Articles

Anastomosis Is Required for Virulence of the Fungal Necrotroph Alternaria brassicicola

Kelly D. Craven, Heriberto Vélëz, Yangrae Cho, Christopher B. Lawrence, Thomas K. Mitchell
DOI: 10.1128/EC.00423-07

Article Figures & Data

Figures

  • FIG. 1.
    FIG. 1.

    Anastomosis phenotypes within and between A. brassicicola strains. (A) Prototrophic growth indicative of anastomosis between complementary nit mutants of the same A. brassicicola isolates (self-pairing). The lack of prototrophic growth between complementary nit mutants of different isolates (non-self-pairing) indicates a failure to anastomose. (B) Anastomosis within and between A. brassicicola strains Ab5 and Ab6. (C) The GFP-labeled strain ATCC 96836 showing “laddering phenotype” of anastomosed hyphae. (D) Rare interisolate fusion between typically incompatible isolates ATCC 96836-GFP and unlabeled Ab4, where the GFP signal is migrating into Ab4 hyphae (arrows). (E) Hyphae of strain Ab5 showing the initiation of anastomosis “pegs” in response to similar structures in adjacent hyphal strands. (F) Pairing of aso1/nit single and/or double mutants on nitrate media. Interacting hyphae involving complementary nit mutants, but both defective in Aso1, show no evidence of prototrophic growth (side arrows), while interacting hyphae of complementary nit mutants do yield prototrophic growth if at least one has a functional Aso1 gene (top arrow).

  • FIG. 2.
    FIG. 2.

    Nucleotide and amino acid sequence of the inferred Aso1 ORF. The predicted translational start and stop codons are in bold and shaded in gray, respectively. The inferred CAAT and TATA promoter elements are in bold italics. Introns are underlined. The double-tryptophan domain is shaded in dark gray, bounded with two tryptophan residues (bold italic).

  • FIG. 3.
    FIG. 3.

    Autoradiograph of a DNA gel blot of XhoI-genomic digests of A. brassicicola wild-type Ab7 and aso1 mutants (aso1-2a, aso1-6a, aso1-10a, and aso1-11a) and the ectopic transformant Aso1-7E probed with the Aso1 gene fragment labeled with an ECL direct nucleic acid labeling and detection system (Amersham Biosciences, Piscataway, NJ).

  • FIG. 4.
    FIG. 4.

    Anastomosis phenotype of A. brassicicola aso1 mutants. (A) Wild type; (B) aso1-1a; (C) aso1-9a; (D) aso1-7E (ectopic). Arrows indicate fusion events (panels A and D) or instances of close contact without fusion (panels B and C).

  • FIG. 5.
    FIG. 5.

    Pathogenicity assay with the A. brassicicola wild type, aso1 mutants, and ectopic transformant. (A, C, and E) Inoculated, abaxial surface at 6, 12 and 18 days postinfection; (B and D) adaxial (uninoculated) surface of the same cabbage leaves at 6 and 12 days postinfection; (F) wild type (wt) and aso1 lesions, at a magnification of ×2. Panel A and C isolate numbers are identified as follows: wild type, 1, 8, 13, and 15; aso1-10a, 2, 7, 14, and 17; aso1-11a, 5, 9, 11, and 19; aso1-14a, 3, 10, 16, and 18; aso1-7E (ectopic), 4, 6, 12, and 20.

  • FIG. 6.
    FIG. 6.

    Anastomosis phenotype of complemented strain aso1-10. Arrows indicate self-fusion events.

Tables

  • TABLE 1.

    Isolates included in this study

    Fungal speciesIsolateOriginMating typeaHostAccession numberb
    A. brassicicola Ab1California MAT1-2 Brassica oleracea NA
    A. brassicicola Ab2California MAT1-2 Brassica oleracea NA
    A. brassicicola Ab4Lampoc, CaliforniaND Brassica oleracea var. botrytisNA
    A. brassicicola Ab5Chualar, California MAT1-2 Brassica oleracea var. botrytisNA
    A. brassicicola Ab6Texas MAT1-2 Brassica oleracea NA
    A. brassicicola Ab7Oregon MAT1-2 Brassica oleracea NA
    A. brassicicola 34622Unknown MAT1-1 Crambe sp. ATCC 34622
    A. brassicicola 96836United States MAT1-1 Brassica oleracea ATCC 96836
    A. japonica Ajap1New ZealandND Brassica pekinensis ATCC 96834
    A. mimicula Amim1Georgia MAT1-1 Lycopersicon esculentum ATCC 12251

    • ↵ a ND, not determined.

    • ↵ b NA, not applicable.

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KEYWORDS

Alternaria

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