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.     

A proteomic study of Xanthomonas oryzae pv. oryzae in rice xylem sap

Xanthomonas oryzae pv. oryzae (Xoo) is the second most important rice pathogen, causing a disease called bacterial leaf blight. Xoo colonizes and infects the vascular tissue resulting in tissue necrosis and wilting causing significant yield losses worldwide. In this study Xoo infected vascular fluid (xylem sap) was recovered and analyzed for secreted Xoo proteins. Three independent experiments resulted in the identification of 324 different proteins, 64 proteins were found in all three samples which included many of the known virulence-associated factors. In addition, 10 genes encoding for the identified proteins were inactivated and one mutant displayed statistically a significant loss in virulence when compared to the wild type Xoo, suggesting that a new virulence-associated factor has been revealed. The usefulness of this approach in understanding the lifestyle and unraveling the virulence-associated factors of phytopathogenic vascular bacteria is discussed.     

Molecular characterization of a pathogenesis-related protein 8 gene encoding a class III chitinase in rice

A cDNA encoding a class III chitinase (Oschib1) was isolated from a cDNA library constructed from rice leaves infected with the blast fungus Magnaporthe grisea. The cDNA contains an open reading frame of 861 nucleotides encoding 286 amino acid residues with a pI of 5.06. The deduced amino acid sequence of Oschibl has a high level of similarity with class IIIb chitinases of Gladiolus gandavensis (46%) and Tulipa bakeri (49%). A high level of Oschibl mRNA was detected after inoculation with M. grisea or Xanthomonas oryzae pv. oryzae. Expression of Oschib1 was induced more rapidly when an avirulent strain of M. grisea was inoculated (incompatible interaction) than when a virulent strain was used (compatible interaction). Expression of Oschibl was also induced by treatment of signaling molecules such as salicylic acid, ethylene, and methyl jasmonic acid, and by treatment with H2O2 or CuSO4. The induction patterns of Oschibl expression suggest that Oschib1 may be involved in defense response against pathogen infections and may be classified as a member of pathogenesis-related protein 8 in rice.     

RIR1 represses plant immunity by interacting with mitochondrial complex I subunit in rice

We previously observed decreased expression of rice OsmiR159a.1 on infection with the bacterial blight-causing pathogen Xanthomonas oryzae pv. oryzae (Xoo), and identified the OsLRR_RLK (leucine-rich repeat_ receptor like kinase) gene as an authentic target of OsmiR159a.1. Here, we found that a Tos17 insertion mutant of LRR_RLK displayed increasing temporal resistance to Xoo, whereas the LRR_RLK overexpression lines were susceptible to the pathogen early on in the infection, indicating that LRR_RLK encodes a repressor of rice resistance to Xoo infection, and it was renamed as RIR1 (Rice Immunity Repressor 1). RIR1 overexpression plants were more susceptible to Xoo at late growth stage, suggesting that RIR1 mRNA levels are negatively correlated with the resistance of rice against Xoo. We discovered that OsmiR159a.1 repression in Xoo-infected plants was largely dependent on the pathogen's type III secretion system. Co-immunoprecipitation, bimolecular fluoresence complementation, and pull-down assays indicated that RIR1 interacted with the NADH-ubiquinone oxidoreductase (NUO) 51-kDa subunit of the mitochondrial complex I through its kinase domain. Notably, impairment of RIR1 or overexpression of NUO resulted in reactive oxygen species accumulation and enhanced expression of pathogen-resistance genes, including jasmonic acid pathway genes. We propose that pathogens may inhibit OsmiR159 to interfere with the RIR1–NUO interaction, and subsequently depression of rice immune signalling pathways. The resistance genes manipulated by Xoo can be a probe to explore the regulatory network during host–pathogen interactions.     

The avrRxo1 gene from the rice pathogen Xanthomonas oryzae pv. oryzicola confers a nonhost defense reaction on maize with resistance gene Rxo1

Maize lines that contain the single dominant gene Rxo1 exhibit a rapid hypersensitive response (HR) after infiltration with the rice bacterial streak pathogen Xanthomonas oryzae pv. oryzicola, but not with the rice bacterial blight pathogen X. oryzae pv. oryzae. The avirulence effector gene that corresponds to Rxo1, designated avrRxo1, was identified in an X. oryzae pv. oryzicola genomic library. When introduced into X. oryzae pv. oryzae, clones containing avrRxo1 induced an HR on maize with Rxo1, but not on maize without Rxo1. The avrRxo1 gene is 1,266 bp long and shows no significant homology to any database sequences. When expressed in an X. oryzae pv. oryzae hrpC mutant that is deficient in the type III secretion system, avrRxo1 did not elicit the HR, indicating that the avrRxo1-Rxo1 interaction is dependent on type III secretion. Transient expression of avrRxo1 in onion cells after biolistic delivery revealed that the protein product was associated with the plasma membrane. Transient expression in maize lines carrying Rxo1 resulted in cell death, suggesting that AvrRxo1 functions from inside maize cells to elicit Rxo1-dependent pathogen recognition.     

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.     

Dynamic and Coordinated Expression Changes of Rice Small RNAs in Response to Xanthomonas oryzae pv.oryzae

Endogenous small RNAs are newly identified players in plant immune responses, yet their roles in rice(Oryza sativa) responding to pathogens are still less understood, especially for pathogens that can cause severe yield losses. We examined the small RNA expression profiles of rice leaves at 2, 6, 12, and 24 hours post infection of Xanthomonas oryzae pv. oryzae(Xoo) virulent strain PXO99, the causal agent of rice bacterial blight disease. Dynamic expression changes of some mi RNAs and trans-acting si RNAs were identified, together with a few novel mi RNA targets, including an RLK gene targeted by osa-mi R159 a.1. Coordinated expression changes were observed among some small RNAs in response to Xoo infection, with small RNAs exhibiting the same expression pattern tended to regulate genes in the same or related signaling pathways, including auxin and GA signaling pathways, nutrition and defense-related pathways. These findings reveal the dynamic and complex roles of small RNAs in rice-Xoo interactions, and identify new targets for regulating plant responses to Xoo.     

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.     

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.     

转录调控因子OxyRxoo对水稻白叶枯病菌H2O2降解途径的调控作用

摘要：【目的】为了阐明水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae, 简称Xoo)转录调控因子OxyRxoo对过氧化氢(H2O2) 降解途径的调控作用。【方法】本研究对推导的H2O2识别调控基因oxyRxoo进行了基因克隆、序列分析、缺失突变和互补试验及其相关表型的鉴定。【结果】克隆的oxyRxoo基因序列与其它几种病原黄单胞菌的同源序列高度保守。OxyRxoo是LysR家族成员之一,具有PBPb结构域和DNA结合保守结构域(HTH)。用标记交换法构建了△oxyRxoo基因缺失突变体。与野生型菌株PXO99A相比,尽管△oxyRxoo在离体培养条件下的生长无明显变化,但H2O2抗性显著地降低,过氧化物酶(CAT)活性明显下降,基因互补可以使之恢复; 过氧化物酶基因表达下调, oxyRxoo自身表达显著上调。【结论】OxyRxoo作为一个重要转录调控因子,调控了Xoo的 H2O2降解途径。     

Pdk1 kinase regulates basal disease resistance through the OsOxi1-OsPti1a phosphorylation cascade in rice

The AGC kinase OsOxi1, which has been isolated as an interactor with OsPti1a, positively regulates basal disease resistance in rice. In eukaryotes, AGC kinase family proteins are regulated by 3-phosphoinositide-dependent protein kinase 1 (Pdk1). In Arabidopsis, AtPdk1 directly interacts with phosphatidic acid, which functions as a second messenger in both biotic and abiotic stress responses. However, the functions of Pdk1 are poorly understood in plants. We show here that OsPdk1 acts upstream of the OsOxi1-OsPti1a signal cascade in disease resistance in rice. OsPdk1 interacts with OsOxi1 and phosphorylates the Ser283 residue of OsOxi1 in vitro. To investigate whether OsPdk1 is involved in immunity that is triggered by microbial-associated molecular patterns, we analyzed the phosphorylation status of OsPdk1 in response to chitin elicitor. Like OsOxi1, OsPdk1 is rapidly phosphorylated in response to chitin elicitor, suggesting that OsPdk1 participates in signal transduction through pathogen recognition. The overexpression of OsPdk1 enhanced basal resistance against a blast fungus, Magnaporthe oryzae, and a bacterial pathogen, Xanthomonas oryzae pv. oryzae (Xoo). Taken together, these results suggest that OsPdk1 positively regulates basal disease resistance through the OsOxi1-OsPti1a phosphorylation cascade in rice.