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.     

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.     

水稻类Tubby蛋白质在叶片生长和白叶枯病抗性反应中的表达

类Tubby蛋白质（Tubby-like protein,TLP）在动植物中广泛存在,暗示其在生命过程中发挥重要的作用。水稻（Oryza sativa）基因组中有14个TLP家族成员,首先制备了这些蛋白质的抗体,用免疫印迹方法检测了它们在水稻叶片不同生长时期的表达情况,揭示其表达模式;然后对Xa21介导的水稻白叶枯病抗性反应不同时间点进行检测,发现OsTLP2、OsTLP7、OsTLP8和OsTLP9等4个蛋白质的表达发生了变化;进一步比较它们在抗病、感病反应和对照处理中的表达情况,发现不同反应间的表达也有区别。该研究结果为阐释水稻TLP在叶片生长过程中的功能,尤其是在水稻-白叶枯病菌互作过程中的作用提供了重要线索。     

A paralog of the MtN3/saliva family recessively confers race-specific resistance to Xanthomonas oryzae in rice

Approximately one third of the identified 34 rice major disease resistance (R) genes conferring race-specific resistance to different strains of Xanthomonas oryzae pv. oryzae (Xoo), which causes rice bacterial blight disease, are recessive genes. However, only two of the recessive resistance genes have been characterized thus far. Here we report the characterization of another recessive resistance gene, xa25, for Xoo resistance. The xa25, localized in the centromeric region of chromosome 12, mediates race-specific resistance to Xoo strain PXO339 at both seedling and adult stages by inhibiting Xoo growth. It encodes a protein of the MtN3/saliva family, which is prevalent in eukaryotes, including mammals. Transformation of the dominant Xa25 into a resistant rice line carrying the recessive xa25 abolished its resistance to PXO339. The encoding proteins of recessive xa25 and its dominant allele Xa25 have eight amino acid differences. The expression of dominant Xa25 but not recessive xa25 was rapidly induced by PXO339 but not other Xoo strain infections. The nature of xa25-encoding protein and its expression pattern in comparison with its susceptible allele in rice-Xoo interaction indicate that the mechanism of xa25-mediated resistance appears to be different from that conferred by most of the characterized R proteins.     

Xa26, a gene conferring resistance to Xanthomonas oryzae pv. oryzae in rice, encodes an LRR receptor kinase-like protein

Rice bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most serious rice diseases worldwide. A rice gene, Xa26, conferring resistance against Xoo at both seedling and adult stages was isolated by map-based cloning strategies from the rice cultivar Minghui 63. Xa26 belongs to a multigene family consisting of four members. It encodes a leucine-rich repeat (LRR) receptor kinase-like protein and is constitutively expressed. Sequence analysis revealed that IRBB3 and Zhachanglong lines that are resistant to a broad range of Xoo strains, also carry Xa26. However, significant difference in lesion length was observed among these lines after inoculation with a set of Xoo strains. Moreover, transgenic plants carrying Xa26 showed enhanced resistance compared with the donor line of the gene in both seedling and adult stages. These results suggest that the resistance conferred by Xa26 is influenced by the genetic background.     

水稻抗白叶枯病基因Xa21的研究进展

Xa21是最早克隆的水稻抗病基因,作为类受体激酶类广谱抗病基因它受到广泛的关注。转基因Xa21材料,很可能成为世界上第一个被批准进行大田释放的水稻转基因材料。本文在简要回顾Xa21的发现、定位和克隆过程之后,总结了目前Xa21基因的抗病作用机理和育种应用研究现状,包括XA21蛋白质激酶的生化特性、AvrXa21的鉴别、Xa21介导的抗病途径、抗病机理等,并对今后的研究进行了展望。     

水稻类钙调磷酸酶亚基B蛋白质在叶片生长和白叶枯病抗性反应中的表达

钙信号介导植物对多种外部刺激的反应并参与调控广泛的生理学过程。钙离子结合蛋白质, 如类钙调磷酸酶亚基B蛋白质(calcineurin B-like protein, CBL), 对感受和传递钙信号具有重要作用。根据基因组测序及注释分析, 水稻(Oryza sativa)基因组中有10个CBL家族成员。采用基于抗体的蛋白质组学策略, 利用免疫印迹方法研究了水稻CBL蛋白质在叶片生长不同时期的表达, 揭示了其在正常发育过程中的表达模式。然后对Xa21介导的水稻白叶枯病抗性反应不同时间点的蛋白质表达进行检测, 发现OsCBL-1、OsCBL-3、OsCBL-5、OsCBL-9和OsCBL-10这5个蛋白质的表达发生了变化; 进一步比较它们在抗病、感病和对照处理中的表达情况, 发现其在不同反应间的表达也有区别, 提示了CBL蛋白质在水稻-白叶枯病菌互作过程中的功能。该研究为深入了解水稻CBL蛋白质的功能提供了有价值的线索。     

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.     

Are the dominant and recessive plant disease resistance genes similar? A case study of rice R genes and Xanthomonas oryzae pv. oryzae races.

The resistance of rice to its bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) has both qualitative and quantitative components that were investigated using three near-isogenic line sets for four resistance (R) genes (Xa4, xa5, xa13, and Xa21) and 12 Xoo races. Our results indicate that these two resistance components of rice plants were associated with the properties of the R genes. The qualitative component of the R genes was reflected by their large effects against corresponding avirulent Xoo races. The quantitative component of the R genes was their residual effects against corresponding virulent races and their epistatic effects, which together could lead to high-level resistance in a race-specific manner. Our results revealed important differences between the different types of R genes. Two R genes, Xa4 and Xa21, showed complete dominance against the avirulent Xoo races and had large residual effects against virulent ones. They acted independently and cumulatively, suggesting they are involved in different pathways of the rice defensive system. The third R gene, xa5, showed partial dominance or additivity to the avirulent Xoo races and had relatively small but significant residual effects against the virulent races. In contrast, xa13 was completely recessive, had no residual effects against the virulent races, and showed more pronounced race specificity. There was a strong interaction leading to increased resistance between xa13 and xa5 and between either of them and Xa4 or Xa21, suggesting their regulatory roles in the rice defensive pathway(s). Our results indicated that high-level and durable resistance to Xoo should be more efficiently achieved by pyramiding different types of R genes.     

无选择标记和载体骨干序列的Xa21转基因水稻的获得

利用双右边界T-DNA载体通过根癌农杆菌介导法将水稻白叶枯病广谱抗性基因Xa21导入杂交稻重要恢复系C418中。T0代共获得27个独立转基因株系,通过田间抗性鉴定与PCR分析,有17个株系的Xa21基因分子鉴定为阳性,且对白叶枯病原菌P6生理小种具有抗性。通过对17个株系的后代植株进行田间抗性鉴定,分子标记辅助选择及Southern杂交分析,结果显示4个株系的T1代植株中能分离出无潮霉素标记基因的Xa21转基因植株。无选择标记Xa21转基因株系的获得率为15%。PCR检测还表明,这些无选择标记的Xa21转基因植株不带有载体骨架序列。通过对转基因后代进一步的抗性鉴定与PCR辅助选择,获得了无选择标记和载体骨架序列的转基因Xa21纯合的抗白叶枯病水稻。     

The Xanthomonas oryzae pv. lozengeoryzae raxP and raxQ genes encode an ATP sulphurylase and adenosine-5'-phosphosulphate kinase that are required for AvrXa21 avirulence activity

Xanthomonas oryzae pv. oryzae (Xoo) Philippine race 6 (PR6) is unable to cause bacterial blight disease on rice lines containing the rice resistance gene Xa21 but is virulent on non-Xa21 rice lines, indicating that PR6 carries avirulence (avrXa21) determinants required for recognition by XA21. Here we show that two Xoo genes, raxP and raxQ, are required for AvrXa21 activity. raxP and raxQ, which reside in a genomic cluster of sulphur assimilation genes, encode an ATP sulphurylase and APS (adenosine-5'-phosphosulphate) kinase. These enzymes function together to produce activated forms of sulphate, APS and PAPS (3'-phosphoadenosine-5'-phosphosulphate). Xoo PR6 strains carrying disruptions in either gene, PR6DeltaraxP or PR6DeltaraxQ, are unable to produce APS and PAPS and are virulent on Xa21-containing rice lines. RaxP and RaxQ are similar to the bacterial symbiont Sinorhizobium meliloti host specificity proteins, NodP and NodQ and the Escherichia coli cysteine synthesis proteins CysD, CysN and CysC. The APS and PAPS produced by RaxP and RaxQ are used for both cysteine synthesis and sulphation of other molecules. Mutation in Xoo xcysI, a homologue of Escherichia coli cysI that is required for cysteine synthesis, blocked APS- or PAPS-dependent cysteine synthesis but did not affect AvrXa21 activity, suggesting that AvrXa21 activity is related to sulphation rather than cysteine synthesis. Taken together, these results demonstrate that APS and PAPS production plays a critical role in determining avirulence of a phytopathogen and reveal a commonality between symbiotic and phytopathogenic bacteria.     

水稻白叶枯病广谱抗性基因Xa21导入两用不育系培矮64S

以克隆的Xa21基因为外源基因,成熟胚愈伤组织为转化受体,应用农杆菌介导法对水稻两用型核不育系培矮64S进行转化,获46株转基因植株。PCR和Southern分析结果表明,Xa21已整合到受体基因组。用稻白叶枯病病原菌(Xanthomonasoryzaepv.oryzae)菲律宾小种6号接种鉴定,结果表明大多数转基因植株获得了抗病性。已整合的Xa21基因能够稳定地遗传,在所检测转基因株系的T1代中,Xa21基因显示3:1的分离。     

The autophosphorylated Ser686, Thr688, and Ser689 residues in the intracellular juxtamembrane domain of XA21 are implicated in stability control of rice receptor-like kinase

The rice gene Xa21 confers resistance against Xanthomonas oryzae pv. oryzae (Xoo). Xa21 encodes a receptor-like kinase (XA21). We demonstrate that XA21 autophosphorylates residues Ser686, Thr688 and Ser689 in vitro. Substitution of these residues with alanines did not affect the autophosphorylation function of this kinase, but specifically destabilized the resistance protein in vitro and in vivo. Plants carrying these same substitutions in XA21 were compromised in their resistance to the normally avirulent Xoo Philippine race 6. Additionally, we show that wild-type XA21 and the kinase-dead mutant with the invariable Lys736 residue mutated to glutamic acid were also proteolytically degraded in protein extracts. Finally, we show a correlation between the in vitro degradation and in vivo instability of the proteins. We propose that autophosphorylation of Ser686, Thr688 and Ser689 functions to stabilize XA21 against the developmentally controlled proteolytic activity.     

Rice NRR, a negative regulator of disease resistance, interacts with Arabidopsis NPR1 and rice NH1

Arabidopsis NPR1/NIM1 is a key regulator of systemic acquired resistance (SAR), which confers lasting broad-spectrum resistance. Over-expression of Arabidopsis NPR1 or the NPR1 homolog 1 (NH1) in rice results in enhanced resistance to the pathogen Xanthomonasoryzae pv. oryzae (Xoo), suggesting the presence of a related defense pathway in rice. We investigated this pathway in rice by identifying proteins that interact with NH1. Here we report the isolation and characterization of a rice cDNA encoding a novel protein, named NRR (for negative regulator of resistance). NRR interacts with NPR1 in the NPR1-interacting domain (NI25) consisting of 25 amino acids. NRR also interacts with NH1; however, NI25 was not sufficient for a strong interaction, indicating a difference between the rice and the Arabidopsis proteins. Silencing of NRR in rice had little effect on resistance to Xoo. When constitutively over-expressed in rice, NRR affected basal resistance, age-related resistance and Xa21-mediated resistance, causing enhanced susceptibility to Xoo. This phenotype was correlated with elevated NRR mRNA and protein levels and increased Xoo growth. Over-expression of NRR suppressed the induction of defense-related genes. NRR:GFP (green fluorescent protein) protein was localized to the nucleus, indicating that NRR may act directly to suppress the activation of defense genes. The fact that NRR compromises Xa21-mediated resistance indicates cross-talk or overlap between NH1- and Xa21-mediated pathways.     

Genetic and physical mapping of a new gene for bacterial blight resistance in rice

The inheritance of resistance for bacterial blight, caused by Xanthomonas oryzae pv. oryzae ( Xoo), was studied in Minghui 63, an elite restorer line for a number of widely used rice hybrids in China. A new dominant gene against a Chinese Xoo strain JL691 in both the seedling and adult stages was identified in Minghui 63 and designated as Xa26( t). Using a total of 477 highly susceptible individuals from an F(2) population, the Xa26( t) locus was mapped to a region of about 1.68 cM. This locus co-segregated with marker R1506 and was 0.21 cM from marker RM224 on one side and 1.47 cM from marker Y6855RA on the other side, in rice chromosome 11. A contig map, composed of five non-redundant bacterial artificial chromosome (BAC) clones and spanning approximately 500 kb in length, was constructed. Analysis of recombination events in the Xa26( t) region with the highly susceptible F(2) individuals anchored the gene locus to a region covered by three overlapped BAC clones. Assay of the lines showing a double crossover in marker loci flanking Xa26( t), in a population of recombinant inbred lines carrying Xa26( t), further delineated the gene to a 20-kb fragment. The Xa26( t) locus is tightly linked to another bacterial blight resistance gene locus, Xa4.