Horizontal chromosome transfer, a mechanism for the evolution and differentiation of a plant-pathogenic fungus

Abstract

The tomato pathotype of Alternaria alternata produces host-specific AAL toxin and causes Alternaria stem canker on tomato. A polyketide synthetase (PKS) gene, ALT1, which is involved in AAL toxin biosynthesis, resides on a 1.0-Mb conditionally dispensable chromosome (CDC) found only in the pathogenic and AAL toxin-producing strains. Genomic sequences of ALT1 and another PKS gene, both of which reside on the CDC in the tomato pathotype strains, were compared to those of tomato pathotype strains collected worldwide. This revealed that the sequences of both CDC genes were identical among five A. alternata tomato pathotype strains having different geographical origins. On the other hand, the sequences of other genes located on chromosomes other than the CDC are not identical in each strain, indicating that the origin of the CDC might be different from that of other chromosomes in the tomato pathotype. Telomere fingerprinting and restriction fragment length polymorphism analyses of the A. alternata strains also indicated that the CDCs in the tomato pathotype strains were identical, although the genetic backgrounds of the strains differed. A hybrid strain between two different pathotypes was shown to harbor the CDCs derived from both parental strains with an expanded range of pathogenicity, indicating that CDCs can be transmitted from one strain to another and stably maintained in the new genome. We propose a hypothesis whereby the ability to produce AAL toxin and to infect a plant could potentially be distributed among A. alternata strains by horizontal transfer of an entire pathogenicity chromosome. This could provide a possible mechanism by which new pathogens arise in nature.

FIG. 1.

Electrophoretic karyotypes of the tomato pathotype of A. alternata and other Alternaria strains. Chromosome-size DNA was separated by PFGE under conditions for <2.0 Mb DNA (a and b) and 1.0 to 6.0 Mb DNA (c and d). The blots were probed with ALT1 (b) and with the 1.0-Mb chromosomal DNA of As-27 (CDC probe). (a and b) Chromosome-size DNA was separated in a 0.8% agarose (pulsed-field certified agarose; Bio-Rad) gel run at 5.5 V/cm with pulse intervals of 120 s for 12 h and 180 s for 12 h. Lanes: M, Saccharomyces cerevisiae; 1, nonpathogenic A. alternata O-94; 2, A. alternata Japanese pear pathotype O-276; 3, A. alternata tangerine pathotype AC320; 4, A. alternata rough lemon pathotype AC325; 5, A. alternata tobacco pathotype O-267; 6, A. alternata strawberry pathotype M-30; 7, A. alternata apple pathotype M-71; 8, A. alternata apple pathotype IFO8984; 9, A. alternata tomato pathotype As-27; 10, A. alternata tomato pathotype VU2001; 11, A. alternata tomato pathotype H6; 12, A. alternata tomato pathotype AL4; 13, A. alternata tomato pathotype O-227. (c and d) Chromosome-size DNA was separated in a 0.8% agarose (pulsed-field certified agarose; Bio-Rad) gel run at 1.5 V/cm for 115 h with pulse intervals of 3,600 to 1,800 s; for 24 h with 1,800 to 1,300 s; at 1.8 V/cm for 28 h with 1,300 to 800 s; and 2.4 V/cm for 28 h with 800 to 600 s. Lanes: M1, S. cerevisiae; 1, O-94; 2, O-276; 3, AC320; 4, AC325; 5, O-267; 6, M-30; 7, M-71; 8, IFO8984; 9, As-27; 10, VU2001; 11, H6; 12, AL4; 13, O-227; M2, Schizosaccharomyces pombe.

FIG. 2.

Transformation-mediated loss of the 1.0-Mb CDC of the A. alternata tomato pathotype. (a) Electrophoretic karyotypes of the CDC-deficient mutant. As-27, the wild-type strain of the A. alternata tomato pathotype; 9-1, the mutant strain; (b) Leaf necrosis bioassay for AAL toxin production by the wild-type and mutant strains. Leaves of the susceptible cultivar Aichi-first and the resistant cultivar Ace were treated with culture filtrates of the strain at 25°C for 3 days. (c) Pathogenicity test of the wild-type and mutant strains. Leaves were inoculated with a spore suspension (10⁵ spores/ml) and incubated in a moist chamber at 25°C for 3 days.

FIG. 3.

Diagram of electrophoretically separated A. alternata chromosomes and the distribution of marker genes on each chromosome. ECs and the 1.0-Mb CDC are indicated by green and red shading, respectively.

FIG. 4.

Phylogenetic tree based on the MAT1-1-1 genes of the A. alternata strains. Sequence alignment and phylogenetic analysis were performed with Genetyx-Mac software. The name of each pathotype of A. alternata is indicated next to the strain names.

FIG. 5.

Telomere fingerprint of HindIII-digested DNA from the A. alternata tomato pathotype and other Alternaria strains. The blots were probed with a telomere repeat sequence (TTAGGG)ⁿ. Lanes: 1, As-27; 2, VU2001; 3, H6; 4, AL4; 5, O-227; 6, O-94; 7, O-276; 8, AC320; 9, AC325; 10, O-267; 11, M-30; 12, M-71; 13, IFO8984; 14, O-159; 15, FIV cont 45; 16, O-210.

FIG. 6.

RFLPs of chromosomal DNA obtained from the A. alternata tomato pathotype. Chromosomal DNA samples were digested in agarose blocks with the rare-cutting enzyme NotI, electrophoresed (a), transferred to a membrane, and hybridized with the following probes: ALT1 (b), 1.0-Mb CDC DNA of As-27 (c), and repetitive DNA of A. alternata pAR274 (d).

FIG. 7.

Production of AAL and AF toxins and pathogenicity of the fusion strain EST6. (a) Leaves of tomato cultivar Aichi-first and strawberry cultivar Morioka-16 were wounded slightly, treated with culture filtrates of the parental and fusion strains, and incubated in a moist chamber at 25°C for 3 days. (b) Leaves were inoculated with mycelial pieces of the strains and incubated in a moist chamber at 25°C for 3 days. 27G-1, Geneticin-resistant parental strain derived from the A. alternata tomato pathotype (As-27) harboring an npt gene; TP1, hygromycin B-resistant parental strain derived from the A. alternata strawberry pathotype (NAF8) harboring an hph gene; EST6, the fusion strain between 27G-1 and TP1.

FIG. 8.

Electrophoretic karyotypes of parental and hybrid strains of A. alternata. Chromosome-size DNA was separated by PFGE under conditions for <2.0 Mb DNA (a, b, and c) and for 1.0 to 6.0 Mb DNA (d). The blots were probed with AFT1 (b) from the strawberry pathotype and ALT1 (c) from the tomato pathotype. Chromosome-size DNA was separated in a 0.8% agarose gel run at 5.5 V/cm with pulse intervals of 120 s for 12 h and 180 s for 12 h (a, b, and c) and at 1.5 V/cm for 48 h (d) with pulse intervals of 1,500 to 3,000 s, at 1.8 V/cm for 40 h with 900 to 1,500 s, and 2.4 V/cm for 57 h with 480 to 900 s. 27G-1, Geneticin-resistant parental strain derived from the A. alternata tomato pathotype (As-27) harboring an npt gene; TP1, hygromycin B-resistant parental strain derived from the A. alternata strawberry pathotype (NAF8) harboring an hph gene; EST6, the fusion strain between 27G-1 and TP1.