Minisymposium 18: Plant defense signaling

Abs # 33002: Dual role of the small GTPase OsRac1 as an inducer of ROS and a suppressor of ROS scavenger

Presenter:	Wong, Hann Ling , h-wong@bs.naist.jp
Authors	Wong, Hann Ling  (A)   Pinontoan, Reinhard  (A)   Sakamoto, Tsuyoshi  (A)   Kawasaki, Tsutomu  (A)   Shimamoto, Ko  (A)
Affiliations:	(A): Nara Institute of Science and Technology (NAIST)

Reactive oxygen species (ROS) produced by NADPH oxidase are thought to play critical roles in the plant defense against pathogens.In human neutrophil, the NADPH oxidase complex, consists of the plasma membrane proteins gp91phox and p22phox, and is regulated by the cytosolic proteins p40phox, p67phox, and p47phox, and the small GTPase Rac. Rac regulates the acivation of the neutrophil NADPH oxidase complex through interaction with p67phox, which also interacts with gp91phox, the catalytic subunit. In plants, only the homologs for gp91phox and Rac have been found. In rice, we found that overexpression of the constitutively active form of the small GTPase OsRac1 enhanced resistance to virulent rice fungus and bacterial blight, and enhanced elicitor-induced hydrogen peroxide (H₂O₂) production. To examine if plant Rac could directly interact with the plant gp91phox homologs, which are known as RBOHs, we used yeast two hybrid assays study the interaction between rice Racs and RBOHs from rice and potato. We found that constitutively active form of most rice Racs, but not their dominant negative form, interacted with the N-terminus of rice and potato RBOHs, through two Ca²⁺-binding EF-hands domains. This suggests that unlike in the human NADPH oxidase, plant Rac directly interacts with RBOH, to regulate ROS production. Presently, we are studying the OsRac1-RBOH interaction using a single molecular mutant GFP-tagged fluorescence resonance energy transfer (FRET) system. In addition, we found that the expression of a metallothionein gene OsMT2b was synergically downregulated by OsRac1 and rice blast-derived elicitors. Elicitor-induced H₂O₂ production was reduced in OsMT2b-overexpressing cells. In contrast, homozygous OsMT2b::Tos17-inserted mutant and OsMT2b-RNAi-silenced transgenic cells showed significantly enhanced elicitor-induced H₂O₂ production. In vitro assays using recombinant OsMT2b fusion protein showed that OsMT2b is an ROS scavenger. Transient transformation of GFP-OsMT2b fusion into rice protoplast showed that the GFP fluorescence was localized in the cytoplasm, suggesting that OsMT2b function as an ROS scavenger mainly in the cytoplasm. Taken together, the results suggest that OsRac1 plays a dual role as an inducer of ROS and a suppressor of ROS scavenger.