Activation of a Rac GTPase by the NLR Family Disease Resistance Protein Pit Plays a Critical Role in Rice Innate Immunity

Abstract

The nucleotide-binding domain and leucine-rich repeat-containing (NLR) family proteins recognize pathogen-derived molecules and trigger immune responses in both plants and animals. In plants, the direct or indirect recognition of specific pathogen effectors by NLRs culminates in a hypersensitive response (HR) and the production of reactive oxygen species (ROS), key components of the plant defense response. However, the molecules activated by NLRs and how they induce immune responses are largely unknown. We found that the rice GTPase OsRac1 at the plasma membrane interacts directly with Pit, an NLR protein that confers resistance to the rice blast fungus. OsRac1 contributes to Pit-mediated ROS production as well as the HR and is required for Pit-mediated disease resistance in rice. Furthermore, the active form of Pit induces the activation of OsRac1 at the plasma membrane. Thus, OsRac1 is activated by Pit during pathogen attack and plays a critical role in Pit-mediated immunity in rice.

Rice RING protein OsBBI1 with E3 ligase activity confers broad-spectrum resistance against Magnaporthe oryzae by modifying the cell wall defence

“…Two R genes, Pi9 and Pi21, confer broad-spectrum resistance against multiple races of M. oryzae [9,10]. Rac GTPasemediated events (e.g., generation of ROS), activation of MAP kinase, and other key components, such as RAR1, SGT1, Hsp90, and WRKY45, play important roles in regulating the immune response against M. oryzae [11][12][13][14][15][16][17][18][19][20][21][22][23][24]. Endogenous SA is involved in defence against M. oryzae [25].…”

In vivomonitoring of plant small GTPase activation using a Förster resonance energy transfer biosensor

“…These methods exploit the selective interaction between the Cdc42/Rac interactive binding (CRIB) domain of the Rac-effector PAK1 (PAK CRIB) and plant and animal Rac GTPases. PAK CRIB shows a high affinity only for the active GTP-bound form of Rac, and not for the inactive GDP-bound form (17,19). PAK CRIB binding suppresses the intrinsic GTPase activity of Rac, and this function provides a useful tool for isolating active GTP-bound forms of Rac/ROP from crude cell lysates and for monitoring the activation state of Rac/ROP in living cells.…”

“…Currently, two alternative non-radioactive techniques are available for monitoring the in vivo activation of Rac/ROP: a PAK CRIB pull-down assay, and FRET sensors (5,(17)(18)(19). These methods exploit the selective interaction between the Cdc42/Rac interactive binding (CRIB) domain of the Rac-effector PAK1 (PAK CRIB) and plant and animal Rac GTPases.…”

“…Rice protoplasts also display a high growth rate and a high transfection rate, and we can control the expression levels of FRET sensors in plant cells without difficulty. Our work has pioneered the monitoring of spatiotemporal activities of plant small GTPases in living cells, which had been impossible by conventional biochemical methods (19). We have observed the resistance (R) protein Pit, an immune receptor that activates OsRac1 at the plasma membrane (19) (27), and reported that OsRac1 is activated within 3 min after treatment with a fungal cell wall component, chitin (28), which is a microbe-associated molecular pattern (MAMP) that triggers plant immunity.…”

RhoGTPases — NODes for effector-triggered immunity in animals

“…npg Together this emerging body of data support the idea that the activity of GTPases is monitored by NLR and related pathways, and used as a cue to augment ongoing immune responses during pathogen invasion. Interestingly, Kawano and colleagues have shown that resistance to the rice blast fungus also involves activation of Rac (OsRac1) downstream of Pit, a plant nucleotidebinding site-leucine-rich repeat (NBS-LRR) receptor [10]. OsRac1 contributes to NBS-LRR-mediated production of ROS and induction of a hypersensitive response for the purpose of destroying infected tissue and preventing dissemination.…”