Identification and characterization of Arabidopsis ecotypes and mutants deficient in crown gall tumorigenesis
Abstract
The purpose of the present study was to identify plant factors involved in crown gall tumorigenesis. We screened a large number of Arabidopsis thaliana ecotypes for susceptibility to crown gall disease using an in vitro root inoculation assay. Based on the phenotype of tumorigenesis, we found that there was considerable variation among ecotypes of Arabidopsis thaliana in their susceptibility to Agrobacterium infection. In several ecotypes, recalcitrance to Agrobacterium infection resulted from decreased binding of Agrobacterium tumefaciens to inoculated root explants. The recalcitrance of another ecotype, UE-1 occurred at a late step, T-DNA integration into the plant genome, in the Agrobacterium-mediated transformation process. In crosses with the highly susceptible ecotype Aa-0, the recalcitrant phenotype of UE-1 was recessive. In addition, we screened T-DNA insertion mutant lines of Arabidopsis for resistance in response to Agrobacterium infection. Of approximately 2500 T4 kanamycin-resistant plants investigated, twelve plants were resistant to Agrobacterium infection. The mutants were designated atr mutants (for Agrobacterium tumefaciens resistant). Two mutants, atr1 and atr3, were characterized. They showed low transient GUS activity, very low stable transformation efficiency (tumorigenesis and phosphinothricin-resistance), and reduced bacterial attachment compared to the wild-type plant. Genetic analysis revealed that in both mutants one genetic locus was disrupted by the insertion of T-DNA, although the number of T-DNAs integrated was different in each line. The resistance phenotype of each mutant was linked with the kanamycin-resistant phenotype and segregated as semidominant. Genomic DNA and cDNA clones containing the complete ATR3 gene were identified by using plant DNA flanking the right border of T-DNA rescued from the atr3 mutant. Two highly homologous cDNA clones were found; one (ATR3-1) weakly hybridized with the probe, whereas another (ATR3-2) strongly hybridized with the probe. Both ATR3-1 and ATR3-2 contain a putative signal peptide for secretion, and their deduced molecular weights are 15 kDa. Genomic DNA blot analyses revealed that ATR3-1 and ATR3-2 exist as single copy genes and are tightly linked. Genomic DNA sequencing showed that ATR3-1 and ATR3-2 were tandomly positioned with just 600 bp between them, and ATR3-1 was in the atr3 mutant by a T-DNA integration event in the 3$\sp\prime$ untranslated region. BLAST search showed that both ATR3-1 and ATR3-2 have homology with only the C-terminal region of $\beta$-1,3-glucanase homologues from Triticum and Arabidopsis. These homologues contain unusually long C-terminal regions with no significant homology with other $\beta$-1,3-glucanase genes.
Degree
Ph.D.
Advisors
Gelvin, Purdue University.
Subject Area
Plant pathology|Molecular biology
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