| Xanthomonas citri subsp. citri (Xcc), which causes citrus canker, relies on a type III secretion system (T3SS) to successfully develop disease. Various plant pathogenic bacteria carrying a T3SS inject more than 40 different effector proteins (T3-effectors) into the plant cells via this apparatus. In Xanthomonas, the genes for the T3SS are regulated by HrpX, which is an AraC-type transcriptional regulator. The possible roles that nineteen candidate T3-effector genes play in the ability of Xcc to cause citrus canker were investigated through the use of site-directed mutagenesis. The candidate genes were selected on the basis of having promoter features similar to gene regulated by HrpX or by sequence similarity to known T3-effectors in other plant pathogenic bacteria. Inoculation in grapefruit revealed that none of these mutants were visually impaired for disease development. Unlike the hrpW null mutant, deletion in the harpin domain from hrpW resulted in the loss of Xcc pathogenicity symptoms, while not affecting the ability of of the bacteria to multiply in the plant. The mutants were also assessed for the ability to elicit hypersensitive response (HR) in non-host plant tomato. The Xcc hrp- mutants, in contrast to many pathogenic xanthomonads, retained the ability to trigger HR in the nonhost. Using subcloning procedures and homology search, three candidates open reading frames, XAC3857, XAC3858 and XAC3859, were identified in Xcc that might be responsible for this T3SS-independent HR elicitation. Experiments were also performed to further characterize a new avirulence gene, avrGf1, isolated from Xcc strain A w, which induces HR in grapefruit. The avrGf1 gene was demonstrated to encode a protein that is translocated into plant cells via the T3SS. Additionally, a transient expression on grapefruit leaves was devised using an Agrobacterium-mediated delivery system. Different avrGf1 deletion mutants in the N- and C-terminal coding regions were tested using the system, and the results showed that the first 116 amino acids in the N-terminal and the last 83 amino acids in the C-terminal were crucial for HR elicitation in grapefruit. In summary, this study reported the presence of candidate T3-effector genes that did not affect the disease progress under the experimental conditions and a harpin domain of hrpW gene is essential for symptom development. Additionally, the development of an efficient transient expression in grapefruit leaves revealed specific regions of avrGf1 are required for defense activation. |
