A rice isoflavone reductase-like gene, OsIRL, is induced by rice blast fungal elicitor

We have isolated and characterized a rice isoflavone reductase-like gene, OsIRL, whose expression is induced by a fungal elicitor. The OsIRL cDNA contains 1203 bp with an open reading frame of 942 nucleotides encoding 314 amino acids. The deduced amino acid sequence of OsIRL has a putative pyridine nucleotide binding domain and is 68% homologous with the maize isoflavone reductase-like gene. Southern blot analysis revealed that OsIRL belongs to a small multigene family. Expression of OsIRL was induced by treatment with a fungal elicitor and jasmonic acid as well as by inoculation with rice blast fungus. Cycloheximide (1 microM), strongly inhibited the induction of OsIRL by the fungal elicitor, indicating that new protein synthesis is required. The protein kinase inhibitor, staurosporine (1 microM), had little effect, but the phosphatase inhibitor, calyculin A (1 microM), strongly inhibited induction. Treatment with salicylic acid (SA, 5 mM) strongly inhibited expression of OsIRL in response to fungal elicitor and JA, while abscisic acid (ABA, 200 microM) also strongly antagonized OsIRL induction by JA, but had only a weak effect on induction by the fungal elicitor. These results suggest that the expression of OsIRL is positively regulated by phytohormones such as JA, and negatively by phytohormones such as SA, ABA.     

不结球白菜BcLRK01基因对芜菁花叶病毒及胁迫诱导的响应

通过cDNA-AFLP技术,从芜菁花叶病毒(TuMV)侵染的不结球白菜幼叶中分离到一条差异表达的基因片段,克隆获得其cDNA全长为2 124bp,编码707个氨基酸的富亮氨酸重复类受体激酶,命名为BcLRK01。利用实时定量PCR研究了该基因在TuMV侵染及高盐、冷胁迫、水杨酸(SA)、茉莉酸(JA)、乙烯(ET)等处理下的表达情况,结果显示,TuMV侵染、高盐、冷胁迫、SA、JA和ET等均能诱导BcLRK01不同程度的表达,说明该基因可能是不结球白菜病毒病的病程相关基因,同时也参与高盐和冷胁迫以及SA、JA、ET等的信号途径。     

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.     

Signalling molecules and blast pathogen attack activates rice OsPR1a and OsPR1b genes: A model illustrating components participating during defence/stress response

Recently the rice (Oryza sativa L.) OsPR1a and OsPR1b genes were primarily characterized against jasmonic acid, ethylene and protein phosphatase 2A inhibitors. The dicot PR1 are recognized as reliable marker genes in defence/stress responses, and we also propose OsPR1 as marker genes in rice, a model monocot crop genus. Therefore, to gain further insight into the expression/regulation of OsPR1 genes, we characterized their activation against signalling molecules such as salicylic acid (SA), abscisic acid (ABA) and hydrogen peroxide (H₂O₂), and the blast pathogen Magnaporthe grisea. Here, we report that SA and H₂O₂ strongly induced the mRNA level of both OsPR1 genes, whereas ABA was found to be moderately effective. These inductions were specific in nature and required a de novo synthesized protein factor. A potential interaction amongst the signalling molecules in modulating the expression of OsPR1 genes was observed. Moreover, a specific induction of OsPR1 expression in an incompatible versus compatible host-pathogen interaction was also found. Finally, based on our present and previous results, a model of OsPR1 expression/regulation has been proposed, which reveals their essential role in defence/stress responses in rice and use as potent gene markers.     

水稻病程相关基因OsPR1b启动子的表达特性研究

目的:分析水稻病程相关基因OsPR1b的表达特性,以进一步了解其表达和调控机制。方法:利用PCR技术从水稻日本晴基因组中扩增OsPR1b基因的启动子片段,命名为OsPR1bp,并构建相应的OsPR1bp::GUS融合表达载体,采用农杆菌介导的转基因技术获得转基因植株,进行GUS组织化学分析;利用Real-time PCR对OsPR1b基因在植物激素、非生物因子和水稻白叶枯菌(Xoo)毒性菌株P10(PXO124)处理下的表达水平进行分析。结果:GUS组织染色结果表明OsPR1b在水稻叶片中的表达量较高,而在茎、根、愈伤和花器中的表达量较低;植物激素水杨酸(SA)、茉莉酸甲酯(MeJA)、激动素(KT)、脱落酸(ABA)及NaCl、PEG均可不同程度地提高OsPR1b在叶片中的表达水平,Me-JA、KT和NaCl的处理能提高其在根部的表达水平,但这些激素在诱导OsPR1b在叶片和根部的表达程度上存在明显差异;单独接种Xoo毒性菌株P10 24 h对OsPR1b表达的影响不大,而MeJA与其共同处理后则可显著增强其在叶片中的表达。结论:作为一种防卫基因,OsPR1b在健康植株中的表达水平较低,容易受盐/干旱胁迫及Xoo病原菌的诱导,多种植物激素如JA、KT和ABA很可能作为信号分子参与激活和介导了这种系统性的反应。     

Ethylene and jasmonic acid signaling affect the NPR1-independent expression of defense genes without impacting resistance to Pseudomonas syringae and Peronospora parasitica in the Arabidopsis ssi1 mutant

Salicylic acid (SA), ethylene, and jasmonic acid (JA) are important signaling molecules in plant defense to biotic stress. An intricate signaling network involving SA, ethylene, and JA fine tunes plant defense responses. SA-dependent defense responses in Arabidopsis thaliana are mediated through NPR1-dependent and -independent mechanisms. We have previously shown that activation of an NPR1-independent defense mechanism confers enhanced disease resistance and constitutive expression of the pathogenesis-related (PR) genes in the Arabidopsis ssi1 mutant. In addition, the ssi1 mutant constitutively expresses the defensin gene PDF1.2. Moreover, SA is required for the ssi1-conferred constitutive expression of PDF1.2 in addition to PR genes. Hence, the ssi1 mutant appears to target a step common to SA- and ethylene- or JA-regulated defense pathways. In the present study, we show that, in addition to SA, ethylene and JA signaling also are required for the ssi1-conferred constitutive expression of PDF1.2 and the NPR1-independent expression of PR-1. Furthermore, the ethylene-insensitive ein2 and JA-insensitive jar1 mutants enhance susceptibility of ssi1 plants to the necrotrophic fungus Botrytis cinerea. However, defects in either the ethylene- or JA-signaling pathways do not compromise ssi1-conferred resistance to the bacterial pathogen Pseudomonas synringae pv. maculicola and the oomycete pathogen Peronospora parasitica. Interestingly, ssi1 exhibits a marginal increase in the levels of ethylene and JA, suggesting that low endogenous levels of these phytohormones are sufficient to activate expression of defense genes. Taken together, our results indicate that although cross talk in ssi1 renders expression of ethylene- or JA-responsive defense genes sensitive to SA and vice versa, it does not affect downstream signaling leading to resistance.     

Novel insight into kinetin-inducible stress responses in rice seedlings

Kinetin (KN) action in rice self-defense mechanism was studied using our established 2-week-old rice (Oryza sativa L. japonica-type cv. Nipponbare) seedling in vitro model system. It was strikingly observed that KN caused formation of brownish necrotic microlesions in leaves, suggesting it triggers a stress response in rice. Subsequent northern analyses revealed differential regulation (both up-and down-regulations) of 10 prominent defense/stress-related marker genes, including the critical pathogenesis-related (PR) protein genes of class 1, 5 and 10. A systemic effect of KN in leaves was shown using OsPR1b (basic) and OsPOX (peroxidase) genes as representatives. KN also exclusively triggered potent accumulation of PR proteins (OsPR5 and OsPR10), and a phytoalexin, sakuranetin. Interestingly, as KN failed to induce jasmonic acid (JA) inducible genes (OsPR1a and JIOsPR10), and had almost no effect on accumulated endogenous JA level due to wounding by cut, KN might act through a yet unknown (and JA-independent) pathway. These results provide a new aspect on the role of KN as a potent activator of the stress responses in the rice plant.     

OsCOI1,水稻中一个受茉莉酸甲酯和脱落酸诱导表达的F-box家族基因

利用Blast检索、EST分析和RT-PCR,在水稻中分离到一个与拟南芥COI1同源的新基因,命名为OsCOI1.OsCOI1编码595个氨基酸.推测的OsCOI1编码蛋白有一个F-boxmotif和16个富含亮氨酸的重复序列,这与拟南芥COI1相似.OsCOI1在氨基酸水平上和COI1有很高的同源性(74%).经半定量RT-PCR法和RNA印迹分析,表明水稻中OsCOI1表达水平在经茉莉酸甲酯和脱落酸处理后呈明显变化,但不受水杨酸和乙烯的影响,说明OsCOI1可能在茉莉酸信号途径和脱落酸途径中具有特定功能.