The rice 14-3-3 gene family and its involvement in responses to biotic and abiotic stress.

14-3-3 proteins function as major regulators of primary metabolism and cellular signal transduction in plants. However, their involvement in plant defense and stress responses is largely unknown. In order to better address functions of the rice 14-3-3/GF14 proteins in defense and abiotic stress responses, we examined the rice GF14 family that comprises eight numbers. The phylogenetic comparison with the Arabidopsis 14-3-3 family revealed that the majority of rice GF14s might have evolved as an independent branch. At least four rice GF14 genes, GF14b, GF14c, GF14e and Gf14f were differentially regulated in the interactions of rice-Magnaporthe grisea and rice-Xanthomonas oryzae pv. oryzae, and the incompatible interactions stronger induced the genes than the compatible interactions. These GF14 genes were also induced by the defense compounds, benzothiadiazole, methyl jasmonate, ethephon and hydrogen peroxide. Similarly, they were differentially regulated by salinity, drought, wounding and abscisic acid. Tissue-specific analysis and expression of GF14-YFP fusions revealed that the four GF14 isoforms were expressed with tissue specificity and accumulated differentially in the cytoplasm and nucleus. Our current study provides fundamental information for the further investigation of the rice GF14 proteins.     

OsSERK1 regulates rice development but not immunity to Xanthomonas oryzae pv. oryzae or Magnaporthe oryzae

Somatic embryogenesis receptor kinase (SERK) proteins play pivotal roles in regulation of plant development and immunity. The rice genome contains two SERK genes, OsSerk1 and OsSerk2. We previously demonstrated that OsSerk2 is required for rice Xa21-mediated resistance to Xanthomonas oryzae pv. oryzae (Xoo) and for normal development. Here we report the molecular characterization of OsSerk1. Overexpression of OsSerk1 results in a semi-dwarf phenotype whereas silencing of OsSerk1 results in a reduced angle of the lamina joint. OsSerk1 is not required for rice resistance to Xoo or Magnaporthe oryzae. Overexpression of OsSerk1 in OsSerk2-silenced lines complements phenotypes associated with brassinosteroid (BR) signaling defects, but not the disease resistance phenotype mediated by Xa21. In yeast, OsSERK1 interacts with itself forming homodimers, and also interacts with the kinase domains of OsSERK2 and BRI1, respectively. OsSERK1 is a functional protein kinase capable of auto-phosphorylation in vitro. We conclude that, whereas OsSERK2 regulates both rice development and immunity, OsSERK1 functions in rice development but not immunity to Xoo and M. oryzae.     

转拟南芥AtNPR1基因增强水稻对水稻白叶枯病和稻瘟病的抗性

AtNPR1基因是拟南芥系统获得抗性的一个重要调节基因,在拟南芥中过量表达AtNPR1基因能使拟南芥对细菌和真菌的抗性同时增强.为了研究在水稻中过量表达AtNPR1基因对水稻抗病性的影响,将该基因转入到广西主栽籼稻恢复系品种桂99中.经PCR验证得到了79株转基因植株,DNA斑点杂交表明ATNPR1基因已经整合到桂99染色体DNA中.Northern杂交和RT-PCR分析表明,AtNPR1基因在桂99中已经表达;同时还检测了转基因植株对水稻白叶枯病和稻瘟病的抗性,结果表明转基因植株对该两种病害的抗性均显著增强.     

Proteomic analysis of bacterial-blight defense-responsive proteins in rice leaf blades

Plants exhibit resistance against incompatible pathogens, via localized and systemic responses as part of an integrated defense mechanism. To study the compatible and incompatible interactions between rice and bacteria, a proteomic approach was applied. Rice cv. Java 14 seedlings were inoculated with compatible (Xo7435) and incompatible (T7174) races of Xanthomonas oryzae pv. oryzae (Xoo). Cytosolic and membrane proteins were fractionated from the leaf blades and separated by 2-D PAGE. From 366 proteins analyzed, 20 were differentially expressed in response to bacterial inoculation. These proteins were categorized into classes related to energy (30%), metabolism (20%), and defense (20%). Among the 20 proteins, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RuBisCO LSU) was fragmented into two smaller proteins by T7174 and Xo7435 inoculation. Treatment with jasmonic acid (JA), a signaling molecule in plant defense responses, changed the level of protein accumulation for 5 of the 20 proteins. Thaumatin-like protein and probenazole-inducible protein (PBZ) were commonly up-regulated by T7174 and Xo7435 inoculation and JA treatment. These results suggest that synthesis of the defense-related thaumatin-like protein and PBZ are stimulated by JA in the defense response pathway of rice against bacterial blight.     

Overexpression of (At)NPR1 in rice leads to a BTH- and environment-induced lesion-mimic/cell death phenotype

Systemic acquired resistance (SAR) is an inducible defense response that protects plants against a broad spectrum of pathogens. A central regulator of SAR in Arabidopsis is NPR1 (nonexpresser of pathogenesis-related genes). In rice, overexpression of Arabidopsis NPR1 enhances plant resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae. This report demonstrates that overexpression of (At)NPR1 in rice also triggers a lesion-mimic/cell death (LMD) phenotype. The LMD phenotype is environmentally regulated and heritable. In addition, the development of lesions and death correlates with the expression of rice defense genes and the accumulation of hydrogen peroxide. Application of the salicylic acid (SA) analog, benzo(1,2,3) thiadiazole-7-carbothioc acid S-methyl ester (BTH), potentiates this phenotype Endogenous SA levels are reduced in rice overexpressing (At)NPR1 when compared with wildtype plants, supporting the idea that (At)NPR1 may perceive and modulate the accumulation of SA. The association of (At)NPR1 expression in rice with the development of an LMD phenotype suggests that (At)NPR1 has multiple roles in plant stress responses that may affect its efficacy as a transgenic tool for engineering broad-spectrum resistance.     

Arabidopsis 14-3-3 lambda is a positive regulator of RPW8-mediated disease resistance

The RPW8 locus from Arabidopsis thaliana Ms-0 includes two functional paralogous genes ( RPW8.1 and RPW8.2 ) and confers broad-spectrum resistance via the salicylic acid-dependent signaling pathway to the biotrophic fungal pathogens Golovinomyces spp. that cause powdery mildew diseases on multiple plant species. To identify proteins involved in regulation of the RPW8 protein function, a yeast two-hybrid screen was performed using RPW8.2 as bait. The 14-3-3 isoform lambda (designated GF14λ) was identified as a potential RPW8.2 interactor. The RPW8.2–GF14λ interaction was specific and engaged the C-terminal domain of RPW8.2, which was confirmed by pulldown assays. The physiological impact of the interaction was revealed by knocking down GF14λ by T-DNA insertion, which compromised basal and RPW8-mediated resistance to powdery mildew. In addition, over-expression of GF14λ resulted in hypersensitive response-like cell death and enhanced resistance to powdery mildew via the salicylic acid-dependent signaling pathway. The results from this study suggest that GF14λ may positively regulate the RPW8.2 resistance function and play a role in enhancing basal resistance in Arabidopsis.     

Evidence for a disease-resistance pathway in rice similar to the NPR1-mediated signaling pathway in Arabidopsis

The Arabidopsis NPR1/NIM1 gene is a key regulator of systemic acquired resistance (SAR). Over-expression of NPR1 leads to enhanced resistance in Arabidopsis. To investigate the role of NPR1 in monocots, we over-expressed the Arabidopsis NPR1 in rice and challenged the transgenic plants with Xanthomonas oryzae pv. oryzae (Xoo), the rice bacterial blight pathogen. The transgenic plants displayed enhanced resistance to Xoo. RNA blot hybridization indicates that enhanced resistance requires expression of NPR1 mRNA above a threshold level in rice. To identify components mediating the resistance controlled by NPR1, we used NPR1 as bait in a yeast two-hybrid screen. We isolated four cDNA clones encoding rice NPR1 interactors (named rTGA2.1, rTGA2.2, rTGA2.3 and rLG2) belonging to the bZIP family. rTGA2.1, rTGA2.2 and rTGA2.3 share 75, 76 and 78% identity with Arabidopsis TGA2, respectively. In contrast, rLG2 shares highest identity (81%) to the maize liguleless (LG2) gene product, which is involved in establishing the leaf blade-sheath boundary. The interaction of NPR1 with the rice bZIP proteins in yeast was impaired by the npr1-1 and npr1-2 mutations, but not by the nim1-4 mutation. The NPR1-rTGA2.1 interaction was confirmed by an in vitro pull-down experiment. In gel mobility shift assays, rTGA2.1 binds to the rice RCH10 promoter and to a cis-element required sequence-specifically for salicylic acid responsiveness. This is the first demonstration that the Arabidopsis NPR1 gene can enhance disease resistance in a monocot plant. These results also suggest that monocot and dicot plants share a conserved signal transduction pathway controlling NPR1-mediated resistance.     

The molecular basis of disease resistance in rice

The rice gene Xa21 conferring resistance to Xanthomonas oryzae pv. oryzae (Xoo), was isolated using a map-based cloning strategy. Compared with previously cloned genes, the structure of Xa21 represents a novel class of plant disease R genes encoding a putative receptor kinase (RK). This article proposes a model for the mode of action of Xa21 and summarizes our current knowledge of the modular basis of resistance in rice to bacterial leaf blight and blast.     

Overexpression of the Arabidopsis 14-3-3 protein GF14 lambda in cotton leads to a "stay-green" phenotype and improves stress tolerance under moderate drought conditions

The Arabidopsis gene GF14 lambda that encodes a 14-3-3 protein was introduced into cotton plants to explore the physiological roles that GF14 lambda might play in plants. The expression level of GF14 lambda under the control of the cauliflower mosaic virus 35S promoter varied in transgenic cotton plants, and lines that expressed GF14 lambda demonstrated a "stay-green" phenotype and improved water-stress tolerance. These lines wilted less and maintained higher photosynthesis than segregated non-transgenic control plants under water-deficit conditions. Stomatal conductance appears to be the major factor for the observed higher photosynthetic rates under water-deficit conditions. The stomatal aperture of transgenic plants might be regulated by GF14 lambda through some transporters such as H(+)-ATPase whose activities are controlled by their interaction with 14-3-3 proteins. However, since 14-3-3 proteins interact with numerous proteins in plant cells, many metabolic processes could be affected by the GF14 lambda overexpression. Whatever the mechanisms, the traits observed in the GF14 lambda-expressing cotton plants are beneficial to crops under certain water-deficit conditions.     

Sucrose-mediated priming of plant defense responses and broad-spectrum disease resistance by overexpression of the maize pathogenesis-related PRms protein in rice plants

Expression of pathogenesis-related (PR) genes is part of the plant's natural defense response against pathogen attack. The PRms gene encodes a fungal-inducible PR protein from maize. Here, we demonstrate that expression of PRms in transgenic rice confers broad-spectrum protection against pathogens, including fungal (Magnaporthe oryzae, Fusarium verticillioides, and Helminthosporium oryzae) and bacterial (Erwinia chrysanthemi) pathogens. The PRms-mediated disease resistance in rice plants is associated with an enhanced capacity to express and activate the natural plant defense mechanisms. Thus, PRms rice plants display a basal level of expression of endogenous defense genes in the absence of the pathogen. PRms plants also exhibit stronger and quicker defense responses during pathogen infection. We also have found that sucrose accumulates at higher levels in leaves of PRms plants. Sucrose responsiveness of rice defense genes correlates with the pathogen-responsive priming of their expression in PRms rice plants. Moreover, pretreatment of rice plants with sucrose enhances resistance to M. oryzae infection. Together, these results support a sucrose-mediated priming of defense responses in PRms rice plants which results in broad-spectrum disease resistance.     

转拟南芥NPR1基因桂99 T3代植株的抗病性及农艺性状表现

以转拟南芥AtNPR1基因的恢复系品种桂99T3代纯合株系为材料,考查其农艺性状及其抗病性,并比较转基因植株与桂99侵染水稻白叶枯病菌后的农艺性状。结果表明:转基因植株表现出对水稻白叶枯病的抗性显著增强77%以上;穗长、剑叶长、有效穗数、一次枝梗数、每穗实粒数、单株产量和谷粒宽等农艺性状与未转基因桂99无显著差别。在受到水稻白叶枯病菌侵染后,转基因植株的一次枝梗数、每穗粒数、每穗实粒数和单株产量等方面均比对照桂99高出13%～78%。说明AtNPR1基因增强了水稻的抗病能力,从而降低了病害引起的产量损失。转基因植株的恢复力不受影响,稻米品质比桂99更加优良。本工作为转基因水稻抗病育种的研究奠定了基础。     

Fusicoccin, 14-3-3 proteins, and defense responses in tomato plants

Fusicoccin (FC) is a fungal toxin that activates the plant plasma membrane H+-ATPase by binding with 14-3-3 proteins, causing membrane hyperpolarization. Here we report on the effect of FC on a gene-for-gene pathogen-resistance response and show that FC application induces the expression of several genes involved in plant responses to pathogens. Ten members of the FC-binding 14-3-3 protein gene family were isolated from tomato (Lycopersicon esculentum) to characterize their role in defense responses. Sequence analysis is suggestive of common biochemical functions for these tomato 14-3-3 proteins, but their genes showed different expression patterns in leaves after challenges. Different specific subsets of 14-3-3 genes were induced after treatment with FC and during a gene-for-gene resistance response. Possible roles for the H+-ATPase and 14-3-3 proteins in responses to pathogens are discussed.     

LMM5.1 and LMM5.4,two eukaryotic translation elongation factor 1Alike gene family members,negatively affect cell death and disease resistance in rice

Lesion mimic mutant(LMM) genes, stimulating lesion formation in the absence of pathogens, play significant roles in immune response. In this study, we characterized a rice lesion mimic mutant, lmm5,which displayed light-dependent spontaneous lesions. Additionally, lmm5 plants exhibited enhanced resistance to all of the tested races of Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae(Xoo) by increasing the expression of defense-related genes and the accumulation of hydrogen peroxide. Genetic analysis showed that the lesion mimic phenotype of lmm5 was controlled by two genes, lmm5.1 and lmm5.4, which were isolated with a map-based cloning strategy. Remarkably, LMM5.1 and LMM5.4 share a 97.4% amino acid sequence identity, and they each encode a eukaryotic translation elongation factor 1A(e EF1A)-like protein. Besides, LMM5.1 and LMM5.4 were expressed in a tissue-specific and an indicaspecific manner, respectively. In addition, high-throughput m RNA sequencing analysis confirmed that the basal immunity was constitutively activated in the lmm5 mutant. Taken together, these results suggest that the homologous e EF1A-like genes, LMM5.1 and LMM5.4, negatively affect cell death and disease resistance in rice.     

水稻黄单胞菌黄色素合成相关基因的克隆与鉴定

用硫酸二乙酯(DES)诱变水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae,简称Xoo)和条斑病细菌(Xanthomonas oryzae pv. oryzicola, 简称Xooc),分别得到5株和13株黄色素缺失突变体,其中来自Xooc的M6和M12 还丧失了对水稻的致病性和在烟草上激发过敏反应的能力。以Xooc黄色素缺失突变体M51为受体菌交叉互补从Xoo JXOIII基因文库中筛选出一个黄色素合成相关的基因克隆pA341,以Xoo黄色素缺失突变体M1071为受体菌,从Xooc RS105基因文库中获得了一个黄色素合成相关的基因克隆pA270。功能互补显示,18株黄色素缺失突变体中的10株能分别被pA341和 pA270互补后正常产生黄色素,但这两个克隆不能同时互补同一株黄色素缺失突变体。能被pA341互补的黄色素缺失突变体M6没有恢复对水稻的致病性和在烟草上激发过敏反应,表明黄色素合成相关基因与hrp基因间不存在相关性。斑点杂交结果表明,pA270与pA341之间没有同源性。pA270亚克隆结果显示,与黄色素合成相关的基因约11.6kb大小,以基因簇的形式存在,不仅决定了黄色素的产生,还影响黄色素合成的数量和质量（吸收峰）。在紫外光条件下,黄色素能够提高菌体的存活率,提示黄色素对病原细菌有保护作用。