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 .