Partial characterization of the PDA genes in Nectria haematococca MP VI.

Persistent Link:
http://hdl.handle.net/10150/186744
Title:
Partial characterization of the PDA genes in Nectria haematococca MP VI.
Author:
Hirschi, Kendal Dee.
Issue Date:
1994
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Isolates of the plant pathogenic fungus Nectria haematococca can demethylate pisatin, a phytoalexin produced by pea. Pisatin demethylation ability (pda) in this fungus is accomplished by a cytochrome P450 and specific PDA genes encode different enzymatic activities. The objective of this study was to further characterize the different PDA genes. A survey of 130 field isolates using Southern analysis demonstrated that PDA genes are conserved and can be divided into two categories: 1) high activity PDA genes referred to as PDAᴴ; and 2) low activity PDA genes termed PDAᴸ. This analysis also demonstrated that there is a nonfunctional PDA homolog (Phda) in some isolates. The distribution of PDA genes suggest that PDAᴴ genes confer a selective advantage in N. haematococca on pea: every isolate pathogenic on pea contained a PDAᴴ gene. Sequence analysis demonstrated that two PDAᴴ genes and Phda are 96% identical at the deduced amino acid level and more similar to one another than to a PDAᴸ gene. Phda is unable to exhibit pda due to a 84 amino acid addition at the carboxy terminus. Biochemical analysis of PDA gene products showed that PDAᴴ encodes a protein with a high substrate specificity for pisatin while the PDAᴸ gene product is slightly less specific for pisatin. Chimeric PDA constructs between the PDAᴴ and PDAᴸ genes demonstrated that the carboxy terminus did not define the majority of the substrate differences. Expression of the different PDA genes in cultures showed PDA transcripts of PDAᴴ and Phda genes were detected within two hours of addition of pisatin. In contrast, PDAᴸ transcripts were not detected until ten hours. Chimeric gene constructs which fused the promoter region from PDAᴴ onto a PDAL structural gene alter the apparent pda phenotype of these genes to a PDAᴴ-type. Northern analysis of PDA transcripts in plant tissue demonstrated that PDAᴴ is expressed at detectable levels whereas PDAᴸ transcripts are not. After twelve hours of infection, using the polymerase chain reaction, the transcripts of both the PDAᴴ gene and a plant gene which encodes isoflavone reductase, involved in pisatin biosynthesis, can be detected.
Type:
text; Dissertation-Reproduction (electronic)
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Plant Pathology; Graduate College
Degree Grantor:
University of Arizona
Committee Chair:
VanEtten, Hans D.

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titlePartial characterization of the PDA genes in Nectria haematococca MP VI.en_US
dc.creatorHirschi, Kendal Dee.en_US
dc.contributor.authorHirschi, Kendal Dee.en_US
dc.date.issued1994en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractIsolates of the plant pathogenic fungus Nectria haematococca can demethylate pisatin, a phytoalexin produced by pea. Pisatin demethylation ability (pda) in this fungus is accomplished by a cytochrome P450 and specific PDA genes encode different enzymatic activities. The objective of this study was to further characterize the different PDA genes. A survey of 130 field isolates using Southern analysis demonstrated that PDA genes are conserved and can be divided into two categories: 1) high activity PDA genes referred to as PDAᴴ; and 2) low activity PDA genes termed PDAᴸ. This analysis also demonstrated that there is a nonfunctional PDA homolog (Phda) in some isolates. The distribution of PDA genes suggest that PDAᴴ genes confer a selective advantage in N. haematococca on pea: every isolate pathogenic on pea contained a PDAᴴ gene. Sequence analysis demonstrated that two PDAᴴ genes and Phda are 96% identical at the deduced amino acid level and more similar to one another than to a PDAᴸ gene. Phda is unable to exhibit pda due to a 84 amino acid addition at the carboxy terminus. Biochemical analysis of PDA gene products showed that PDAᴴ encodes a protein with a high substrate specificity for pisatin while the PDAᴸ gene product is slightly less specific for pisatin. Chimeric PDA constructs between the PDAᴴ and PDAᴸ genes demonstrated that the carboxy terminus did not define the majority of the substrate differences. Expression of the different PDA genes in cultures showed PDA transcripts of PDAᴴ and Phda genes were detected within two hours of addition of pisatin. In contrast, PDAᴸ transcripts were not detected until ten hours. Chimeric gene constructs which fused the promoter region from PDAᴴ onto a PDAL structural gene alter the apparent pda phenotype of these genes to a PDAᴴ-type. Northern analysis of PDA transcripts in plant tissue demonstrated that PDAᴴ is expressed at detectable levels whereas PDAᴸ transcripts are not. After twelve hours of infection, using the polymerase chain reaction, the transcripts of both the PDAᴴ gene and a plant gene which encodes isoflavone reductase, involved in pisatin biosynthesis, can be detected.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplinePlant Pathologyen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.chairVanEtten, Hans D.en_US
dc.contributor.committeememberNelson, Merritt R.en_US
dc.contributor.committeememberHawes, Martha C.en_US
dc.contributor.committeememberWard, Samuelen_US
dc.contributor.committeememberWeinert, Teden_US
dc.identifier.proquest9426573en_US
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