Subcellular localization of Pik1-H4 and Pik2-H4 in planta
As NLR pairs, Pik1-H4 and Pik2-H4 collaborate to counter rice blast, invoking HR. In the previous study, we found that Pik-H4 maintained some expression levels and was blast-induced. To clarify how Pik-H4 changes between the resting and active stages, our focus turned to observing the subcellular localization of Pik1-H4 and Pik2-H4 singletons or as a pair within leaf sheaths of LTH or Pik-H4 NILs plants. Interestingly, Pik1-H4-GFP (Figure 6A, B) and Pik2-H4-GFP (Figure 6E, F) exhibited varying subcellular localization patterns across different cells. Pik1-H4-GFP and Pik2-H4-GFP were observed in the plasma membrane and cytoplasm, with the GFP signals partially coinciding. In OE-Pik1-H4 -GFP /LTH plants, Pik1-H4-GFP localization extended to the nucleus and vesicle-like particles (Figure 6C), while the Pik2-H4-GFP singleton predominantly resided within the nucleus. Upon M. oryzae invasion of leaf sheaths, Pik1-H4-GFP relocated to vesicles (Figure 6D), whereas Pik2-H4-GFP translocated to the nucleus (Figure 6H). Notably, as a sensor, Pik1-H4 singleton may be transported towards M. oryzae via vesicles (Figure S5A, B). Our observations revealed Pik1-H4 within numerous minute vesicles (Figure S5C-E) in cells neighboring blast-invaded or normal cells. These findings suggest that Pik1-H4 plays a vital role in recognizing Avr via vesicle transportation, possibly originating from smaller particles in healthier cells and converging into larger vesicles in the presence of rice blast fungus.
Upon overexpression of Pik1-H4 orPik2-H4 in Pik-H4 NILs, their subcellular localization was similar to Pik-H4 singletons in the absence of rice blast fungus (Figure 6I, K), with fewer instances of Pik1-H4-GFP residing in vesicles. Given the resistance of Pik-H4 NILs against rice blast, we did not find severe invasion but only several cells with a single spore or short hypha (Figure 6J, L). The appearance of M. oryzae significantly attenuated the Pik1-H4-GFP or Pik2-H4-GFP and chlorophyll signals in cells inhabited by the fungus and their neighboring cells, indicative of cell death. Although the GFP signals were weak, the Pik-H4 were mainly found in the membrane or cytoplasm. Interestingly, Pik1-H4 and Pik2-H4 were found in chloroplasts with weak GFP signals (Figure S6A-H) in planta .