Progress 08/15/24 to 08/14/25

Outputs
Target Audience:The main target audience for the reporting period are undergraduate students that the PD mentored or is mentoring. These students have an interest in pursuing a career in the life sciences and were trained in skills important for becoming an independent researcher. The PD also shared results from the reporting period with an international audience of molecular plant pathologists at a major scientific conference. Changes/Problems:Objective [1.7] has been modified due to logistical concerns. Instead of traveling to Madagascar to assess Xo1 deployment potential, collect Xoc isolates and gain field experience, the PD has instead developed a collaboration with the Adachi Lab at Hokkaido University, where she will conduct a 3 month research stay to gain structural insights into Xo1 and its potential interactions with TALEs and truncTALEs. This work will lay the foundation for potential Xo1 engineering and a better understanding of how this NLR is activated and inhibited by Xoc TALEs and truncTALEs. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?The PD presented a poster, "Activation of nuclear NLR Xo1 by TALEs and inhibition by truncTALEs depend on a repeat-for-repeat interface", at the 2025 International Society for Molecular Plant-Microbe Interactions Congress (~1,300 attendees). What do you plan to do during the next reporting period to accomplish the goals?Objectives [1.2], [1.4], [1.6] and [2.2] are not yet met. Experiments to address all other objectives in Goal 1 are currently underway. Objectives [1.2] and [1.6] will be carried out as planned using the protoplast transient expression system that was successfully established during this reporting period. Objective [1.4] uses methods optimized previously in the lab and should also proceed as planned. Objective [1.7] has been modified as described in the Changes/Problems section. The travel budget under [1.7] will now be used for a 3-month research stay at Hokkaido University to expand Objective [2.2], to include structural characterization of Xo1 with TALEs or truncTALEs. The PD will be hosted by Hiroaki Adachi, whose lab has expertise in expressing plant immune receptors in Nicotiana benthamiana plants to analyze their biochemical and structural features.

Impacts
What was accomplished under these goals? 1. Problem the project addresses: Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak disease in rice. BLS occurs in most rice growing regions of the world and can impact yield. This project seeks to address this threat to food security by learning how a rice protein called Xo1, found in an American rice variety, defends the plant against the pathogen, and how some strains of the pathogen overcome Xo1. Xo1 is a rice immune receptor that confers disease resistance against Xoc by detecting transcription activator-like effector proteins (TALEs) Xoc secretes into rice cells. Some Xoc strains carry TALE variants called truncated TALEs (truncTALEs) that inhibit Xo1 immunity. How TALEs trigger and truncTALEs inhibit Xo1 immunity is largely unknown. 2. What work has been done in the project: Under Goal 1, the PD investigated how Xo1 detects TALEs and is inhibited by truncTALEs by testing mutated versions of the proteins in plant disease assays and protoplast (defense-associated) cell death assays. Under Goal 2, the PD mentored undergraduate research assistants and presented at a plant pathology conference. 3. What has resulted from the project: In alignment with Objective [1.1], the PD found that TALE C-term domain and truncTALE N-term domain are important for Xo1 activation and inhibition, respectively. Objective [1.3] was also accomplished and revealed that TALEs require their nuclear localization signals (NLS) to activate Xo1 but truncTALEs can still inhibit Xo1 without a functional NLS. TruncTALE inhibition of Xo1 is disrupted by fusion to a nuclear export signal and actively exclusion from the nucleus. Research on Objective [1.5] is ongoing and the PD has found that specific residues in the super-repeat domain and the zfBED domain of Xo1 are critical for triggering immunity against TALEs. This was accomplished by optimizing a protocol for a protoplast cell death assay. In alignment with the objectives under Goal 2, the PD mentored one graduating undergraduate student, preparing her for her career in the life sciences. The PD also disseminated knowledge gained from objectives under Goal 1 at the 2025 International Society for Molecular Plant-Microbe Interactions Congress. In addition, the PD established a new collaboration with the lab of Hiroaki Adachi at Hokkaido University towards a structural and biochemical characterization of Xo1 and TALEs/truncTALEs. 4. Why it matters: Data obtained under Goal 1 reveals new insights into how Xo1 detects TALEs and how it is inhibited by truncTALEs. The optimization of the protoplast cell death assay that was achieved will expedite further investigation of parts of Xo1 required for immunity. Understanding how TALE activation and truncTALE inhibition of Xo1 works at the molecular level can serve as a foundation for engineering Xo1 to better detect TALEs or be immune to truncTALE inhibition. Such engineering, in turn, may provide a new source of resistance to Xoc. The understanding gained by these data contribute to the plant health and production priority area and address questions relevant for NLR and effector biology, such as how an under characterized NLR with non-canonical domains detect its cognate effectors and how effector variants can subvert this defense mechanism.

Publications