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In addition to well-known roles in RNA metabolism, the nucleolus and Cajal bodies (CBs), both located within the nucleus, are involved in plant responses to biotic and abiotic stress. Previously we showed that plants in which expression of the CB protein coilin is downregulated are more susceptible to certain viruses including tobacco rattle virus (TRV), suggesting a role of coilin in antiviral defence. Experiments with coilin-deficient plants and the deletion mutant of the TRV 16K protein showed that both 16K and coilin are required for restriction of systemic TRV infection. The potential mechanisms of coilin-mediated antiviral defence were elucidated via experiments involving co-immunoprecipitation, use of NahG transgenic plants deficient in salicylic acid (SA) accumulation, measurement of endogenous SA concentrations and assessment of SA-responsive gene expression. Here we show that TRV 16K interacts with and relocalizes coilin to the nucleolus. In wild-type plants these events are accompanied by activation of SA-responsive gene expression and restriction of TRV systemic infection. By contrast, viral systemic spread was enhanced in NahG plants, implicating SA in these processes. Our findings suggest that coilin is involved in plant defence, responding to TRV infection by recognition of the TRV-encoded 16K protein and activating SA-dependent defence pathways.

Precise expression of a transgene in the desired manner is important for plant genetic engineering and gene function deciphering, but it is a challenge to obtain specific transgene expression free from the interference of the constitutive promoters used to express the selectable marker gene, such as the Cauliflower mosaic virus (CaMV) 35S promoter. So, the solutions to avoid these inappropriate regulations are largely demanded. In this study, we report the characterization of a callus strong promoter (CSP1) in rice and its application for accurate transgene expression. Our results indicate that the high expression of theCSP1promoter in the callus enables efficient selection of hygromycin equivalent to that provided by the CaMV 35S promoter, whereas its expression in other tissues is low. To evaluate possible leaky effects, the expression of a β-glucuronidase reporter driven by six specific promoters involving hormone signaling, pathogen response, cell fate determination, and proliferation was observed in transgenic rice plants generated byCSP1-mediated selection. Distinct β-glucuronidase expression was found consistently in most of the transgenic lines obtained for each promoter. In addition, we applied these specific marker lines to investigate the root cellular responses to exogenous cytokinin and auxin treatment. The results reveal that the root growth inhibition by cytokinin was differently regulated at high and low concentrations. In summary, we have established the feasibility of using callus-specific promoter-dependent selection to mitigate the transgene misexpression in rice. By enabling efficient transformation, rice plants with reliable transgene expression will be easily acquired for broad applications.

P3N-PIPO (P3 N-terminal fused with Pretty Interesting Potyviridae ORF), the movement protein of potyviruses, is expressed as a translational fusion with the N-terminus of P3 in potyviruses. As reported in previous studies, P3N-PIPO is expressed via transcriptional slippage at a conserved G2A6 slippery site in the genus Potyvirus. However, it is still unknown whether a similar expression mechanism of P3N-PIPO is used in the other genera of the family Potyviridae. Moreover, due to the extremely low expression level of P3N-PIPO in natural virus-infected plants, the peptides spanning the slippery site which provide direct evidence of the slippage at the protein level, have not been identified yet. In this study, a potato virus X (PVX)-based expression vector was utilized to investigate the expression mechanism of P3N-PIPO. A high expression level of the P3N-PIPO(WT) of turnip mosaic virus (TuMV, genus Potyvirus) was observed based on the PVX expression vector. For the first time, we successfully identified the peptides of P3N-PIPO spanning the slippery site by mass spectrometry. Likewise, the P3N-PIPO(WT) of wheat yellow mosaic virus (WYMV, genus Bymovirus) was also successfully expressed using the PVX expression vector. Integrated proteome and transcriptome analyses revealed that WYMV P3N-PIPO was expressed at the conserved G2A6 site through transcriptional slippage. Moreover, as revealed by mutagenesis analysis, Hexa-adenosine of the G2A6 site was important for the frameshift expression of P3N-PIPO in WYMV. According to our results, the PVX-based expression vector might be used as an excellent tool to study the expression mechanism of P3N-PIPO in Potyviridae. To the best of our knowledge, this is the first experimental evidence dissecting the expression mechanism of a bymovirus P3N-PIPO in the experimental host Nicotiana benthamiana.

Oryza meyeriana is a wild species of rice with high resistance to Xanthomonas oryzae pv. oryzae (Xoo), but the detailed resistance mechanism is unclear. Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) activase (RCA) is an important enzyme that regulates photosynthesis by activating Rubisco. We have previously reported that Xoo infection induced the relocation of RCA from the chloroplast stroma to the thylakoid membrane in O. meyeriana, but the underlying regulating mechanism and physiological significance of this association remains unknown. In this study, &ldquo, H2O2 burst&rdquo, with rapid and large increase in the amount of H2O2 was found to be induced by Xoo invasion in the leaves of O. meyeriana. 3, 3-diaminobenzidine (DAB) and oxidative 2, 7-Dichlorodi-hydrofluorescein diacetate (H2DCFDA) staining experiments both showed that H2O2 was generated in the chloroplast of O. meyeriana, and that this H2O2 generation as well as Xoo resistance of the wild rice were dramatically dependent on light. H2O2, methyl viologen with light, and the xanthine-xanthine oxidase system all induced RCA to associate with the thylakoid membrane in vitro, which showed that H2O2 could induce the relocation of RCA. In vitro experiments also showed that H2O2 induced changes in both the RCA and thylakoid membrane that were required for them to associate and that this association only occurred in O. meyeriana and not in the susceptible cultivated rice. These results suggest that the association of RCA with the thylakoid membrane helps to protect the thylakoid membrane against oxidative damage from H2O2. Therefore, in addition to its universal function of activating Rubisco, RCA appears to play a novel role in the resistance of O. meyeriana to Xoo.

Background Rice bacterial blight (BB) caused by Xanthomonas oryzae pv.oryzae (Xoo) is one of the most devastating bacterial diseases in rice-growing regions worldwide. The rice-Xoo interaction is a classical model for studying the interaction between plants and pathogens. Secreted proteins play important roles in plant-bacterial interactions, but are poorly studied in the rice-Xoo system. Rice cv. Nipponbare is highly susceptible to Xoo. Here, we used two-dimensional difference gel electrophoresis (2D-DIGE) coupled with MALDI-TOF/TOF mass spectrometry (MS), to investigate secreted proteins in Nipponbare embryo cell suspension culture infected by Xoo. Results A total of 32 protein spots changed significantly (p

Previously, we successfully introduced the bacterial blight (BB) resistance trait from Oryza meyeriana into Oryza sativa using asymmetric somatic hybridization with O. meyeriana as the donor species. After years of breeding, a near-isogenic line named Y73 was generated with recurrent parent O. sativa L. ssp. japonica cv. Dalixiang, and it shows high resistance to a broad-spectrum BB pathogens Xanthomonas oryzae pv. oryzae. To provide insights into the high-resistance phenotype of these plants, we examined the transcriptome response in leaves of Y73 to BB infection 24 h postinoculation using Affymetrix Rice GeneChip microarrays. Microarray analysis revealed that there was little impact on the transcriptome of inoculated leaves compared with controls, with only 0.22% of genes that were differentially regulated. However, several interesting genes, including a NAC domain–containing protein, an elicitor-responsive protein, involved in ubiquitination, and a glycosyl transferase gene, two transcription factors (TFs) and two unknown genes were up-regulated more than five-fold. Genes for metabolic and cellular components, TFs and defence signalling, such as the mitogen-activated protein kinase cascade, were also significantly induced after Xoo infection. This study provides a genome-wide view of the initial reaction of rice (Y73) to Xoo infection and elucidates some of the genes that may play an important role in disease resistance.

Nucleotide-binding site leucine-rich repeat (NB-LRR) genes play important roles in plant disease resistance. Due to its similarity to Rp1 in maize (Zea mays), a novel NB-LRR gene was isolated from rice and designated as OsRP1L1 (Oryza sativa Rp1-like 1). Analysis of expression of a super green fluorescent protein (sGFP) fusion gene revealed that the OsRP1L1 protein localizes to the nucleolus. Expression patterns suggested that OsRP1L1 was involved in responses to several plant growth regulators (PGRs) and environmental stresses. To explore its function in bacterial blight (BB) resistance, OsRP1L1 was cloned and overexpressed in a susceptible japonica rice cultivar. Overexpression of OsRP1L1 moderately elevated the resistance of plants to Xanthomonas oryzae pv. oryzae strains PXO86 and PXO341. Transcriptome analysis showed that plants overexpressing OsRP1L1 had small changes in their transcriptome, with 26 genes showing statistically significant changes of more than two-fold. The most up-regulated transcripts—a DIN1-like gene and a terpene synthase (TPS) gene—were up-regulated 7.9- and 7.6-fold, respectively. It was also noted that seven of the up-regulated genes have also reportedly been induced by overexpression of OsbZIP46, which has been identified recently as a player in ABA sensing. All these findings suggest that OsRP1L1 plays an important role in rice BB resistance, and may be useful as a genetic resource for engineering disease resistance in plants.

Y73 is a progeny of asymmetric somatic hybridization from an elite japonica rice cultivar (Dalixiang) and a wild rice ( Oryza meyeriana ), which shows high resistance to bacterial blight. It has a similar genetic background to its recurrent parent, Dalixiang, but Y73 has a high resistance to both bacterial blight and Agrobacterium . The transformation efficiency of Y73 was 35.7%, while Dalixiang had higher transformation efficiency (71.2%) under the same co-cultivation temperature (25 °C). These results indicate that the resistance to Agrobacterium tumefaciens in Y73 was linked with its characteristic of bacterial blight resistance, and was obtained from its donor parent, O. meyeriana . Further studies also showed that the resistance to A. tumefaciens was weakened at a lower temperature (20 °C). To study its molecular mechanism, the expression levels of genes associated with Agrobacterium -mediated transformation ( OsMPK s, OsVIP1s and OsPR1s ) in rice were investigated by Quantitative real-time PCR (qRT-PCR) analysis. The expression levels of OsMPK genes remained unchanged at different temperatures, while all OsVIP1 s and almost all OsPR1s were up-regulated and transformation efficiency of Y73 was increased notably when infected by Agrobacterium at 20 °C compared with 25 °C. This study suggests that the bacterial blight resistance genes in Y73 also have an effect on Agrobacterium- mediated transformation and that the response to temperature is associated with the regulation of MAPK/VIP1 defense signaling pathway.

Oryza meyeriana is a wild species of rice with high resistance to Xanthomonas oryzae pv. oryzae (Xoo), but the resistance mechanism is poorly understood. Protein gel blot analysis and immuno-gold electron microscopy showed that Xoo infection induced an association of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (RCA) with the thylakoid membrane in O. meyeriana, which led to considerable decline in the initial activity and the activation state of Rubisco. In susceptible cultivated rice, RCA remained in the chloroplast stroma. RCA may play a role in resistance to Xoo in O. meyeriana that differs from its well-known role in activating Rubisco, perhaps by protecting the thylakoid membrane against damage from Xoo.

Oryza meyeriana, a wild species of rice from China, shows high resistance to Xanthomonas oryzae pv. oryzae (Xoo), the cause of rice bacterial blight, one of the most serious rice pathogens. To better understand the resistance mechanism, a proteomic study was conducted to identify changes in the proteins secreted in embryo cell suspension cultures in response to Xoo. After two-dimensional difference gel electrophoresis (2D-DIGE), 72 differentially expressed protein spots corresponding to 34 proteins were identified by Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry. Of the 34 proteins, 10 were up regulated and 24 down regulated. The secreted proteins identified were predicted to be involved in various biological processes, including signal transduction, defense, ROS and cell wall modification. 77% of the 34 proteins were predicted to have a signal peptide by Signal P. Quantitative Real-Time PCR showed that transcript levels of 14 secreted proteins were not well correlated with secreted protein levels. Peroxidase activity was up regulated in both O. meyriana and susceptible rice but was about three times higher in O. meyeriana. This suggests that peroxidases may play an important role in the early response to Xoo in O. meyeriana. These results not only provide a better understanding of the resistance mechanism of O. meyeriana, but have implications for studies of the interactions between other plants and their pathogens.

Variation of transgene expression caused by either position effect at the insertion site or the promoter/enhancer elements employed for the expression of selectable marker genes has complicated phenotype characterization and caused misinterpretation. We have developed a reporter system in rice to analyze the influence of vector configuration, spacer and selectable marker gene promoter on the expression of the promoterless GUS reporter and DR5 promoter. Our results indicate that a spacer inserted between the reversed 35S promoter and the GUS reporter could reduce leaky expression of the reporter but was unable to block the nonspecific expression of DR5::GUS. Stacking the selectable marker unit in head to tail with the GUS reporter aided the gene specific expression of the GUS reporter under the DR5 promoter even when the 35S promoter is used for expression of the selectable marker. Compared to 35S under this configuration, a quick and distinctive expression of DR5::GUS was observed in the root cap, quiescent center and xylem cells in the root apical meristem by using the tCUP derived promoter (tCUP1) for selection, that is similar to the pattern obtained by a sensitive DR5 variant (DR5rev) in Arabidopsis. These data suggest a conserved property of the tCUP promoter in preventing enhancer-promoter interactions in rice as it does in Arabidopsis, and also demonstrate that an analogous distal auxin maximum exists in roots of rice. Therefore, the tCUP promoter based selection system provides a new strategy for specific expression of transgenes in rice.

Artificial microRNA (amiRNA) technology is used for gene silencing in Arabidopsis. We describe a method for constructing amiRNA vectors that requires only one PCR and one ligation reaction. Vectors produced by this method are the same as those from the method of Schwab et al. (Plant Cell 2006, 18:1121–1133). Transgenic plants created by this method can therefore be tested in the same way or compared with existing transgenic material without the risk of alteration to the amiRNA skeleton. With optimized parameters, 36–42 % colonies had the insertion in the expected orientation and 85–95 % of these had the correct sequence. Using this method, a transient gene knock-down analysis in Arabidopsis could be completed in 4–5 days.