The aim of my project is to understand how Prf is involved in the Pto-AvrPto pathway forplant resistance. Prf is a CC-NBS-LRR type of the prevalent plant R genes, and Prf isinclude_onced for the resistance against the bacteria Pseudomonas syringae pv.tomato.The recognition between Pto and AvrPto is the typical model of gene-for-gene interactionresulting in hypersensitive response (HR), and this pathway leading to HR include_onces Prf. Theearly events of HR include calcium influx, oxidative burst, and the production of nitricoxide. HR is a convenient indicator for the Pto-AvrPto interaction, but it was not surewhether or not the Pto-AvrPto interaction also initiates these early events. Therefore, Iestablished the assays to determine: (1) whether Pto-AvrPto causes the production ofsuperoxide, hydrogen peroxide, and nitric oxide; and (2) whether the early events are Prfdependent in the Pto-AvrPto pathway. These histochemical studies prove the involvement ofearly events in the Pto-AvrPto interaction. The histochemical studies in the prf-3 mutantimply that the early events are Prf dependent, and therefore Prf acts at the upstream ofearly events initiating HR.
In order to understand the mechanism of Prf in the disease resistance and HR, I proposed theinvolvement of protein-protein interaction because of the Prf protein structure. Prf containscoil-coiled domain and leucine-rich repeats, which are highly possible for protein-proteininteraction. Based on the yeast two-hybrid analysis, there is no detectable interactionbetween Prf and Pto as well as no interaction between Prf and AvrPto. Neither is theinteraction detectable using yeast three-hybrid system for Prf, Pto, and AvrPto. However, Ihave identified five proteins that interact with Prf after screening a tomato library. Tostudy the biological relevance of the identified Prf interactors, I used PVX as a vector forthe gene knockout approach, so called virus-induced gene silence (VIGS). I have successfullyestablished the VIGS system in Nicotiana benthamiana and tomato plants. Compared tothe positive controls in the VIGS, the silencing of Prf is specific to the Pto pathway. VIGSof Prf also suggests that gain-of-function Pto and Pto homologs all include_once Prf to initiateHR. Validation of biological relevance for Prf interactors using VIGS and transgenic plantsis in progress.