Ectopic Expression of Rice OsBIANK1, Encoding an Ankyrin Repeat‐Containing Protein,in Arabidopsis Confers Enhanced Disease Resistance to Botrytis cinerea and Pseudomonas syringae

Ankyrin repeat‐containing proteins comprise a large family whose members have been shown to play important roles in various aspects of biological processes in plant growth and development as well as in responses to biotic and abiotic stresses. We previously identified a rice gene, OsBIANK1, encoding an ankyrin repeat‐containing protein and found that expression of OsBIANK1 can be induced by defence signalling molecules and by infection of Magnaporthe oryzae, the causal agent of blast disease. To better understand the possible function of OsBIANK1 in disease resistance, we generated transgenic Arabidopsis plants that constitutively overexpress the OsBIANK1 gene. Results from disease assays revealed that the OsBIANK1‐overexpressing plants display increased resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000 as compared with the wild‐type plants. In OsBIANK1‐overexpressing plants, expression of some of well‐known defence genes (e.g. PR‐1, PR‐2 and PDF1.2) was up‐regulated after infection with B. cinerea or P. syringae pv. tomato DC3000. Furthermore, the OsBIANK1‐overexpressing plants showed decreased levels of reactive oxygen species (i.e. superoxide anion and H₂O₂) after Botrytis infection. Thus, our present results further support the role of OsBIANK1 in regulation of defence responses against different types of pathogens.     

Molecular characterization and expression analysis of <Emphasis Type="Italic">OsBISERK1</Emphasis>, a gene encoding a leucine-rich repeat receptor-like kinase,during disease resistance responses in rice

A rice gene, OsBISERK1, encoding a protein belonging to SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) type of leucine-rich repeat receptor-like kinases (LRR-RLKs) was identified. The OsBISERK1 encodes a 624 aa protein with high level of identity to known plant SERKs. OsBISERK1 contains a hydrophobic signal peptide, a leucine zipper, and five leucine-rich repeat motifs in the extracellular domain; the cytoplasmic region carries a proline-rich region and a single transmembrane domain, as well as a conserved intracellular serine/threonine protein kinase domain. OsBISERK1 has a low level of basal expression in leaf tissue. However, expression of OsBISERK1 was induced by treatment with benzothiadiazole (BTH), which is capable of inducing disease resistance in rice, and also up-regulated after inoculation with Magnaporthe grisea in BTH-treated rice seedlings and during incompatible interaction between a blast-resistant rice genotype and M. grisea. The results suggest that OsBISERK1 may be involved in disease resistance responses in rice.     

Molecular cloning and expression analysis of rice OsTVLP1, encoding a protein with similarity to TGF-beta receptor interacting proteins and vacuolar assembly Vam6p/Vps39p proteins.

We describe the cloning and identification of a rice cDNA, OsTVLP1, encoding a protein with similarity to TGF-beta receptor interacting proteins and vacuolar assembly Vam6p/Vps39p proteins. OsTVLP1 has an open reading frame of 2955 bp, which encodes a 984 amino acid protein, containing a citron homology (CNH) domain at its N-terminal and a clathrin heavy-chain repeat homology (CLH) domain at its C-terminal. The expression of OsTVLP1 was induced by treatments with benzothiadiazole (BTH), a chemical activator of plant disease resistance responses, and by infection of the blast fungus, Magnaporthe grisea. Importantly, the expression of OsTVLP1 was activated specifically in disease resistance response induced by BTH and in an incompatible interaction between rice and the blast fungus. Our observations suggest that OsTVLP1 may play a role in rice disease resistance response against pathogen infection.     

Overexpression of a rice defense-related F-box protein gene OsDRF1 in tobacco improves disease resistance through potentiation of defense gene expression

F-box proteins play important roles in plant growth/development and responses to environmental stimuli through targeting substrates into degradation machinery. A rice defense-related F-box protein gene, OsDRF1, was cloned and identified during a course of study aimed at elucidating the molecular basis of induced immunity in rice. OsDRF1 encodes a protein of 328 amino acids, containing a highly conserved F-box domain. Expression of OsDRF1 was induced upon treatment with benzothiadiazole (BTH), a chemical inducer of defense responses in rice. Moreover, in BTH-treated rice seedlings, expression of OsDRF1 was further induced by infection with Magnaporthe grisea, the rice blast fungus, compared with those in water-treated seedlings. OsDRF1 was also upregulated in rice seedlings after treatment with ABA. Overexpression of OsDRF1 in transgenic tobacco resulted in enhanced disease resistance against tomato mosaic virus (ToMV) and Pseudomonas syringae pv. tabaci and strengthened expression of defense-related genes after salicylic acid treatment or ToMV infection. Root elongation of the OsDRF1-overexpressing transgenic seedlings was significantly inhibited by ABA, indicating that overexpression of OsDRF1 resulted in increased ABA sensitivity. The results suggest that OsDRF1 plays a role in disease resistance via upregulating defense-related gene expression and that OsDRF1 may also be involved in the response to ABA.     

OsBISAMT1, a gene encoding S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase, is differentially expressed in rice defense responses

We isolated and identified a full-length cDNA, OsBISAMT1 [Oryza sativa L. benzothiadiazole (BTH)-induced SAMT 1], which encodes a putative S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase (SAMT) from rice. OsBISAMT1 contains an ORE of 1128 bp, which predicts to encode a 375 aa protein. The OsBISAMT1 protein sequence shows a high level of identity to known plant SAMTs and contains a conserved characteristic methyltransferase domain. OsBISAMT1 is a member of a small gene family in the rice genome. Expression of OsBISAMT1 in rice leaves was induced by treatments with benzothiadiazole and salicylic acid, which are capable of inducing rice disease resistance. OsBISAMT1 was also up-regulated in both incompatible and compatible interactions between rice and the blast fungus, Magnaporthe grsiea, but the induced expression of OsBISAMT1 was greater and more rapid in the incompatible interaction than that in the compatible one. Moreover, mechanical wounding also activated OsBISAMT1 expression. The results suggest that OsBISAMT1 may be involved in disease resistance responses as well as in wound response in rice.     

Overexpression of a rice diacylglycerol kinase gene OsBIDK1 enhances disease resistance in transgenic tobacco

A rice diacylglycerol kinase (DGK) gene, OsBIDK1, which encodes a 499-amino acid protein, was cloned and characterized. OsBIDK1 contains a conserved DGK domain, consisting of a diacylglycerol kinase catalytic subdomain and a diacylglycerol kinase accessory subdomain. Expression of OsBIDK1 in rice seedlings was induced by treatment with benzothiadiazole (BTH), a chemical activator of the plant defense response, and by infection with Magnaporthe grisea, causal agent of blast disease. In BTH-treated rice seedlings, expression of OsBIDK1 was induced earlier and at a higher level than in water-treated control seedlings after inoculation with M. grisea. Transgenic tobacco plants that constitutively express the OsBIDK1 gene were generated and disease resistance assays showed that overexpression of OsBIDK1 in transgenic tobacco plants resulted in enhanced resistance against infection by tobacco mosaic virus and Phytophthora parasitica var. nicotianae. These results suggest that OsBIDK1 may play a role in disease resistance responses.     

Over-expression of rice leucine-rich repeat protein results in activation of defense response, thereby enhancing resistance to bacterial soft rot in Chinese cabbage

Pectobacterium carotovorum subsp. carotovorum causes soft rot disease in various plants, including Chinese cabbage. The simple extracellular leucine-rich repeat (eLRR) domain proteins have been implicated in disease resistance. Rice leucine-rich repeat protein (OsLRP), a rice simple eLRR domain protein, is induced by pathogens, phytohormones, and salt. To see whether OsLRP enhances disease resistance to bacterial soft rot, OsLRP was introduced into Chinese cabbage by Agrobacterium-mediated transformation. Two independent transgenic lines over-expressing OsLRP were generated and further analyzed. Transgenic lines over-expressing OsLRP showed enhanced disease resistance to bacterial soft rot compared to non-transgenic control. Bacterial growth was retarded in transgenic lines over-expressing OsLRP compared to non-transgenic controls. We propose that OsLRP confers enhanced resistance to bacterial soft rot. Monitoring expression of defense-associated genes in transgenic lines over-expressing OsLRP, two different glucanases and Brassica rapa polygalacturonase inhibiting protein 2, PDF1 were constitutively activated in transgenic lines compared to non-transgenic control. Taken together, heterologous expression of OsLRP results in the activation of defense response and enhanced resistance to bacterial soft rot.     

OsCPK20 positively regulates Arabidopsis resistance against Pseudomonas syringae pv. tomato and rice resistance against Magnaporthe grisea

Calcium-dependent protein kinases are important decoders of calcium signals in plants, which are involved in plant immunity. We report isolation and functional characterization of a pathogen-responsive OsCPK20 gene in rice. The expression of OsCPK20 in rice was significantly induced following treatment with a Magnaporthe grisea elicitor. Overexpression of constitutively active OsCPK20 in Arabidopsis enhanced the resistance to infection with Pseudomonas syringae pv. tomato, associated with elevated expression of both SA- and JA-related defense genes. Similarly, transgenic rice plants containing constitutively active OsCPK20 exhibited enhanced resistance to blast fungus M. grisea. The enhanced resistance in the transgenic Arabidopsis and rice was associated with activated expression of both SA- and JA-related defense genes. We also found that OsCPK20 was significantly induced by drought stress, indicating that OsCPK20 might be involved in plant response to drought stress. Taken together, our results indicate that rice OsCPK20 positively regulates Arabidopsis resistance against Pseudomonas syringae pv. tomato and rice resistance against M. grisea, and that it may enhance disease resistance by activating both SA- and JA-dependent defense responses.     

The Blast Resistance Gene Pi54of Cloned from Oryza officinalis Interacts with Avr-Pi54 through Its Novel Non-LRR Domains

The dominant rice blast resistance gene Pi54 cloned by map-based cloning approach from indica rice cultivar Tetep confers broad spectrum resistance to Magnaporthe oryzae. In this investigation, an orthologue of Pi54 designated as Pi54of was cloned from Oryza officinalis conferring high degree of resistance to M. oryzae and is functionally validated. We have also characterized the Pi54of protein and demonstrate its interaction with AVR-Pi54 protein. The Pi54of encoded ∼43 kDa small and unique cytoplasmic LRR family of disease resistance protein having unique Zinc finger domain overlapped with the leucine rich repeat regions. Pi54of showed Magnaporthe-induced expression. The phylogenetic and western blot analysis confirmed orthologous nature of Pi54 and Pi54of genes, whereas the identity of protein was confirmed through MALDI-TOF analysis. The in silico analysis showed that Pi54of is structurally more stable than other cloned Pi54 proteins. The molecular docking revealed that Pi54of protein interacts with AVR-Pi54 through novel non-LRR domains such as STI1 and RhoGEF. The STI1 and GEF domains which interact with AVR-Pi54 are also components of rice defensome complex. The Pi54of protein showed differential domain specificity while interacting with the AVR protein. Functional complementation revealed that Pi54of transferred in two rice lines belonging to indica and japonica background imparts enhanced resistance against three highly virulent strains of M. oryzae. In this study, for the first time, we demonstrated that a rice blast resistance gene Pi54of cloned from wild species of rice provides high degree of resistance to M. oryzae and might display different molecular mechanism involved in AVRPi54-Pi54of interaction.     

The XA21 binding protein XB25 is required for maintaining XA21‐mediated disease resistance

Plant genomes encode a large number of proteins that potentially function as immune receptors in the defense against pathogen invasion. As a well‐characterized receptor kinase consisting of 23 tandem leucine‐rich repeats, a transmembrane domain and a serine/threonine kinase, the rice (Oryza sativa) protein XA21 confers resistance to a broad spectrum of Xanthomonas oryzae pv. oryzae (Xoo) races that cause bacterial blight disease. We report here that XA21 binding protein 25 (XB25) belongs to the plant‐specific ankyrin‐repeat (PANK) family. XB25 physically interacts, in vitro, with the transmembrane domain of XA21 through its N–terminal binding to transmembrane and positively charged domain (BTMP) repeats. In addition, XB25 associates with XA21 in planta. The downregulation of Xb25 results in reduced levels of XA21 and compromised XA21‐mediated disease resistance at the adult stage. Moreover, the accumulation of XB25 is induced by Xoo infection. Taken together, these results indicate that XB25 is required for maintaining XA21‐mediated disease resistance.     

Molecular dissection of the response of a rice leucine-rich repeat receptor-like kinase (LRR-RLK) gene to abiotic stresses

Leucine-rich repeat (LRR) receptor-like kinase (RLK) proteins play key roles in a variety of biological pathways. In a previous study, we analyzed the members of the rice LRR-RLK gene family using in silico analysis. A total of 23 LRR-RLK genes were selected based on the expression patterns of a genome-wide dataset of microarrays. The Oryza sativa gamma-ray induced LRR-RLK1 (OsGIRL1) gene was highly induced by gamma irradiation. Therefore, we studied its expression pattern in response to various different abiotic and phytohormone treatments. OsGIRL1 was induced on exposure to abiotic stresses such as salt, osmotic, and heat, salicylic acid (SA), and abscisic acid (ABA), but exhibited downregulation in response to jasmonic acid (JA) treatment. The OsGIRL1 protein was clearly localized at the plasma membrane. The truncated proteins harboring juxtamembrane and kinase domains (or only harboring a kinase domain) exhibited strong autophosphorylation. The biological function of OsGIRL1 was investigated via heterologous overexpression of this gene in Arabidopsis plants subjected to gamma-ray irradiation, salt stress, osmotic stress, and heat stress. A hypersensitive response was observed in response to salt stress and heat stress, whereas a hyposensitive response was observed in response to gamma-ray treatment and osmotic stress. These results provide critical insights into the molecular functions of the rice LRR-RLK genes as receptors of external signals.     

Rice blast resistance gene Pikahei-1(t), a member of a resistance gene cluster on chromosome 4, encodes a nucleotide-binding site and leucine-rich repeat protein

Pikahei-1(t) is the strongest quantitative trait locus (QTL) for blast resistance in upland rice cv. Kahei, which has strong field resistance to the rice blast disease. A high-quality bacterial artificial chromosome library was used to fine-map Pikahei-1(t) within ~300 kb on the 31-Mb region on rice chromosome 4. Of the 42 predicted open reading frames, seven resistance gene analogs (RGAs) with the nucleotide-binding site and leucine-rich repeat (NBS-LRR) domain were identified. Among these, RGA1, 2, 3, 5, and 7, but not RGA4 and 6, were found to be expressed in Kahei and monogenic lines containing Pikahei-1(t). Blast inoculation of transgenic rice lines carrying the genomic fragment of each RGA revealed that only RGA3 was associated with blast resistance. On the basis of these results, we concluded that RGA3 is the Pikahei-1(t) and named it Pi63. Pi63 encoded a typical coiled-coil-NBS-LRR protein and showed isolate-specificity. These results suggest that Pi63 behaves like a typical Resistance (R) gene, and the strong and broad-spectrum resistance of Kahei is dependent on natural pyramiding of multiple QTLs. The blast resistance levels of Pi63 were closely correlated with its gene expression levels, indicating a dose-dependent response of Pi63 function in rice resistance. Pi63 is the first cloned R gene in the R gene cluster on rice chromosome 4, and its cloning might facilitate genomic dissection of this cluster region.     

RD19, an Arabidopsis cysteine protease required for RRS1-R-mediated resistance, is relocalized to the nucleus by the Ralstonia solanacearum PopP2 effector

Bacterial wilt, a disease impacting cultivated crops worldwide, is caused by the pathogenic bacterium Ralstonia solanacearum. PopP2 (for Pseudomonas outer protein P2) is an R. solanacearum type III effector that belongs to the YopJ/AvrRxv protein family and interacts with the Arabidopsis thaliana RESISTANT TO RALSTONIA SOLANACEARUM 1-R (RRS1-R) resistance protein. RRS1-R contains the Toll/Interleukin1 receptor–nucleotide binding site–Leu-rich repeat domains found in several cytoplasmic R proteins and a C-terminal WRKY DNA binding domain. In this study, we identified the Arabidopsis Cys protease RESPONSIVE TO DEHYDRATION19 (RD19) as being a PopP2-interacting protein whose expression is induced during infection by R. solanacearum. An Arabidopsis rd19 mutant in an RRS1-R genetic background is compromised in resistance to the bacterium, indicating that RD19 is required for RRS1-R–mediated resistance. RD19 normally localizes in mobile vacuole-associated compartments and, upon coexpression with PopP2, is specifically relocalized to the plant nucleus, where the two proteins physically interact. No direct physical interaction between RRS1-R and RD19 in the presence of PopP2 was detected in the nucleus as determined by Förster resonance energy transfer. We propose that RD19 associates with PopP2 to form a nuclear complex that is required for activation of the RRS1-R–mediated resistance response.     

Palmitoylation-dependent Membrane Localization of the Rice Resistance Protein Pit Is Critical for the Activation of the Small GTPase OsRac1

Nucleotide binding domain and leucine-rich repeat (NLR)-containing family proteins function as intracellular immune sensors in both plants and animals. In plants, the downstream components activated by NLR family proteins and the immune response mechanisms induced by these downstream molecules are largely unknown. We have previously found that the small GTPase OsRac1, which acts as a molecular switch in rice immunity, is activated by Pit, an NLR-type resistance (R) protein to rice blast fungus, and this activation plays critical roles in Pit-mediated immunity. However, the sites and mechanisms of activation of Pit in vivo remain unknown. To clarify the mechanisms involved in the localization of Pit, we searched for consensus sequences in Pit that specify membrane localization and found a pair of potential palmitoylation sites in the N-terminal coiled-coil region. Although wild-type Pit was localized mainly to the plasma membrane, this membrane localization was compromised in a palmitoylation-deficient mutant of Pit. The palmitoylation-deficient Pit displayed significantly lower affinity for OsRac1 on the plasma membrane, thereby resulting in failures of the Pit-mediated cell death, the production of reactive oxygen species, and disease resistance to rice blast fungus. These results indicate that palmitoylation-dependent membrane localization of Pit is required for the interaction with and the activation of OsRac1 and that OsRac1 activation by Pit is vital for Pit-mediated disease resistance to rice blast fungus.     

Two novel protein kinase genes,OsMSRPK1 andOsMSURPK2, are regulated by diverse environmental stresses in rice

Two novel rice (Oryza sativa L.) protein kinase (PK) genes have been isolated.OsMSRPK1 andOsMSURPK2, which most likely exist as single-copy genes in the rice genome, encode 693 and S03 amino acids polypeptide, respectively, and have the serine/threonine kinase domain of cyclin dependent protein kinase (OsMSRPK1), or the serine/threonine kinase domain and NAF domain (OsMSURPK2). Steady-state mRNA analyses of these PKs, with constitutive expression in the leaves of two-week-old seedlings, revealed thatOsMSRPK1 is up-regulated upon exposure to environmental stresses, whereasOsMSVRPK2 is down-regulated by these same stresses. Furthermore, the two PKs are developmentally regulated in both young and mature rice plants, including in the panicles. These results strongly suggest that the genes have roles in both plant development and in their defense/stress-signaling pathways.     

OsCDPK13, a calcium-dependent protein kinase gene from rice,is induced in response to cold and gibberellin

Ca²⁺-dependent protein kinases (CDPKs) (EC 2.7.1.37) are the predominant Ca²⁺-regulated serine/threonine protein kinase in plants and their genes are encoded by a multigene family. CDPKs are important components in signal transduction, but the precise role of each individual CDPK is still largely unknown. A CDPK gene designated as OsCDPK13 was cloned from rice seedlings and it showed a high level of sequence similarities to rice and other plant CDPK genes. OsCDPK13 contains all conserved regions found in CDPKs. It was a single copy gene and was highly expressed in root and leaf sheath tissues of rice seedlings. OsCDPK13 expression was increased in leaf sheath segments treated with gibberellin or subjected to cold stress. The results in this investigation, together with our previous studies, suggest that OsCDPK13 may be an important signaling component in rice seedlings under cold stress condition and in response to gibberellin.