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A gene for maackiain detoxification from a dispensable chromosome ofNectria haematococca

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Abstract

InNectria haematococca theMAK1 gene product converts a chick-pea (Cicer arietinum) phytoalexin, maackiain, into a less toxic compound. The presence ofMAK1 in this fungal pathogen is also correlated with high virulence on chick-pea. Previous genetic analysis suggested thatMAK1 is located on a meiotically unstable, dispensable chromosome. The unstable nature of this chromosome facilitatedMAK1 cloning by allowing us to identify a subset of genomic cosmid clones likely to containMAK1. Truncated forms of the chromosome, generated during meiosis, were isolated from strains either able (Mak+) or unable (Mak−) to metabolize maackiain and used to probe a chromosome-specific cosmid library. Only clones that hybridized exclusively to the chromosome from the Mak+ strain were then screened for their ability to transform a Mak− isolated to the Mak+ phenotype. A 2.7 kbHindIII-PstI fragment was subcloned from a cosmid conferring MAK1 activity, and its nucleotide sequence determined. BecauseMAK1 transcription is not induced strongly by maackiain, a reverse transcriptase-polymerase chain reaction was required to detectMAK1 transcription in a Mak+ strain, and to isolateMAK1 cDNA fragments. Comparison of the genomic and cDNA sequences ofMAK1 revealed the presence of three introns and an open reading frame encoding a protein 460 amino acids in length. Two diagnostic domains in its deduced amino acid sequence suggestMAK1 encodes a flavin-containing mono-oxygenase.MAK1 is the first gene encoding maackiain detoxification to be cloned, and is the second functional gene cloned from this dispensable chromosome. Southern analysis of genomic DNA from ascospore isolates containingMAK2, MAK3, andMAK4 indicated thatMAK1 is not homologous to other known maackiain-detoxifying genes.

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Authors and Affiliations

  1. Daniel B. Warnell School of Forest Resources, University of Georgia, 30602-2152, Athens, GA, USA

    S. F. Covert

  2. Department of Botany, University of Georgia, 30602, Athens, GA, USA

    J. Enkerli

  3. 13750-31 Avenue, V4P 2B7, Surrey, BC, Canada

    V. P. W. Miao

  4. Department of Plant Pathology, University of Arizona, 85721, Tucson, AZ, USA

    H. D. VanEtten

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  1. S. F. Covert
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  2. J. Enkerli
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  3. V. P. W. Miao
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  4. H. D. VanEtten
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Covert, S.F., Enkerli, J., Miao, V.P.W. et al. A gene for maackiain detoxification from a dispensable chromosome ofNectria haematococca . Molec. Gen. Genet. 251, 397–406 (1996). https://doi.org/10.1007/BF02172367

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  • DOI: https://doi.org/10.1007/BF02172367

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