Ralstonia solanacearum, the causal agent of bacterial wilt disease, has been found to affect numerous economically important plants. Understanding the molecular basis of resistance, tolerance and susceptibility of plants to pathogens such as R. solanacearum is a major goal of molecular plant pathologists. Prior to this study it was thought that Arabidopsis accession Kil-0 shows gene-for-gene “resistance” to an African Eucalyptus isolate of R. solanacearum, BCCF402. However, a subsequent preliminary study indicated that Kil-0 may exhibit “tolerance” which is defined as the plant’s ability to support high pathogen numbers without displaying disease symptoms or a reduction in host fitness. The aim of this study was to determine if Kil-0 was tolerant to R. solanacearum BCCF402. The bacterial load of R. solanacearum was quantified in accessions Kil-0 and Be-0 using dilution plating and quantitative PCR methods. The cytC gene region was used to quantify R. solanacearum in Arabidopsis plants and the amount of bacterial DNA was normalized to “alien” DNA that was spiked into each sample. High bacterial concentrations of BCCF402 were found in Kil-0 but plants exhibited no wilting symptoms. Additionally, Kil-0 plants inoculated with BCCF402 showed no significant reduction in fitness compared to control Kil-0 plants. In contrast, high bacterial numbers and severe disease symptoms were observed in the susceptible Be-0 plants, whereas Nd1 plants contained a low number of bacteria and no disease symptoms indicative of a resistance response. These results illustrated that Kil-0 is tolerant to R. solanacearum isolate BCCF402. A tool for the visualization of R. solanacearum in Arabidopsis plants was designed. R. solanacearum isolate BCCF402 was tagged with two mCherry-containing plasmids under the constitutive expression of the tac promoter. The expression levels of mCherry were suitable for successful visualization in planta. BCCF402 cells transformed with the mCherry-containing plasmids were not affected in terms of virulence or disease progression compared to wildtype BCCF402 cells. A plasmid loss of 30-35% was observed in mCherry-tagged BCCF402 cells at later stages of Arabidopsis infection. mCherry-tagged BCCF402 was successfully visualized in Kil-0 leaves at early infection stages. / Dissertation (MSc)--University of Pretoria, 2014. / gm2014 / Plant Science / unrestricted

<p dir="ltr">Bacterial Wilt, caused by <i>Ralstonia solanacearum</i>, is among the most devastating plant diseases in the world. This pathogen causes significant loss in crops such as tobacco, potato, and tomato. <i>R. solanacearum</i> root infection and xylem colonization determine disease outcome. To date, little is known about the defense mechanism mediated by roots to prevent <i>R. solanacearum</i> vascular colonization during the initial infection stages. Plant early responses are important since they may impact disease outcomes<i>.</i><i> </i>Here, we report the formation of root hairs and primary root growth inhibition in tomato seedlings as <i>Ralstonia</i>-induced phenotypes that depend on tomato genotype and <i>Ralstonia</i> species. The <i>Ralstonia</i>-induced root phenotypes are independent of a functional type III secretion system and exopolysaccharide production (EPS). We also found that <i>R. solanacearum</i><i> </i>K60 infection increased auxin levels throughout the root meristem in wilt-susceptible tomato roots. Our data suggest proper auxin signaling and transport are important for susceptibility to <i>R. solanacearum</i> K60. Blocking auxin transport pharmacologically or genetically led to fewer wilting symptoms, suggesting that auxin is important during early infection stages and disease outcomes in tomato. We previously found that a tomato mutant defective in auxin transport and signaling, known as <i>diageotropica</i> (<i>dgt</i>), has enhanced resistance to <i>R. solanacearum</i> K60. We characterized the resistant response in the <i>dgt</i> mutant, and we found that the resistant response in the <i>dgt</i> mutant may be due to increased lignin content preventing pathogen vasculature colonization. <i>DGT</i> encodes a cyclophilin protein that regulates auxin transport and signaling. Mutations in the cyclophilin DGT promote resistance to <i>R. solanacearum</i> K60. DGT has been reported to regulate auxin transport and signaling. However, the molecular mechanism regarding how DGT mediates these processes is still unknown. We used Yeast Two-Hybrid to identify candidate protein interactors, and we found that SlbZIP1/SlbZIP29, Sl14-3-3, and SlMYB110 may interact with DGT to regulate both development and defense responses. Understanding the role of DGT, auxin, and lignin in defense responses to <i>R. solanacearum</i> K60 in tomato is necessary for Solanaceae crop improvement.</p>