Date of Degree

2-2015

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Theodore R. Muth

Subject Categories

Agricultural Science | Agriculture | Microbiology | Plant Biology

Keywords

Agrobacterium, Arabidopsis, microscopy, qPCR, transformation

Abstract

Agrobacterium tumefaciens is able to infect a diverse array of plants and causes crown gall disease. Typically these bacteria attach to plant roots and transform the plant cells to induce tumors. The mechanism of this attachment in the infection process is not yet fully understood. Using wild type Arabidopsis thaliana, Columbia-0, and several Arabidopsis mutant lines as a binding target, we screened for A. thaliana mutants with altered adhesion.

The A. thaliana mutant lines were selected in The Arabidopsis Information Resource (TAIR) according to possible location of the resulting protein and similarity to known transformation mutants. Of these mutants nine showed a variation in attachment from the wild type, of which two were known transformation mutants rat1 and rat3. Of these, the two were higher and seven were lower. Two mutants showed a growth phenotype with one having more roots and the other having wavy root hair growth, but both had wildtype attachment.

I also attempted to quantify the adhesion in these mutants using several approaches. However, I was not able to find a quantitative method that correlated well with microscopic observations of adhesion. Real-time PCR (qPCR) assay showed measurable differences between the mutants lines and the wildtype, suggesting some effect of the mutation on the interaction of A. thaliana and A. tumefaciens. Using this assay the level of bacterial attachment to the root surface can be indirectly measured. In the process of selecting this method several other approaches were attempted. These included flow cytometry of bacterial cells and of cells bound to beads, 96-well plate binding assay and the previously used plate colony counting. Mutants used in this study were also evaluated for transformation efficiency. Most of the mutants had not been previously tested for attachment or transformation. The attachment and transformation phenotypes provide a better understanding of the gene that has been affected by these mutant Arabidopsis lines.

The affected gene sequence and the data available on that gene were used to analyze the functional domains of the proteins showing an altered phenotype. There should be specific results here, rather than generalizations. These showed that kinase, extensin and heat shock protein domains were present in low attachment mutants and fasciclin, CDC48 and VirB2 domains were in high attachment mutants. The leucine-rich repeat (LRR) domains were strongly represented in all of the attachment mutants.. The SALK_ 040891C and SALK_085076C mutants that had high clumping but low attachment had heat shock, extensin and LRR domains. The putative protein functional domains may give insight to the possible function of the gene in both Arabidopsis and in possible interaction with A. tumefaciens. From these phenotypes, along with bioinformatic analysis, we can analyze mutant plant lines that exhibit enhanced or inhibited attachment. The combination of these methods may yield insight on the attachment mechanism as well as the infection process as a whole.

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