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.     

水稻抗稻瘟病基因资源与分子育种策略

稻瘟病是水稻主要病害之一。利用抗病品种是防治稻瘟病最经济、有效和安全的措施。近年来,随着植物先天免疫机制、抗病分子生物学以及水稻和稻瘟菌基因组学研究的不断深入,一系列参与病原菌识别和防卫信号传导与应答,以及外源抗菌蛋白、病原菌激发子等抗病相关基因陆续被鉴定和克隆,为提高水稻抗稻瘟病能力提供了一些新的基因资源、育种策略和技术。本文概述了国内外近年来克隆的主要抗稻瘟病基因资源及其在分子育种研究中应用的进展,提出了通过转基因手段整合不同防卫反应关键调控基因的抗稻瘟病聚合育种策略。     

水稻广谱抗稻瘟病基因研究进展

稻瘟病是水稻生产中的最严重病害之一,由于稻瘟菌小种的高度变异性,垂直抗性基因难以持续控制稻瘟病的危害,因此,克隆和利用广谱持久抗瘟基因被认为是解决稻瘟病问题最经济有效的策略。本文从广谱抗源的筛选与利用,广谱抗瘟基因的定位、克隆与应用等方面对水稻广谱抗稻瘟病基因研究取得的进展进行了概述,并介绍了广谱抗性分子机理的最新研究进展。基于国内外稻瘟病抗性基因研究的现状及趋势,以及我国丰富的抗瘟水稻种质资源,克隆越来越多的广谱抗瘟基因具有重要的理论与应用价值。     

The defense-responsive genes showing enhanced and repressed expression after pathogen infection in rice (Oryza sativa L.)

Despite large numbers of studies about defense response, processes involved in the resistance of plants to incompatible pathogens are still largely uncharacterized. The objective of this study was to identify genes involved in defense response by cDNA array analysis and to gain knowledge about the functions of the genes involved in defense response. Approximately 20000 rice cDNA clones were arrayed on nylon filters. RNA samples isolated from different rice lines after infection with incompatible strains or isolates of Xanthomonas oryzae pv. oryzae or Pyricularia grisea, respectively, were used to synthesize cDNA as probes for screening the cDNA arrays. A total of 100 differentially expressed unique sequences were identified from 5 pathogen-host combinations. Fifty-three sequences were detected as showing enhanced expression and 47 sequences were detected as showing repressed expression after pathogen infection. Sequence analysis revealed that most of the 100 sequences had various degrees of homology with     

Rice 14-3-3 protein (GF14e) negatively affects cell death and disease resistance

Plant 14-3-3 proteins regulate important cellular processes, including plant immune responses, through protein-protein interactions with a wide range of target proteins. In rice (Oryza sativa), the GF14e gene, which encodes a 14-3-3 protein, is induced during effector-triggered immunity (ETI) associated with pathogens such as Xanthomonas oryzae pv. oryzae (Xoo). To determine whether the GF14e gene plays a direct role in resistance to disease in rice, we suppressed its expression by RNAi silencing. GF14e suppression was correlated with the appearance of a lesion-mimic (LM) phenotype in the transgenic plants at 3 weeks after sowing. This indicates inappropriate regulation of cell death, a phenotype that is frequently associated with enhanced resistance to pathogens. GF14e-silenced rice plants showed high levels of resistance to a virulent strain of Xoo compared with plants that were not silenced. Enhanced resistance was correlated with GF14e silencing prior to and after development of the LM phenotype, higher basal expression of a defense response peroxidase gene (POX22.3), and accumulation of reactive oxygen species (ROS). In addition, GF14e-silenced plants also exhibit enhanced resistance to the necrotrophic fungal pathogen Rhizoctonia solani. Together, our findings suggest that GF14e negatively affects the induction of plant defense response genes, cell death and broad-spectrum resistance in rice.     

Constitutive heterologous expression of avrXa27 in rice containing the R gene Xa27 confers enhanced resistance to compatible Xanthomonas oryzae strains

The vascular pathogen Xanthomonas oryzae pv. oryzae ( Xoo ) and nonvascular pathogen Xanthomonas oryzae pv. oryzicola ( Xoc ) cause bacterial blight (BB) and bacterial leaf streak (BLS) diseases of rice, respectively. We have previously identified the avirulence gene avrXa27 from Xoo PXO99^A, which specifically induces the expression of the rice resistance gene Xa27 , ultimately leading to resistance against BB disease in rice. In this study, we have generated a transgenic rice line (L24) that expresses avrXa27 constitutively under the control of the PR1 promoter, and have examined its role in the host–pathogen interaction. L24 is not more susceptible to BB, indicating that avrXa27 does not contribute to virulence. AvrXa27 retains avirulence activity in L24 and, after crossing with a line containing Xa27 , progeny display phenotypic changes including inhibition of tillering, delay in flowering, stiff leaves, early leaf senescence and activation of pathogenesis-related ( PR ) genes. On challenge with a variety of compatible strains of Xoo and Xoc strain L8, lines with both avrXa27 and Xa27 also show enhanced resistance to bacterial infection. The induction of Xa27 and subsequent inhibition of Xoc growth in Xa27 plants are observed on inoculation with Xoc L8 harbouring avrXa27 . Our results indicate that the heterologous expression of avrXa27 in rice containing Xa27 triggers R gene-specific resistance and, at the same time, confers enhanced resistance to compatible strains of Xoo and Xoc . The expression of AvrXa27 and related proteins in plants has the potential to generate broad resistance in plants.     

Screening for resistance against Pseudomonas syringae in rice-FOX Arabidopsis lines identified a putative receptor-like cytoplasmic kinase gene that confers resistance to major bacterial and fungal pathogens in Arabidopsis and rice

Approximately 20,000 of the rice-FOX Arabidopsis transgenic lines, which overexpress 13,000 rice full-length cDNAs at random in Arabidopsis, were screened for bacterial disease resistance by dip inoculation with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). The identities of the overexpressed genes were determined in 72 lines that showed consistent resistance after three independent screens. Pst DC3000 resistance was verified for 19 genes by characterizing other independent Arabidopsis lines for the same genes in the original rice-FOX hunting population or obtained by reintroducing the genes into ecotype Columbia by floral dip transformation. Thirteen lines of these 72 selections were also resistant to the fungal pathogen Colletotrichum higginsianum. Eight genes that conferred resistance to Pst DC3000 in Arabidopsis have been introduced into rice for overexpression, and transformants were evaluated for resistance to the rice bacterial pathogen, Xanthomonas oryzae pv. oryzae. One of the transgenic rice lines was highly resistant to Xanthomonas oryzae pv. oryzae. Interestingly, this line also showed remarkably high resistance to Magnaporthe grisea, the fungal pathogen causing rice blast, which is the most devastating rice disease in many countries. The causal rice gene, encoding a putative receptor-like cytoplasmic kinase, was therefore designated as BROAD-SPECTRUM RESISTANCE 1. Our results demonstrate the utility of the rice-FOX Arabidopsis lines as a tool for the identification of genes involved in plant defence and suggest the presence of a defence mechanism common between monocots and dicots.     

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.     

水稻抗白叶枯病基因及其应用研究进展

由黄单胞菌水稻变种Xanthomonas oryzae pv.Oryzae（Xoo）引起的白叶枯病是水稻重要病害之一。目前,已有37个水稻白叶枯抗性基因被鉴定并报道,其中28个被定位到染色体上,7个被克隆。本文简要综述了水稻白叶枯抗性基因的鉴定、定位和克隆的进展,并讨论了合理利用抗性基因防治白叶枯病的前景。     

DNA markers and marker-assisted breeding for durable resistance to bacterial blight disease in rice

Bacterial leaf blight caused by the bacterial pathogen Xanthomonas oryzae pv oryzae (Xoo) limits rice yield in all major rice-growing regions of the world, especially in irrigated lowland and rainfed conditions where predisposition factors favor disease development to epidemic proportions. Since bacterial pathogens are difficult to manage, development of host plant resistance is the most effective means of disease management. As many as 24 major genes conferring resistance to various races of the pathogen have been identified and utilized in rice breeding programs. However, large-scale and long-term cultivation of varieties carrying a single gene for resistance resulted in a significant shift in pathogen race frequency with consequent breakdown of resistance in these cultivars. To combat the problem of resistance breakdown, pyramiding of resistance genes into different cultivars is being carried out. Pyramiding of resistance genes is now possible with molecular markers that are developed for individual genes. This review discusses the various bacterial blight resistance genes identified and their corresponding molecular markers developed for breeding durable resistance into modern rice cultivars.     

水稻病程相关PR1家族蛋白质在叶片生长及与白叶枯病菌互作反应中的表达

病程相关(PR)蛋白质经常被用作抗病反应的分子标记。利用免疫印迹(WB)技术检测了7个PR1家族蛋白质在水稻(Oryza sativa)叶片生长及与白叶枯病菌互作反应过程中的表达,发现6个PR1家族蛋白质在叶片生长中有表达。检测PR1蛋白质在Xa21介导的抗白叶枯病过程中的表达,结果显示PR1#052、PR1#072、PR1#073和PR1#121四个蛋白质在抗病反应后期呈上调或诱导表达,PR1#071则表达下调。进一步比较它们在抗病、感病和对照(Mock)反应中的表达丰度,发现在抗病和感病反应中的变化幅度均明显大于对照反应,推测这些PR蛋白质在水稻-白叶枯病菌互作反应中发挥作用。另外,对PR1基因上游启动子区的cis元件进行了分析。该研究初步揭示了水稻PR1家族蛋白质的表达谱,为进一步了解PR1蛋白质的功能提供了线索。     

水稻白叶枯病菌EAL结构域蛋白VieAxoo基因缺失突变和功能分析

摘要：【目的】旨在揭示水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae, 简称Xoo) 环鸟苷二磷酸（c-di-GMP）信号蛋白VieAxoo的生物学功能。【方法】本研究通过标记置换法对vieAxoo基因(PXO_04753)进行了缺失突变研究,采用表性测定进行了部分功能鉴定。【结果】从野生型菌株PXO99A中克隆的vieAxoo基因序列与其它病原黄单胞菌的同源序列高度保守。VieAxoo具有参与c-di-GMP降解的磷酸二酯酶（PDE）EAL结构域和磷酸信号识别受体REC结构域     

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.     

Three types of defense-responsive genes are involved in resistance to bacterial blight and fungal blast diseases in rice

Bacterial blight and fungal blast diseases of rice, caused by Xanthomonas oryzae pv. oryzae and Pyricularia grisea Sacc., respectively, are two of the most devastating diseases in rice worldwide. To study the defense responses to infection with each of these pathogens, expression profiling of 12 defense-responsive genes was performed using near-isogenic rice lines that are resistant or susceptible to bacterial blight and fungal blast, respectively, and rice cultivars that are resistant or susceptible to both pathogens. All 12 genes showed constitutive expression, but expression levels increased in response to infection. Based on their expression patterns in 12 host-pathogen combinations, these genes could be classified into three types, pathogen non-specific (6), pathogen specific but race non-specific (4) and race specific (2). Most of the 12 genes were only responsive during incompatible interactions. These results suggest that bacterial blight and fungal blast resistances share common pathway(s), but are also regulated by different defense pathways in rice. Activation of the corresponding R gene is the key step that initiates the action of these genes in defense responses. The chromosomal locations and pathogen specificities of seven of the 12 genes were consistent with those of previously identified quantitative trait loci for rice disease resistance, which indicates that some of the 12 genes studied may have a phenotypic impact on disease resistance in rice.     

水稻抗白叶枯病基因的鉴定、定位和克隆进展

由水稻黄单胞菌水稻变种Xoo引起的水稻白叶枯病是全球性的重要病害之一。已有31个水稻白叶枯抗性基因被鉴定并报道,其中18个被定位到染色体上,5个被克隆。简要综述了水稻白叶枯抗性基因的鉴定、定位和克隆的进展,并讨论了合理利用抗性基因防治白叶枯病的前景。     

Engineering broad-spectrum disease-resistant rice by editing multiple susceptibility genes

Rice blast and bacterial blight are important diseases of rice (Oryza sativa) caused by the fungus Magnaporthe oryzae and the bacterium Xanthomonas oryzae pv. oryzae (Xoo), respectively. Breeding rice varieties for broad-spectrum resistance is considered the most effective and sustainable approach to controlling both diseases. Although dominant resistance genes have been extensively used in rice breeding and production, generating disease-resistant varieties by altering susceptibility (S) genes that facilitate pathogen compatibility remains unexplored. Here, using CRISPR/Cas9 technology, we generated loss-of-function mutants of the S genes Pi21 and Bsr-d1 and showed that they had increased resistance to M. oryzae. We also generated a knockout mutant of the S gene Xa5 that showed increased resistance to Xoo. Remarkably, a triple mutant of all three S genes had significantly enhanced resistance to both M. oryzae and Xoo. Moreover, the triple mutant was comparable to the wild type in regard to key agronomic traits, including plant height, effective panicle number per plant, grain number per panicle, seed setting rate, and thousand-grain weight. These results demonstrate that the simultaneous editing of multiple S genes is a powerful strategy for generating new rice varieties with broad-spectrum resistance.