A novel rice C2H2-type zinc finger protein lacking DLN-box/EAR-motif plays a role in salt tolerance

A cDNA for the gene ZFP182, encoding a C2H2-type zinc finger protein, was cloned from rice by RT-PCR. ZFP182 codes an 18.2 kDa protein with two C2H2-type zinc finger motifs, one nuclear localization signal and one Leu-rich domain. The DLN-box/EAR-motif, which exists in most of plant C2H2-type zinc finger proteins, does not exist in ZFP182. The expression analysis showed that ZFP182 gene was constitutively expressed in leaves, culms, roots and spikes at the adult rice plants, and markedly induced in the seedlings by cold (4 degrees C), 150 mM NaCl and 0.1 mM ABA treatments. The approximate 1.4 kb promoter region of ZFP182 gene was fused into GUS reporter gene and transformed into tobacco. The histochemical analysis revealed that GUS expression could not be detected in transformed tobacco seedlings under normal conditions, but strongly observed in tobacco leaf discs and the vascular tissue of roots treated with NaCl or KCl. Expression of ZFP182 in transgenic tobacco and overexpression in rice increased plant tolerance to salt stress. These results demonstrated that ZFP182 might be involved in plant responses to salt stress.     

Functional analysis reveals pleiotropic effects of rice RING-H2 finger protein gene OsBIRF1 on regulation of growth and defense responses against abiotic and biotic stresses

RING finger proteins comprise a large family and play key roles in regulating growth/developmental processes, hormone signaling and responses to biotic and abiotic stresses in plants. A rice gene, OsBIRF1, encoding a putative RING-H2 finger protein, was cloned and identified. OsBIRF1 encodes a 396 amino acid protein belonging to the ATL family characterized by a conserved RING-H2 finger domain (C-X2-C-X15-C-X1-H-X2-H-X2-C-X10-C-X2-C), a transmembrane domain at the N-terminal, a basic amino acid rich region and a characteristic GLD region. Expression of OsBIRF1 was up-regulated in rice seedlings after treatment with benzothaidiazole, salicylic acid, l-aminocyclopropane-1-carboxylic acid and jasmonic acid, and was induced differentially in incompatible but not compatible interactions between rice and Magnaporthe grisea, the causal agent of blast disease. Transgenic tobacco plants that constitutively express OsBIRF1 exhibit enhanced disease resistance against tobacco mosaic virus and Pseudomonas syringae pv. tabaci and elevated expression levels of defense-related genes, e.g. PR-1, PR-2, PR-3 and PR-5. The OsBIRF1-overexpressing transgenic tobacco plants show increased oxidative stress tolerance to exogenous treatment with methyl viologen and H2O2, and up-regulate expression of oxidative stress-related genes. Reduced ABA sensitivity in root elongation and increased drought tolerance in seed germination were also observed in OsBIRF1 transgenic tobacco plants. Furthermore, the transgenic tobacco plants show longer roots and higher plant heights as compared with the wild-type plants, suggesting that overexpression of OsBIRF1 promote plant growth. These results demonstrate that OsBIRF1 has pleiotropic effects on growth and defense response against multiple abiotic and biotic stresses.     

<Emphasis Type="Italic">OsBIMK1</Emphasis>, a rice MAP kinase gene involved in disease resistance responses

The activation of mitogen-activated protein kinases (MAPKs) has been previously implicated in signal transduction during plant responses to pathogen attack as well as to various environmental stresses. We have isolated from rice a new MAPK cDNA, OsBIMK1 ( O ryza s ativa L. BTH-induced MAPK 1), which encodes a 369-amino-acid protein with moderate to high nucleotide sequence similarity to previously reported plant MAPK genes. OsBIMK1 contains all 11 of the MAPK conserved subdomains and the phosphorylation-activation motif, TEY. We analyzed in detail the expression of OsBIMK1 upon treatment with various chemical and biological inducers of resistance responses in rice and in both incompatible and compatible interactions between rice and Magnaporthe grisea. Expression of OsBIMK1 was activated rapidly upon treatment with benzothiadiazole (BTH) as well as with dichloroisonicotinic acid, probenazole, jasmonic acid and its methyl ester, Pseudomonas syringae pv. syringae, or wounding. Expression of OsBIMK1 was induced rapidly during the first 36 h after inoculation with M. grisea in BTH-treated rice seedlings and in an incompatible interaction between M. grisea and a blast-resistant rice genotype. BTH treatment induced a systemic activation of OsBIMK1 expression. These results suggest that OsBIMK1 plays an important role in rice disease resistance.     

A novel rice C2H2-type zinc finger protein lacking DLN-box/EAR-motif plays a role in salt tolerance

A cDNA for the gene ZFP182, encoding a C2H2-type zinc finger protein, was cloned from rice by RT-PCR. ZFP182 codes an 18.2 kDa protein with two C2H2-type zinc finger motifs, one nuclear localization signal and one Leu-rich domain. The DLN-box/EAR-motif, which exists in most of plant C2H2-type zinc finger proteins, does not exist in ZFP182. The expression analysis showed that ZFP182 gene was constitutively expressed in leaves, culms, roots and spikes at the adult rice plants, and markedly induced in the seedlings by cold (4 °C), 150 mM NaCl and 0.1 mM ABA treatments. The approximate 1.4 kb promoter region of ZFP182 gene was fused into GUS reporter gene and transformed into tobacco. The histochemical analysis revealed that GUS expression could not be detected in transformed tobacco seedlings under normal conditions, but strongly observed in tobacco leaf discs and the vascular tissue of roots treated with NaCl or KCl. Expression of ZFP182 in transgenic tobacco and overexpression in rice increased plant tolerance to salt stress. These results demonstrated that ZFP182 might be involved in plant responses to salt stress.     

A novel rice MAPK gene, OsBIMK2, is involved in disease-resistance responses

The mitogen-activated protein kinase (MAPK) cascades play important roles in transmission of extracellular signals to the downstream effector proteins through a mechanism of protein phosphorylation. In this study, we isolated and identified a novel rice MAPK gene, OSBIMK2 ( ORYZAE SATIVA L. BTH-Induced MAP Kinase 2). The OSBIMK2 encodes a 506 amino acid protein with molecular weight of 63 kD. The recombinant OSBIMK2 expressed in ESCHERICHIA COLI showed an autophosphorylation activity IN VITRO. OSBIMK2 is a single-copy gene in the rice genome. Expression of OSBIMK2 was activated upon treatment with benzothiadiazole (BTH), which is capable of inducing disease resistance in rice. Expression of OsBIMK2 was also up-regulated during early stage after inoculation with MAGNAPORTHE GRISEA in BTH-treated rice seedlings and during an incompatible interaction between M. GRISEA and a blast-resistant rice genotype. Over-expression of the rice OSBIMK2 gene in transgenic tobacco resulted in an enhanced disease resistance against tomato mosaic virus and a fungal pathogen, ALTERNARIA ALTERNATA. These results suggest that OSBIMK2 plays a role in disease resistance responses.     

Novel rice MAP kinases OsMSRMK3 and OsWJUMK1 involved in encountering diverse environmental stresses and developmental regulation

We report isolation of two novel rice (Oryza sativa L.) mitogen-activated protein kinases (MAPKs), OsMSRMK3 (multiple stress responsive) and OsWJUMK1 (wound- and JA-uninducible) that most likely exist as single copy genes in its genome. OsMSRMK3 and OsWJUMK1 encode 369 and 569 amino acid polypeptides having the MAPK family signature and phosphorylation activation motifs TEY and TDY, respectively. Steady state mRNA analyses of these MAPKs with constitutive expression in leaves of two-week-old seedlings revealed that OsMSRMK3 was up-regulated upon wounding (by cut), jasmonic acid (JA), salicylic acid (SA), ethylene, abscisic acid, hydrogen peroxide (H(2)O(2)), protein phosphatase inhibitors, chitosan, high salt/sugar, and heavy metals, whereas OsWJUMK1 not induced by either wounding, JA or SA, showed up-regulation only by H(2)O(2), heavy metals, and cold stress (12 degrees C). Moreover, these MAPKs were developmentally regulated. These results strongly suggest a role for OsMSRMK3 and OsWJUMK1 in both stress-signalling pathways and development 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.     

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.     

BWMK1 Responds to Multiple Environmental Stresses and Plant Hormones

Many plant mltogen-actlvated protein klnases （MAPKs） play an important role In regulating responses to both ablotlc and biotic stresses. The first reported rice MAPK gene BWMK1 Is Induced by both rice blast （Magnaporthe grisea） Infection and mechanical wounding. For further analysis of Its response to other environmental cues and plant hormones, such as jasmonlc acid （JA）, salicylic acid （SA）, and benzothladlazole （BTH）, the promoter of BWMKf was fused with the coding region of the β-glucuronldase （GUS） reporter gene. Two promoter-GUS constructs with a 1.0- and 2.5-kb promoter fragment, respectively, were generated and transformed into the Japonica rice cultIvars TP309 and Zhonghua 11. Expression of GUS was Induced in the transgenic lines by cold, drought, dark, and JA. However, light, SA, and BTH treatments suppressed GUS expression. These results demonstrate that BWMK1 Is responsive to multiple ablotlc stresses and plant hormones and may play a role In cross-talk between different signaling pathways.     

A novel rice PR10 protein, RSOsPR10, specifically induced in roots by biotic and abiotic stresses, possibly via the jasmonic acid signaling pathway

Plant roots have important roles not only in absorption of water and nutrients, but also in stress tolerance such as desiccation, salt, and low temperature. We have investigated stress-response proteins from rice roots using 2-dimensional polyacrylamide-gel electrophoresis and found a rice protein, RO-292, which was induced specifically in roots when 2-week-old rice seedlings were subjected to salt and drought stress. The full-length RO-292 cDNA was cloned, and was determined to encode a protein of 160 amino acid residues (16.9 kDa, pI 4.74). The deduced amino acid sequence showed high similarity to known rice PR10 proteins, OsPR10a/PBZ1 and OsPR10b. RO-292 mRNA accumulated rapidly upon drought, NaCl, jasmonic acid and probenazole, but not by exposure to low temperature or by abscisic acid and salicylic acid. The RO-292 gene was also up-regulated by infection with rice blast fungus. Interestingly, induction was observed almost exclusively in roots, thus we named the gene RSOsPR10 (root specific rice PR10). The present results indicate that RSOsPR10 is a novel rice PR10 protein, which is rapidly induced in roots by salt, drought stresses and blast fungus infection possibly through activation of the jasmonic acid signaling pathway, but not the abscisic acid and salicylic acid signaling pathway.     

The PP2C-type phosphatase AP2C1, which negatively regulates MPK4 and MPK6, modulates innate immunity, jasmonic acid, and ethylene levels in Arabidopsis

Wound signaling pathways in plants are mediated by mitogen-activated protein kinases (MAPKs) and stress hormones, such as ethylene and jasmonates. In Arabidopsis thaliana, the transmission of wound signals by MAPKs has been the subject of detailed investigations; however, the involvement of specific phosphatases in wound signaling is not known. Here, we show that AP2C1, an Arabidopsis Ser/Thr phosphatase of type 2C, is a novel stress signal regulator that inactivates the stress-responsive MAPKs MPK4 and MPK6. Mutant ap2c1 plants produce significantly higher amounts of jasmonate upon wounding and are more resistant to phytophagous mites (Tetranychus urticae). Plants with increased AP2C1 levels display lower wound activation of MAPKs, reduced ethylene production, and compromised innate immunity against the necrotrophic pathogen Botrytis cinerea. Our results demonstrate a key role for the AP2C1 phosphatase in regulating stress hormone levels, defense responses, and MAPK activities in Arabidopsis and provide evidence that the activity of AP2C1 might control the plant's response to B. cinerea.     

A rice serine carboxypeptidase-like gene OsBISCPL1 is involved in regulation of defense responses against biotic and oxidative stress

Serine carboxypeptidase-like proteins (SCPLs) comprise a large family of protein hydrolyzing enzymes that play roles in multiple cellular processes. During the course of study aimed at elucidating the molecular basis of induced immunity in rice, a gene, OsBISCPL1, encoding a putative SCPL, was isolated and identified. OsBISCPL1 contains a conserved peptidase S10 domain, serine active site and a signal peptide at N-terminus. OsBISCPL1 is expressed ubiquitously in rice, including roots, stems, leaves and spikes. Expression of OsBISCPL1 in leaves was significantly up-regulated after treatments with benzothiadiazole, salicylic acid, jasmonic acid and 1-amino cyclopropane-1-carboxylic acid, and also up-regulated in incompatible interactions between rice and the blast fungus, Magnaporthe grisea. Transgenic Arabidopsis plants with constitutive expression of OsBISCPL1 were generated and disease resistance assays indicated that the OsBISCPL1-overexpressing plants showed an enhanced disease resistance against Pseudomonas syringae pv. tomato and Alternaria brassicicola. Expression levels of defense-related genes, e.g. PR1, PR2, PR5 and PDF1.2, were constitutively up-regulated in transgenic plants as compared with those in wild-type plants. Furthermore, the OsBISCPL1-overexpressing plants also showed an increased tolerance to oxidative stress and up-regulated expression of oxidative stress-related genes. The results suggest that the OsBISCPL1 may be involved in regulation of defense responses against pathogen infection and oxidative stress.     

Wounding increases salt tolerance in tomato plants: evidence on the participation of calmodulin-like activities in cross-tolerance signalling

Cross-tolerance is the phenomenon by which a plant resistance to a stress results in resistance to another form of stress. It has previously been shown that salt stress causes the accumulation of proteinase inhibitors and the activation of other wound-related genes in tomato plants (Solanum lycopersicum). However, very little is known about how different stresses interact with one another, and which are the signalling components that interrelate the responses triggered by different stress types. In the present work, it is shown that mechanical wounding increases salt-stress tolerance in tomato plants through a mechanism that involves the signalling peptide systemin and the synthesis of JA. Data are also provided indicating that calmodulin-like activities are necessary for the downstream signalling events that lead to cross-tolerance between wounding and salt stress. Finally, evidence was gathered supporting the hypothesis that LeCDPK1, a Ca2+ -dependent protein kinase from tomato previously described in our laboratory, could participate in this cross-tolerance mechanism interrelating the signalling responses to wounding and salt stress.