Identification of New Pathogenicity Genes in Magnaporthe Oryzae through the Construction of an Agrobacterium Tumefacines-Mediated Insertion Mutant Library

Persistent Link:
http://hdl.handle.net/10150/194453
Title:
Identification of New Pathogenicity Genes in Magnaporthe Oryzae through the Construction of an Agrobacterium Tumefacines-Mediated Insertion Mutant Library
Author:
Betts, Melania Figueroa
Issue Date:
2007
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:
An understanding of plant pathogen-host interactions is essential to design efficient strategies to control disease in crops. Magnaporthe oryzae, an ascomyceteous fungus and causal agent of rice blast disease, is a model organism to study host-microbe interactions. The overall aim of this dissertation project was to identify genes involved in pathogenicity through the construction and characterization of a random insertional mutagenesis library. In order to saturate the genome with DNA inserts, a collection of >54,000 insertion lines of the M. oryzae strain 70-15 was generated via two transformation methods, PEG/CaCl2 (polyethylene glycol)-mediated protoplast transformation and Agrobacterium tumefaciens-mediated transformation. The first part of this dissertation describes the optimization of both transformation approaches, compares their efficiency and provides a description of the high-throughput processing and phenotypic analysis of the insertion lines. An in vitro appressorium assay of 12,000 T-DNA insertion strains allowed the identification of 135 lines that were classified as morphologically or functionally different than wild-type. Rice infection assays demonstrated that 112 of these strains exhibited defects in pathogenicity.The second part of this dissertation project analyzed the T-DNA integration patterns in a subset of pathogenicity mutants. This section aimed to identify the disrupted genes via recovery of M. oryzae sequences adjacent to the sites of T-DNA insertion. Genomic mapping of 61 T-DNA insertions in pathogenicity mutants via rescuing M. oryzae chromosomal T-DNA flanking sequences using inverse PCR resulted in the identification of 22 conserved hypothetical genes with predicted function, 11 predicted open reading frames without a GenBank significant match, two unannotated regions of the genome assembly and seven intergenic regions. The final part of this dissertation describes the characterization of a M. oryzae pathogenicity mutant that contains a T-DNA insertion in the upstream region of two divergently transcribed genes that encode the vacuolar type-ATPase subunit c`` and the general transcription factor TFIIA subunit γ. Genetic complementation demonstrated the insertion of the T-DNA in the promoter region of the general transcription factor TFIIA subunit γ is responsible for observed defects in conidiation, appressorium morphogenesis, and appressorium function. This is the first report relating the function of TFIIA subunit γ to pathogenicity.
Type:
text; Electronic Dissertation
Keywords:
Magnaporthe; Transformation; Agrobacterium; Transcription factor IIA
Degree Name:
PhD
Degree Level:
doctoral
Degree Program:
Plant Pathology; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Orbach, Marc J.
Committee Chair:
Orbach, Marc J.

Full metadata record

DC FieldValue Language
dc.language.isoENen_US
dc.titleIdentification of New Pathogenicity Genes in Magnaporthe Oryzae through the Construction of an Agrobacterium Tumefacines-Mediated Insertion Mutant Libraryen_US
dc.creatorBetts, Melania Figueroaen_US
dc.contributor.authorBetts, Melania Figueroaen_US
dc.date.issued2007en_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.abstractAn understanding of plant pathogen-host interactions is essential to design efficient strategies to control disease in crops. Magnaporthe oryzae, an ascomyceteous fungus and causal agent of rice blast disease, is a model organism to study host-microbe interactions. The overall aim of this dissertation project was to identify genes involved in pathogenicity through the construction and characterization of a random insertional mutagenesis library. In order to saturate the genome with DNA inserts, a collection of >54,000 insertion lines of the M. oryzae strain 70-15 was generated via two transformation methods, PEG/CaCl2 (polyethylene glycol)-mediated protoplast transformation and Agrobacterium tumefaciens-mediated transformation. The first part of this dissertation describes the optimization of both transformation approaches, compares their efficiency and provides a description of the high-throughput processing and phenotypic analysis of the insertion lines. An in vitro appressorium assay of 12,000 T-DNA insertion strains allowed the identification of 135 lines that were classified as morphologically or functionally different than wild-type. Rice infection assays demonstrated that 112 of these strains exhibited defects in pathogenicity.The second part of this dissertation project analyzed the T-DNA integration patterns in a subset of pathogenicity mutants. This section aimed to identify the disrupted genes via recovery of M. oryzae sequences adjacent to the sites of T-DNA insertion. Genomic mapping of 61 T-DNA insertions in pathogenicity mutants via rescuing M. oryzae chromosomal T-DNA flanking sequences using inverse PCR resulted in the identification of 22 conserved hypothetical genes with predicted function, 11 predicted open reading frames without a GenBank significant match, two unannotated regions of the genome assembly and seven intergenic regions. The final part of this dissertation describes the characterization of a M. oryzae pathogenicity mutant that contains a T-DNA insertion in the upstream region of two divergently transcribed genes that encode the vacuolar type-ATPase subunit c`` and the general transcription factor TFIIA subunit γ. Genetic complementation demonstrated the insertion of the T-DNA in the promoter region of the general transcription factor TFIIA subunit γ is responsible for observed defects in conidiation, appressorium morphogenesis, and appressorium function. This is the first report relating the function of TFIIA subunit γ to pathogenicity.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectMagnaportheen_US
dc.subjectTransformationen_US
dc.subjectAgrobacteriumen_US
dc.subjectTranscription factor IIAen_US
thesis.degree.namePhDen_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplinePlant Pathologyen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorOrbach, Marc J.en_US
dc.contributor.chairOrbach, Marc J.en_US
dc.contributor.committeememberPierson III, Leland S.en_US
dc.contributor.committeememberVanEtten, Hansen_US
dc.contributor.committeememberWeinert, Teden_US
dc.contributor.committeememberSchumaker, Karen S.en_US
dc.identifier.proquest2358en_US
dc.identifier.oclc659748240en_US
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