In the natural environment, plants are often bombarded by a combination of abiotic (such as drought, salt, heat or cold) and biotic (necrotrophic and biotrophic pathogens) stresses simultaneously. It is critical to understand how the various response pathways to these stresses interact with one another within the plants, and where the points of crosstalk occur which switch the responses from one pathway to another. Calcium sensors are often regarded as the first line of response to external stimuli to trigger downstream signaling. Abscisic acid (ABA) is a major phytohormone regulating stress responses, and it interacts with the jasmonic acid (JA) and salicylic acid (SA) signaling pathways to channel resources into mitigating the effects of abiotic stresses versus defending against pathogens. The signal transduction in these pathways are often carried out via GTP-binding proteins (G-proteins) which comprise of a large group of proteins that are varied in structures and functions. Deciphering the combined actions of these different signaling pathways in plants would greatly enhance the ability of breeders to develop food crops that can thrive in deteriorating environmental conditions under climate change, and that can maintain or even increase crop yield.

YchF is a subfamily of the Obg family in the TRAFAC class of P-loop GTPases. The wide distribution of YchF homologues in both eukarya and bacteria suggests that they are descendents of an ancient protein, yet their physiological roles remain unclear. Using the OsYchF1-OsGAP1 pair from rice as the prototype, we provide evidence for the regulation of GTPase/ATPase activities and RNA binding capacity of a plant YchF (OsYchF1) by its regulatory protein (OsGAP1). The effects of OsGAP1 on the subcellular localization/cycling and physiological functions of OsYchF1 are also discussed. The finding that OsYchF1 and OsGAP1 are involved in plant defense response might shed light on the functional roles of YchF homologues in plants. This work suggests that during evolution, an ancestral P-loop GTPase/ ATPase may acquire new regulation and function(s) by the evolution of a lineage-specific regulatory protein.

Summary• G-proteins (guanine nucleotide-binding proteins that usually exhibit GTPase activities) and related signal transduction processes play important roles in mediating plant defense responses; here, a rice (Oryza sativa) cDNA clone, OsGAP1, encoding a GTPase-activating protein (GAP) that also contains a protein kinase C conserved region 2 (C2) domain is reported.• An interacting G-protein partner for the OsGAP1 protein was identified by yeast two-hybrid library screening and confirmed by co-immunoprecipitation; the GTPaseactivation activity of OsGAP1 on this interacting G-protein was demonstrated using in vitro assays.• OsGAP1 was induced by wounding in rice and the presence of the R locus Xa14 enhances such induction.• Gain-of-function tests in transgenic rice and Arabidopsis thaliana showed that constitutive expression of OsGAP1 led to increased resistance to bacterial pathogens in both monocots and dicots.

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Receptor-like protein kinases (RLKs) containing an extracellular leucine-rich repeat (eLRR) domain, a transmembrane domain and a cytoplasmic kinase domain play important roles in plant disease resistance. Simple eLRR domain proteins structurally resembling the extracellular portion of the RLKs may also participate in signalling transduction and plant defence response. Yet the molecular mechanisms and subcellular localization in regulating plant disease resistance of these simple eLRR domain proteins are still largely unclear. We provided the first experimental evidence to demonstrate the subcellular localization and trafficking of a novel simple eLRR domain protein (OsLRR1) in the endosomal pathway, using both confocal and electron microscopy. Yeast two-hybrid and in vitro pull-down assays show that OsLRR1 interacts with the rice hypersensitive-induced response protein 1 (OsHIR1) which is localized on plasma membrane. The interaction between LRR1 and HIR1 homologs was shown to be highly conserved among different plant species, suggesting a close functional relationship between the two proteins. The function of OsLRR1 in plant defence response was examined by gain-of-function tests using transgenic Arabidopsis thaliana. The protective effects of OsLRR1 against bacterial pathogen infection were shown by the alleviating of disease symptoms, lowering of pathogen titres and higher expression of defence marker genes.

The BURP-domain protein family comprises a diverse group of plant-specific proteins that share a conserved BURP domain at the C terminus. However, there have been only limited studies on the functions and subcellular localization of these proteins. Members of the RD22-like subfamily are postulated to associate with stress responses due to the stress-inducible nature of some RD22-like genes. In this report, we used different transgenic systems (cells and in planta) to show that the expression of a stress-inducible RD22-like protein from soybean (GmRD22) can alleviate salinity and osmotic stress. We also performed detailed microscopic studies using both fusion proteins and immuno-electron microscopic techniques to demonstrate the apoplast localization of GmRD22, for which the BURP domain is a critical determinant of the subcellular localization. The apoplastic GmRD22 interacts with a cell wall peroxidase and the ectopic expression of GmRD22 in both transgenic Arabidopsis thaliana and transgenic rice resulted in increased lignin production when subjected to salinity stress. It is possible that GmRD22 regulates cell wall peroxidases and hence strengthens cell wall integrity under such stress conditions.

YchF proteins are a group of mysterious but ubiquitous unconventional G-proteins found in all kingdoms of life except Archaea. Their functions have been documented in microorganisms, protozoa and human, but those of plant YchF homologues are largely unknown. Our group has previously shown that OsYchF1 and its interacting protein, OsGAP1, play opposite roles in plant defense responses. OsGAP1 was found to stimulate the GTPase/ATPase activities of OsYchF1 and regulate its subcellular localization. In this report, we demonstrate that both OsYchF1 and OsGAP1 are localized mainly in the cytosol under NaCl treatment. The ectopic expression of OsYchF1 in transgenic Arabidopsis thaliana leads to reduced tolerance towards salinity stress, while the ectopic expression of OsGAP1 has the opposite effect. Similar results were also obtained with the Arabidopsis homologues, AtYchF1 and AtGAP1, by using AtGAP1 overexpressors and underexpressors, as well as an AtYchF1-knockdown mutant. OsYchF1 and OsGAP1 also exhibit highly significant effects on salinity-induced oxidative stress tolerance. The expression of OsYchF1 suppresses the anti-oxidation enzymatic activities and increases lipid peroxidation in transgenic Arabidopsis, and leads to the accumulation of reactive oxygen species (ROS) in tobacco BY-2 cells, while the ectopic expression of OsGAP1 has the opposite effects in these two model systems.

BackgroundIn plants, HIR (Hypersensitive Induced Reaction) proteins, members of the PID (Proliferation, Ion and Death) superfamily, have been shown to play a part in the development of spontaneous hypersensitive response lesions in leaves, in reaction to pathogen attacks. The levels of HIR proteins were shown to correlate with localized host cell deaths and defense responses in maize and barley. However, not much was known about the HIR proteins in rice. Since rice is an important cereal crop consumed by more than 50% of the populations in Asia and Africa, it is crucial to understand the mechanisms of disease responses in this plant. We previously identified the rice HIR1 (OsHIR1) as an interacting partner of the OsLRR1 (rice Leucine-Rich Repeat protein 1). Here we show that OsHIR1 triggers hypersensitive cell death and its localization to the plasma membrane is enhanced by OsLRR1.ResultThrough electron microscopy studies using wild type rice plants, OsHIR1 was found to mainly localize to the plasma membrane, with a minor portion localized to the tonoplast. Moreover, the plasma membrane localization of OsHIR1 was enhanced in transgenic rice plants overexpressing its interacting protein partner, OsLRR1. Co-localization of OsHIR1 and OsLRR1 to the plasma membrane was confirmed by double-labeling electron microscopy. Pathogen inoculation studies using transgenic Arabidopsis thaliana expressing either OsHIR1 or OsLRR1 showed that both transgenic lines exhibited increased resistance toward the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. However, OsHIR1 transgenic plants produced more extensive spontaneous hypersensitive response lesions and contained lower titers of the invading pathogen, when compared to OsLRR1 transgenic plants.ConclusionThe OsHIR1 protein is mainly localized to the plasma membrane, and its subcellular localization in that compartment is enhanced by OsLRR1. The expression of OsHIR1 may sensitize the plant so that it is more prone to HR and hence can react more promptly to limit the invading pathogens' spread from the infection sites.