of substrates to alcohols (Jez et al., 1997). The ORFS1-encoded protein shows significant similarity to fungal enzymes of this superfamily, such as sterigmatocystin biosynthesis dehydrogenase (StcV) of Aspergillus nidulans (Brown et al., 1996) and aryl-alcohol dehydrogenase (Aad) of Phanerochaete chrysosporium (Reiser et al., 1994) (Fig. 2B). ORFS1 is also similar to a hypothetical ORF (EAA29581.1) present in the Neurospora crassa genome (Galagan et al., 2003). The genomes of Schizosaccharomyces pombe and Saccharomyces cerevisiae also contain hypothetical ORFs similar to ORFS1 (data not shown). Although the functions of these hypothetical ORFs have not been identified, they have been classified as putative oxidoreductases or aldo-ketoreductases.

The structures of members of the aldo-ketoreductase superfamily indicate special conservation of the active site residues Asp, Tyr, Lys and His (Jez et al., 1997; Sanli and Blaber, 2001). The ORFS1 product possesses the four amino acids, which are Asp-75, Tyr-80, Lys-108 and His-172 (Fig. 2B). Thus, it is likely that ORFS1 encodes an enzyme belonging to this superfamily.

ORFS1 is encoded by a CD chromosome

We found previously that all AFT genes examined are present on a 1.05 Mb CD chromosome in the strawberry pathotype strains (Hatta et al., 2002). To investigate the genomic distribution of ORFS1, chromosome-sized DNA of strain NAF8 was separated by PFGE, and the blot was probed with the ORFS1 fragment from pORFS1 (Fig. 2A). To compare the distribution pattern with the AFT genes, the blot was also probed with the AFT1-1 fragment from pAFT1-1 (Fig. 2A).

Under the electrophoresis conditions for separating DNA in the range 1.0–6.0 Mb, at least 10 chromosomal DNAs of about 1.0–5.7 Mb were separated (Fig. 3A). Both probes hybridized to a single band of about 1.0 Mb (Fig. 3A). Chromosome-sized DNA was also separated by PFGE under the conditions for resolving DNA of 0.5–2.0 Mb. Under these conditions, the 1.0-Mb DNA (Fig. 3A) was resolved into two chromosomal DNAs of about 0.95 and 1.05 Mb (Fig. 3B). The ORFS1 probe hybridized to the 1.05 Mb DNA, as did the AFT1-1 probe (Fig. 3B). These results demonstrate that ORFS1 is also present only on the 1.05 Mb CD chromosome.

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Figure 3. Genomic distribution of ORFS1 in strain NAF8. A and B. Chromosome-sized DNA molecules of strain NAF8 were separated by PFGE under the conditions for DNA of 1.0–6.0 Mb (A) and of 0.5–2.0 Mb (B). The blots were probed with the AFT1-1 and ORFS1 fragments from pAFT1-1 and pORFS1 respectively (Fig. 2A). Sizes (in kilobases) of chromosomes of Schizosaccharomyces pombe and Saccharomyces cerevisiae (A) and chromosomes of S. cerevisiae (B) are indicated on the left. C. Total DNA (1 µg) of NAF8 was digested with ClaI (lane 1), EcoRV (lane 2), HindIII (lane 3), XbaI (lane 4) or XhoI (lane 5) and separated in a 0.8% agarose gel. The blot was probed with the ORFS1 fragment from pORFS1 (Fig. 2A). Sizes (in kilobases) of marker DNA fragments (HindIII-digested λDNA) are indicated on the left.

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We also showed previously that the strawberry pathotype strains have multiple copies of the AFT1-1, AFT3-1 and AFTR-1 homologues (Hatta et al., 2002). DNA of NAF8 was digested separately with five restriction enzymes (ClaI, EcoRV, HindIII, XbaI and XhoI) and probed with the ORFS1 fragment from pORFS1 (Fig. 2A). The ORFS1 fragment has a single ClaI site and no sites for other enzymes (Fig. 2A). The probe hybridized to two bands from the ClaI-digested DNA and a single band from DNA digested with the other enzymes (Fig. 3C). Thus, it was concluded that NAF8 has a single copy of ORFS1.

The ORFS1 homologue is present in the apple pathotype

The homologues of three AFT genes identified previously are present in three pathotypes: strawberry, Japanese pear and tangerine (Hatta et al., 2002). Distribution of the ORFS1 homologues in A. alternata pathotypes was determined by DNA gel blot analysis. DNA from 10 strains of the strawberry pathotype was digested with EcoRV, and the blot was probed with the ORFS1 fragment from pORFS1 (Fig. 2A). The probe hybridized to 4.4 kb bands in all strains (Fig. 4).

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Figure 4. Distribution of the ORFS1 homologues in A. alternata. Total DNA (1 µg) of each strain was digested with EcoRV and separated in a 0.8% agarose gel. The blots were probed with the ORFS1 fragment from pORFS1 (Fig. 2A). Sizes (in kilobases) of marker DNA fragments (HindIII-digested λDNA) are indicated on the left. Pathotypes: S, strawberry; A, apple; Tb, tobacco; R, rough lemon; Ta, tangerine; Jp, Japanese pear; Tm, tomato.

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Nineteen strains from six other pathotypes and five non-pathogenic strains of A. alternata were also analysed for distribution of the ORFS1 homologues. The ORFS1 homologues were not detected in the Japanese pear and tangerine pathotypes (Fig. 4), suggesting that ORFS1 is not involved in the biosynthetic pathway of EDT, a common moiety in AF-toxin, AK-toxin and ACT-toxin. Unexpectedly, the probe hybridized to 5.0 kb bands in all strains of the apple pathotype (Fig. 4). The structure and function of the ORFS1 homologue of the apple pathotype are described below.

Mutation of ORFS1 diminishes host range

To determine the function of ORFS1 in AF-toxin biosynthesis, homologous recombination was used to replace ORFS1 with the plasmid pGDTS1, which contains ORFS1 interrupted with the hygromycin B phosphotransferase gene (hph) cassette (Fig. 5A). The wild-type strain NAF8 was transformed with pGDTS1, and 133 transformants were selected. AF-toxin production of transformants was evaluated on the basis of toxicity of culture filtrates to leaves of strawberry cultivar Morioka-16 and Japanese pear cultivar Nijisseiki. Culture filtrates of 131 transformants showed toxicity to both strawberry and pear leaves, as did that of NAF8 (Fig. 6A). However, culture filtrates of two transformants (GDS1-1 and GDS1-2) showed toxicity to pear leaves, but not to strawberry leaves (Fig. 6A and Table 1).

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Figure 5. Transformation-mediated mutation of ORFS1. A. Structure of ORFS1 before and after homologous integration of the ORFS1-targeting vector pGDTS1. The targeting vector pGDTS1 was made by cloning the hph cassette into the BamHI site within ORFS1 in pORFS1 (Fig. 2A). B, BamHI; H, HindIII. B. DNA gel blot analysis of pGDTS1 transformants. Total DNA (1 µg) of the wild-type strain NAF8 (W) and the transformants GDS1-1 and GDS1-2 was digested with EcoRV and fractionated in a 0.8% agarose gel. The blot was probed with the ORFS1 fragment from pORFS1 (Fig. 2A). Sizes (in kilobases) of marker DNA fragments (HindIII-digested λDNA) are indicated on the left. C. Detection of ORFS1 transcripts in pGDTS1 transformants. Total RNA of NAF8 (W) and transformants GDS1-1 and GDS1-2 was used as a template for RT-PCR with primers S1-5′ and S1-3′ (Fig. 2A). Total DNA of NAF8 was used as a control (lane C). The RT-PCR products were electrophoresed in a 1.2% agarose gel. Sizes (in kilobases) of marker DNA fragments (a 100 bp ladder; lane M) are indicated on the left.

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Figure 6. AF-toxin production and pathogenicity of pGDTS1 transformant. A. Leaves of strawberry cultivar Morioka-16 (left) and Japanese pear cultivar Nijisseiki (right) were wounded slightly, treated with culture filtrates of the wild-type strain NAF8 (W) and the pGDTS1 transformant GDS1-1 (S1-1) and incubated in a moist box at 25°C for 20 h. B. Leaves were spray inoculated with conidial suspensions of NAF8 (W) and GDS1-1 (S1-1) and incubated in a moist box at 25°C for 20 h.

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Table 1. . AF-toxin production and pathogenicity of the wild-type strain NAF8 and its transformants.
StrainaProduction (µg ml−1)bPathogenicityc
AF-toxin IAF-toxin IIStrawberryJapanese pear
  • a

    . NAF8, wild-type strain; GDS1-1 and GDS1-2, pGDTS1 (Fig. 5A) transformants from NAF8; GDS1-1C1 to GDS1-1C4, pAM-AFTS1h (Fig. 8A) transformants from GDS1-1.

  • b

    . AF-toxins I and II in culture filtrates were analysed by reverse-phase HPLC. Each value represents the average of three determinations. ND, not detected.

  • c

    .+, pathogenic; –, non-pathogenic.

NAF82.380.26++
GDS1-1ND6.86+
GDS1-2ND7.58+
GDS1-1C11.290.79++
GDS1-1C22.180.23++
GDS1-1C33.170.49++
GDS1-1C43.650.52++

The strawberry pathotype strains also produce AF-toxins during conidial germination (Yamamoto et al., 1984; Hayashi et al., 1990). Conidial germination fluid of NAF8 had toxicity to both strawberry and pear leaves, but those of GDS1-1 and GDS1-2 were toxic only to pear leaves (data not shown). These results suggested that mutation of ORFS1 modified the biosynthetic pathway of AF-toxins.

These transformants were tested for pathogenicity to leaves of strawberry and Japanese pear by spray inoculation of conidial suspensions. The wild type caused a number of lesions on both strawberry and pear leaves (Fig. 6B). Transformants GDS1-1 and GDS1-2 caused lesions on pear leaves, but no lesions on strawberry leaves, showing that they preserved pathogenicity to pear and completely lost pathogenicity to strawberry (Fig. 6B and Table 1). These transformants were phenotypically indistinguishable from the Japanese pear pathotype strains: the number and size of lesions were almost the same on leaves inoculated with the transformants and the Japanese pear pathotype strains (data not shown).

The mode of integration of the vector pGDTS1 in transformants GDS1-1 and GDS1-2 was analysed by DNA gel blot hybridization (Fig. 5B). Total DNA of NAF8 and transformants was digested with HindIII, which has no site in ORFS1 or the hph cassette, and the blot was probed with the ORFS1 fragment from pORFS1 (Fig. 2A). The probe hybridized to an expected band of 4.3 kb in NAF8 (Fig. 5B). However, transformants lost the 4.3 kb bands and had 7.3 kb bands, resulting from homologous integration of pGDTS1 (Fig. 5). GDS1-1 also had a band of more than 23 kb, probably resulting from ectopic integration of pGDTS1 (Fig. 5B). RT-PCR amplification of ORFS1 cDNA with primers S1-5′ and S1-3′ (Fig. 2A) produced no DNA fragment from total RNA of these transformants (Fig. 5C). These results demonstrated that ORFS1 of these transformants was inactivated by homologous integration of pGDTS1. Thus, the gene encoding ORFS1 was designated AFTS1 (AF-toxin-specific gene 1).

The ΔaftS1 mutants lack AF-toxin I

The strawberry pathotype strains produce two related molecular species, AF-toxins I and II (Fig. 1), with toxin I being the predominant species with respect to both yield and biological activity (Maekawa et al., 1984; Yamamoto et al., 1984; Nakatsuka et al., 1986; Hayashi et al., 1990; Hatta et al., 2002). Toxins I and II in culture filtrates were quantified by reverse-phase high-performance liquid chromatography (HPLC). Culture filtrates of NAF8 contained toxins I and II, with toxin I being more abundant (Fig. 7A and Table 1). Culture filtrates of GDS1-1 and GDS1-2 contained toxin II, but no detectable toxin I (Fig. 7A and Table 1). These transformants produced larger amounts of toxin II than NAF8 (Fig. 7A and Table 1). The toxin fractions separated by HPLC were assayed for toxicity to strawberry and pear leaves. The fraction of NAF8 was toxic to both strawberry and pear, but those of GDS1-1 and GDS1-2 were toxic only to pear (Fig. 7B). Conidial germination fluids of GDS1-1 and GDS1-2 also contained only toxin II (data not shown). Thus, it appears that a mutation in AFTS1 causes loss of pathogenicity to strawberry, resulting from loss of AF-toxin I production.

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Figure 7. AF-toxin production by pGDTS1 transformants. A. AF-toxins I and II in culture filtrates of the wild-type strain NAF8 (W) and the pGDTS1 transformant GDS1-1 (S1-1) were detected by reverse-phase HPLC. Toxin fraction (TF) was collected and tested for toxicity in (B). B. Leaves of strawberry cultivar Morioka-16 (left) and Japanese pear cultivar Nijisseiki (right) were wounded slightly, treated with TF of NAF8 (W) and GDS1-1 (S-1) and incubated in a moist box at 25°C for 20 h.

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GDS1-1 and GDS1-2 were prototrophic. These transformants and the wild type were identical in growth rate, pigmentation, conidiation, conidial germination and appressorium formation (data not shown). However, they lost the ability to cause lesions on strawberry leaves (Fig. 6B). These results reveal that AFTS1 is specifically required for AF-toxin I production and pathogenicity to strawberry.

The AFTS1 homologue of the apple pathotype

In DNA gel blot analysis, the AFTS1 homologues were also present in the apple pathotype strains (Fig. 4). The cyclic depsipeptide AM-toxin of the apple pathotype has been characterized as three related molecular species, AM-toxins I, II and III (Fig. 1B), with toxin I (alternariolide) being the most abundant and toxic (Okuno et al., 1974; Ueno et al., 1977). Although AF-toxin and AM-toxin are classified into different chemical groups, we found similarity in the structure of AF-toxin I and AM-toxins. AM-toxins consist of four components, and one of the components is 2-hydroxy-isovaleric acid (2-HIV) (Fig. 1B). AF-toxin I contains 2,3-dihydroxy-isovaleric acid (2,3-DHIV), which is probably synthesized by the addition of the 3-hydroxy group to 2-HIV (Fig. 1A). Thus, 2-HIV is a common precursor of AF-toxin I and AM-toxins, and these two pathotypes should have the genes of the same function required for 2-HIV biosynthesis.

To isolate the AFTS1 homologue of the apple pathotype, a genomic library of strain IFO08984 was screened with the pORFS1 probe (Fig. 2A), and a positive clone, named λAM-AFTS1h, was isolated. A 6.0 kb SalI fragment from λAM-AFTS1h was identified as containing the AFTS1 homologue and cloned in pBluescript KS+ as pAM-AFTS1h (Fig. 8A).

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Figure 8. The AFTS1 homologue of the apple pathotype. A. Structure of the plasmid pAM-AFTS1h containing the AFTS1 homologue (ORFS1h). The plasmid has the 6.0 kb SalI fragment containing ORFS1h in pBluescript KS+. The arrowed bar indicates protein-coding region with introns (white segments). The arrowheads denote the orientation and location of oligonucleotide primers S1h-5′ and S1h-3′ used in PCR and RT-PCR experiments. Plasmid pORFS1h was used as a probe for hybridization experiments. E, EcoRV; K, KpnI; P, PstI; S, SalI; X, XhoI; Xb, XbaI. B. Comparison of structure of AFTS1 and ORFS1h. The arrowed bars indicate protein-coding regions with introns (white segments). Number of nucleotides in each exon and intron is indicated. Identity of nucleotide sequence is shown with that of amino acid sequence in parentheses.

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Sequencing of pAM-AFTS1h detected a putative protein-coding region, designated ORFS1h (Fig. 8A). AFTS1 and ORFS1h both consist of four exons, and only the first exon is different in size (AFTS1 = 219 bp; ORFS1h = 222 bp) (Fig. 8B). Of three introns, the third intron is different in size between AFTS1 (50 bp) and ORFS1h (46 bp) (Fig. 8B). The ORFS1h cDNA was prepared from total RNA of IFO08984 by RT-PCR with primers S1h-5′ and S1h-3′ (Fig. 8A). The cDNA sequencing confirmed that three introns are spliced. AFTS1 and ORFS1h are 74.9% identical in nucleotide sequence of the exon plus intron regions and 78.7% identical in deduced amino acid sequence (Fig. 8B). As with AftS1, the ORFS1h-encoded protein reveals significant similarity to members of the aldo-ketoreductase superfamily and contains the active site residues Asp-76, Tyr-81, Lys-109 and His-173. These data suggested that AFTS1 and ORFS1h have the same function.

The apple pathotype gene complements the ΔaftS1 mutation

To assess whether ORFS1h of the apple pathotype has the same function as AFTS1, a genetic complementation experiment was performed. We introduced pAM-AFTS1h, which includes the entire ORFS1h (Fig. 8A), into the AFTS1-targeted mutant GDS1-1 by co-transformation with the plasmid pII99 conferring resistance to geneticin (Inoue et al., 2002). AF-toxin production of 99 transformants was evaluated on the basis of toxicity of culture filtrates to leaves of strawberry and Japanese pear. Culture filtrates of 76 transformants showed toxicity only to pear leaves, as did that of GDS1-1 (Fig. 9A). However, culture filtrates of 23 transformants were toxic to both strawberry and pear leaves, as was that of the wild-type strain NAF8 (Fig. 9A).

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Figure 9. Complementation of the ΔaftS1 mutant with AMT2. The mutant GDS1-1 was transformed with pAM-AFTS1h (Fig. 8A). A and B. AF-toxin production and pathogenicity of pAM-AFTS1h transformants. Leaves of strawberry cultivar Morioka-16 (left) and Japanese pear cultivar Nijisseiki (right) were wounded slightly and treated with culture filtrates (A). Strawberry and Japanese pear leaves were spray inoculated with conidial suspensions (B). Leaves were incubated in a moist box at 25°C for 20 h. W, wild-type strain NAF8; S1-1, GDS1-1; C1 and C2; pAM-AFTS1h transformants GDS1-1C1 and GDS1-1C2. C. DNA gel blot analysis of pAM-AFTS1h transformants. Total DNA (1 µg) of each strain was digested with EcoRV and fractionated in a 0.8% agarose gel. The blot was probed with the ORFS1h fragment from pORFS1h (Fig. 8A). Sizes (in kilobases) of marker DNA fragments (HindIII-digested λDNA) are indicated on the left. W, NAF8; S1-1, GDS1-1; GDS1-1C1 to GDS1-1C4, pAM-AFTS1 transformants producing AF-toxin I; GDS1-1NC1 to GDS1-1NC4, pAM-AFTS1h transformants lacking AF-toxin I.

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All transformants were tested for pathogenicity to leaves of strawberry and Japanese pear. Transformants, culture filtrates of which were toxic only to pear, caused lesions only on pear. However, transformants, culture filtrates of which were toxic to both strawberry and pear, caused lesions on both plants (Fig. 9B and Table 1). AF-toxins I and II in culture filtrates of four transformants (GDS1-1C1 to GDS1-1C4) pathogenic to both plants were quantified by reverse-phase HPLC. Their culture filtrates contained both toxins I and II (Table 1).

We analysed the integration of pAM-AFTS1h in toxin I-producing and toxin I-minus transformants. Total DNA of NAF8, GDS1-1 and transformants was digested with EcoRV, which has no site in ORFS1h, and the blot was probed with the pORFS1h insert (Fig. 8A). The probe detected bands of about 5.0 kb corresponding to the EcoRV fragment containing ORFS1h only in toxin I-producing transformants (Fig. 9C). These results clearly showed that ORFS1h of the apple pathotype complemented the ΔaftS1 mutation of the strawberry pathotype, and that AFTS1 and ORFS1h have the same function. Johnson et al. (2000) reported the AMT gene from the apple pathotype, which encodes a cyclic peptide synthetase required for AM-toxin biosynthesis. Thus, the gene encoding ORFS1h of the apple pathotype was designated AMT2.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary material
  9. References
  10. Supporting Information

Mutation of AFTS1 diminishes host range by modification of secondary metabolism

In this study, we identified the AFTS1 gene from a cosmid clone pcAFT-1, from which AFT1-1, AFT3-1 and AFTR-1 involved in AF-toxin biosynthesis, have been identified previously (Hatta et al., 2002). As the homologues of these three genes are also present in the Japanese pear and tangerine pathotypes, they probably participate in biosynthesis of EDT, a common moiety in AF-toxin, AK-toxin and ACT-toxin (Hatta et al., 2002). In contrast, DNA gel blot analysis could not detect the AFTS1 homologues from the Japanese pear and tangerine pathotypes. This result suggested that AFTS1 does not participate in EDT biosynthesis and is specifically required for the biosynthesis of the unique part of AF-toxin.

Targeted mutation of AFTS1 in the strawberry pathotype produced strains that completely lost the ability to cause lesions on strawberry leaves. However, the ΔaftS1 mutants preserved the ability to cause lesions on Japanese pear leaves and thus became phenotypically indistinguishable from the Japanese pear pathotype. Such diminished host range in the mutants appeared to be attributable to loss of AF-toxin I which is toxic to both strawberry and pear. However, the mutants produced AF-toxin II which is toxic only to pear. These results provide a novel example of a single gene mutation diminishing the host range of a plant pathogenic fungus by modification of its secondary metabolism.

Most host-specific toxins, including AF-toxins, are produced during growth in media and also during conidial germination (Yoder, 1980; Nishimura and Kohmoto, 1983; Kohmoto et al., 1995; Wolpert et al., 2002). The knowledge that host-specific toxins are released from germinating conidia aids our understanding of the early participation of these toxic metabolites in host–parasite interactions (Yoder, 1980; Nishimura and Kohmoto, 1983; Kohmoto et al., 1995; Wolpert et al., 2002). The toxins released during conidial germination cause dysfunction of host cells, suppress defence response and may condition the affected cells to produce an ‘accessible state’ for fungal penetration (Otani et al., 1975; Yoder, 1980; Yamamoto et al., 1984; 2000). Thus, it has been proposed that host-specific toxins are required by the producing fungi to penetrate host cells and colonize tissue (Yoder, 1980; Nishimura and Kohmoto, 1983; Kohmoto et al., 1995; Wolpert et al., 2002). Conidia of the ΔaftS1 mutants germinate and form appressoria at rates that are indistinguishable from the wild-type conidia. However, they cause lesions on pear leaves but not on strawberry leaves. Our finding provides firm evidence that AF-toxins are the critical determinants of host-specific pathogenicity in the strawberry pathotype.

AFTS1 and AMT2 have the same function

In comparison with AF-toxin II, AF-toxin I contains an additional component 2,3-DHIV (Fig. 1A). The ΔaftS1 mutants produced larger amounts of AF-toxin II than the wild type. Thus, AF-toxin II and 2,3-DHIV are the primary precursors of AF-toxin I.

DNA gel blot analysis found a homologue of AFTS1 in the apple pathotype that produces the cyclic depsipeptide AM-toxins (Fig. 1B). This result is consistent with the structural similarity of AM-toxins and AF-toxin I. AM-toxins contain 2-HIV, and AF-toxin I contains 2,3-DHIV, which is the 3-hydroxy derivative of 2-HIV (Fig. 1). We isolated the AFTS1 homologue, named AMT2, from the apple pathotype. Genetic complementation of the ΔaftS1 mutant with AMT2 produced transformants that recovered the ability to produce AF-toxin I, resulting in restoration of pathogenicity to strawberry. These results demonstrate that AFTS1 and AMT2 have the same function.

The common precursor, 2-HIV, of AF-toxin I and AM-toxins is presumed to be synthesized from 2-keto-isovaleric acid (2-KIV) by reduction of its ketone to an alcohol (Fig. 10). 2-KIV is the last intermediate in the biosynthetic pathway of l-valine and can be formed from l-valine by transamination (Fig. 10). AftS1 and Amt2 both have significant similarity to enzymes of the aldo-ketoreductase superfamily and contain the active site residues Asp, Tyr, Lys and His (Jez et al., 1997; Sanli and Blaber, 2001). We propose that these genes encode enzymes catalysing the conversion of 2-KIV to 2-HIV (Fig. 10).

image

Figure 10. Proposed function of AftS1 and Amt2. 2-hydoxy-isovaleric acid (2-HIV) is a component of AM-toxins. 2,3-dihydroxy-isovaleric acid (2,3-DHIV), the 3-hydroxy derivative of 2-HIV, is a component of AF-toxin I. AftS1 and Amt2 are proposed to catalyse the reaction of 2-keto-isovaleric acid (2-KIV) to 2-HIV by reduction of a ketone in 2-KIV to alcohol.

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There are several reports about cyclic depsipeptides that contain 2-HIV, such as enniatin, beauvericin and destruxin, all of which are produced by filamentous fungi (Audhya and Russel, 1973; Païs et al., 1981; Peeters et al., 1983). No genes encoding enzymes catalysing the reaction from 2-KIV to 2-HIV have been reported. However, such an enzyme was biochemically identified as participating in enniatin biosynthesis in Fusarium sambucinum (Lee et al., 1992). This enzyme consists of a single polypeptide chain with a molecular mass of about 53 kDa. It is strictly dependent on NADPH and exhibits a high substrate specificity with respect to 2-KIV (Lee et al., 1992). Thus, it is likely that orthologous genes encoding this enzyme are involved in biosynthetic pathways of various secondary metabolites containing 2-HIV and its derivatives, such as AF-toxin I and AM-toxins. To verify the function of AMT2 in AM-toxin biosynthesis, the targeted mutation of AMT2 in the apple pathotype is under way.

Gene clusters for host-specific toxin biosynthesis

We have identified the gene clusters involved in the biosynthetic pathways of the host-specific toxins in three pathotypes: strawberry, Japanese pear and tangerine (Tanaka et al., 1999; Masunaka et al., 2000; Tanaka and Tsuge, 2000; Tsuge et al., 2001; Hatta et al., 2002). All the genes identified previously are unique to the three pathotypes and probably participate in EDT biosynthesis. AFTS1, however, is not present in the Japanese pear and tangerine pathotypes and is specifically required for AF-toxin I production. We also found the AKTS1 gene, which is present only in the Japanese pear pathotype, from the AKT cluster of this pathotype (Tsuge et al., 2001). Thus, the AFT and AKT clusters consist of pathotype-specific genes as well as genes common to the three pathotypes. The arrangement of common genes in the clusters is different among the three pathotypes (Tanaka et al., 1999; Masunaka et al., 2000; Tanaka and Tsuge, 2000; Hatta et al., 2002). Thus, it is unlikely that the differences among the gene clusters of the three pathotypes originated by simple mutations.

The common genes of the strawberry, Japanese pear and tangerine pathotypes have more than 90% identity with one another (Tanaka et al., 1999; Masunaka et al., 2000; Tanaka and Tsuge, 2000; Hatta et al., 2002). AFTS1 and AMT2, however, have lower sequence identity (about 75%). Thus, the common genes in the strawberry, Japanese pear and tangerine pathotypes are more conserved. These data suggest that the evolutionary relationship between AFTS1 and AMT2 is different from those among the EDT biosynthesis genes in the three pathotypes.

Akamatsu et al. (1999) found an interesting difference in PFGE patterns of chromosomal DNA between pathogenic and non-pathogenic strains of A. alternata: all strains from seven pathotypes had small chromosomes of less than 1.8 Mb, but non-pathogenic strains did not have such small chromosomes. Small chromosomes of several fungi have been identified as supernumerary (dispensable) chromosomes, which are not required for growth in culture and are inherited in a non-Mendelian manner (Covert, 1998). The function of supernumerary chromosomes in most species is still cryptic. However, in the pea pathogen Nectria haematococca, the 1.6 Mb supernumerary chromosome has been characterized as a CD chromosome because it contains genes for phytoalexin detoxification as well as other virulence determinants (Miao et al., 1991; Covert et al., 1996; VanEtten et al., 1998; Han et al., 2001).

In the strawberry pathotype, the AFT genes are on a 1.05 Mb chromosome in all strains tested (Hatta et al., 2002). In the apple pathotype, the AMT and AMT2 genes are on the same chromosome of 1.1–1.7 Mb, depending on the strains (Johnson et al., 2001; T. Tanaka and T. Tsuge, unpublished). In both pathotypes, mutants that have lost the chromosomes encoding toxin biosynthesis genes have been isolated (Johnson et al., 2001; Hatta et al., 2002). Although such mutants completely lost toxin production and pathogenicity, they grew and sporulated normally in culture. Thus, these small chromosomes appear to be CD chromosomes (Johnson et al., 2001; Hatta et al., 2002).

The CD chromosome probes did not hybridize to any other chromosomes of the same genomes in these pathotypes, indicating that these chromosomes are unique in their genomes (Johnson et al., 2001; Hatta et al., 2002). The patterns of repeated DNA sequences on certain supernumerary chromosomes of fungi suggest that they are of a different origin from the essential chromosomes in the same genome, and that they may have been introduced into the genome by horizontal transfer from another species (Enkerli et al., 1997; Covert, 1998; Rosewich and Kistler, 2000; Walton, 2000). We propose a hypothesis whereby the ability to produce a host-specific toxin could potentially be imparted by transfer of a CD chromosome that controls toxin biosynthesis to a strain of A. alternata.

In bacteria, the horizontal transfer of genes, in particular pathogenicity genes, appears to be a common event (Ochman et al., 2000). In Colletotrichum gloeosporioides, supernumerary chromosomes have been shown to have the capacity for transfer between otherwise genetically isolated strains (He et al., 1998). Horizontal transfer has also been proposed for the acquisition of genes for host-specific toxin biosynthesis in Cochliobolus carbonum (Nikolskaya et al., 1995; Walton, 2000) and C. heterostrophus (Yang et al., 1996; Yoder,  1998).  To  understand  the evolution of toxin biosynthesis and the origin of CD chromosomes, we are now focusing on structural and functional analysis of the CD chromosomes that control host-specific toxin biosynthesis and pathogenicity in A. alternata.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary material
  9. References
  10. Supporting Information

Fungal strains

A total of 34 strains from seven pathotypes and non-pathogenic A. alternata were used in this study (Fig. 4). Strain NAF8 of the strawberry pathotype was the source of the AF-toxin biosynthesis genes (Hatta et al., 2002). Strain IFO08984 of the apple pathotype was the source of AMT2. The others were used for analysis of distribution of the AFTS1 homologues. Strains were routinely maintained on potato dextrose agar (PDA; Difco).

Plasmids and genomic library

Integrative transformation vectors pSH75 (Kimura and Tsuge, 1993) and pII99 (Inoue et al., 2002) were used to transform A. alternata. The vectors pSH75 and pII99 carry hph and nptII, respectively, fused to the A. nidulans trpC promoter and terminator.

Plasmid pAFT1-1 contains the 2.0 kb fragment of AFT1-1 in pBluescript KS+ (Stratagene) (Fig. 2A) (Hatta et al., 2002). The exon and intron region of ORFS1 (AFTS1) was amplified from pcAFT-1 by PCR using the primer pair S1-5′ (5′-ATG ACT ACG AAA AAT GGA GA-3′) and S1-3′ (5′-CTA AGC ATG ACC CTG AAT TG-3′) (Fig. 2A). S1-5′ and S1-3′ contain the ORFS1 initiation and termination codons (underlined) respectively. PCR amplification was performed with Taq DNA polymerase (Takara) according to the manufacturer's instructions. The PCR product was cloned in pGEM-T Easy vector (Promega) to make pOFRS1 (Fig. 2A). The 3.0 kb BglII–BamHI fragment containing the hph cassette was cut out from pSH75 and cloned into the BamHI site within ORFS1 in pORFS1 to produce the ORFS1 replacement vector pGDTS1 (Fig. 5A).

Genomic DNA of strain IFO08984 of the apple pathotype was partially digested with Sau3AI to generate fragments of 15–20 kb and cloned in the λDASHII vector (Stratagene) to construct a genomic library according to the manufacturer's instructions. The phage clone λAM-AFTS1h was selected from the library with the pORFS1 probe (Fig. 2A) by the standard plaque hybridization (Sambrook et al., 1989). The 6.0 kb SalI fragment containing the AFTS1 homologue (ORFS1h) was cut out from λAM-AFTS1h and cloned into the SalI site of pBluescript KS+ to make pAM-AFTS1h (Fig. 8A). The exon and intron region of ORFS1h (AMT2) was amplified from pAM-AFTS1h by PCR using the primer pair S1h-5′ (5′-ATG TTG AAC TAT AAA AAA TG-3′) and S1h-3′ (5′-TCA AGA ATG CCC TTG GGG TG-3′) (Fig. 8A). S1h-5′-and S1h-3′ contain the ORFS1h initiation and termination codons (underlined) respectively. The PCR product was cloned in pGEM-T Easy vector to make pOFRS1h (Fig. 8A).

Fungal transformation

Protoplast preparation and transformation of A. alternata were performed by the methods described previously (Tsuge et al., 1990). Transformants carrying the hph gene and the nptII gene were selected on regeneration media containing hygromycin B (Wako) at 100 µg ml−1 and geneticin (Gibco) at 200 µg ml−1 respectively.

Assay for pathogenicity, AF-toxin production and vegetative growth

Strains were grown statically in 5 ml of PDB (Difco) in test tubes at 25°C for 7 days, and culture filtrates and mycelial mats were harvested. Culture filtrates were tested for toxicity to leaves of strawberry cultivar Morioke-16 and Japanese pear cultivar Nijisseiki as described previously (Maekawa et al., 1984; Hatta et al., 2002). AF-toxins I and II in culture filtrates were quantified by reverse-phase HPLC as described previously (Hayashi et al., 1990).

Mycelial mats were used for preparation of conidia as described previously (Hayashi et al., 1990). Conidial suspension (about 5 × 105 conidia ml−1) was sprinkled onto paper towels and incubated in a moist box at 25°C for 20 h. Conidial germination fluids were harvested, and AF-toxins in the fluids were tested by bioassay using Morioke-16 and Nijisseiki leaves and by reverse-phase HPLC analysis. Pathogenicity was assayed by spray inoculation of conidial suspension (about 5 × 105 conidia ml−1) to Morioka-16 and Nijisseiki leaves as described previously (Maekawa et al., 1984; Hatta et al., 2002).

Strains were grown on PDA at 25°C for 4 days. Agar blocks (3 mm in diameter) carrying mycelia were prepared from the resultant colonies and inoculated on PDA. After incubation at 25°C for 4 days, colony growth and morphology were observed. Prototrophy of strains was tested on a minimal agar medium (10 g l−1 KNO3, 5 g l−1 KH2PO4, 2.5 g l−1 MgSO4·7H2O, 0.02 g l−1 FeCl3, 10 g l−1 glucose, 20 g l−1 agar).

Nucleic acid manipulations

Isolation of total DNA and RNA from A. alternata and DNA gel blot hybridization were performed as described previously (Tanaka et al., 1999). Plasmid DNA, cosmid DNA and recombinant λphage DNA were extracted by standard methods (Sambrook et al., 1989).

RT-PCR was performed using the RNA PCR kit, version 2.1 (Takara), according to the manufacturer's instructions. The following primers were used: S1-5′ and S1-3′ for AFTS1 (Fig. 2A), and S1h-5′ and S1h-3′ for AMT2 (Fig. 8A). The RT-PCR products were cloned in pGEM-T Easy vector.

For analysis of nucleotide sequences, DNA was cloned in pBluescript KS+ or pGEM-T Easy vector. DNA sequences were determined with the BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems) and an automated fluorescent DNA sequencer (model 373A; Applied Biosystems) according to the manufacturer's instructions. DNA sequences were analysed with blast (Altschul et al., 1997). Alignment of nucleotide and amino acid sequences was made with the clustal w program (Thompson et al., 1994). The sequence data of AFTS1 and AMT2 have been submitted to the DDBJ/EMBL/GenBank databases under accession numbers AB119280 and AB119281 respectively.

Pulsed-field gel electrophoresis (PFGE)

Chromosome-sized DNA molecules were prepared from fungal protoplasts as described previously (Hatta et al., 2002). PFGE was carried out on a contour-clamped homogeneous electric field (CHEF) apparatus (CHEF-DRII, Bio-Rad Laboratories) using 0.5× TBE (Sambrook et al., 1989) at 8°C in 0.8% agarose gel (Seakem Gold agarose; BioWhittaker Molecular Applications) under the conditions described previously (Hatta et al., 2002). The gels were stained with ethidium bromide for 30 min and destained in distilled water for 30 min. DNA gel blotting and hybridization were performed as described previously (Hatta et al., 2002).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary material
  9. References
  10. Supporting Information

We are grateful to Motoichiro Kodama, Hirofumi Yoshioka, Kazuhito Kawakita and Noriyuki Doke for valuable suggestions, Norio Nakazawa, Fusaharu Nakatani and Yutaka Sato for providing fungal strains, and the Radioisotope Research Center, Nagoya University, for technical assistance. This work was supported by Special Coordination Funds for Promoting Sciences from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, Grant-in-Aid for Scientific Research from the Japanese Society for Promotion of Sciences, and the Daiko Foundation.

Supplementary material

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary material
  9. References
  10. Supporting Information

Fig.S1. Host-specific toxins produced by six pathotypes of A. alternata. AK-toxin, AF-toxin and ACT-toxin have a common moiety, 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid, indicated in shaded boxes. The structure of AT-toxin of the tobacco pathotype has not been determined.

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary material
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary material
  9. References
  10. Supporting Information

Fig. S1. Host-specific toxins produced by six pathotypes of A. alternata. AK-toxin, AF-toxin and ACT-toxin have a common moiety, 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid, indicated in shaded boxes. Structure of AT-toxin of the tobacco pathotype has not been determined.

FilenameFormatSizeDescription
MMI_4004_sm_figS1.TIF7425KSupporting info item

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