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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.

Additional file 1. Supplemental Fig. S1. Zeta potential of GO and ChrGO. Fig. S2. The release of loaded adriamycin at different pH values.

Dual-targeted therapy in HER2-positive breast cancer cells with the combination of carbon dots/HER3 siRNA and trastuzumab resulted in enhanced antitumor activity, which overcomes the resistance to trastuzumab monotherapy. Herein, we have developed branched polyethylenimine-functionalized carbon dot (BP-CD) nanocarriers, which exhibited efficient green fluorescent protein gene delivery and expression. The positively charged BP-CDs allowed for effective nucleic acid binding and displayed a highly efficient small interfering RNA (siRNA)-mediated delivery targeting of cancer cells. The transfection of BP-CDs and HER3 siRNA complexes down-regulated HER3 protein expression and induced significant cell growth inhibition in BT-474 cells. BP-CDs/HER3 siRNA complexes induced cell death of BT-474 cells through G0/G1 cell cycle arrest and apoptosis. The combined treatment of BP-CDs/HER3 siRNA complexes and trastuzumab caused greater cell growth suppression in BT-474 cells when compared to either agent alone. The findings suggest that this dual-targeted therapy with the combination of BP-CDs/HER3 siRNA and trastuzumab represents a promising approach in breast cancer.

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.

Bacterial blight (BB) is one of the major diseases that affect rice productivity. In previous studies, BB resistance was transferred to cultivated rice Oryza sativa from wild rice Oryza meyeriana using asymmetric somatic hybridization. One of the resistant hybrid progenies (Y73) has also been shown to possess novel resistance gene(s) different from any of those previously associated with BB resistance. We have mapped quantitative trait loci (QTLs) for BB resistance in a recombinant inbred line (RIL) population derived from a cross between Y73 and a BB-susceptible cv. IR24. Five QTLs were detected where Y73 alleles contributed to increased BB resistance. Three minor QTLs were identified on chromosomes 3, 10 and 11, and two major QTLs on chromosomes 1 and 5, respectively. QTL on chromosome 5, designated qBBR5, had the strongest effect on BB resistance, explaining approximately 37% of the phenotypic variance. Using the same RIL population, we also mapped QTLs for agronomic traits including plant height (PH), heading date (HD), plant yield (PYD) and PYD component traits. A total of 21 QTLs were identified, of which four were detected for PH, six for HD, three for panicle number per plant (PNPP), one for spikelets per panicle (SPP), six for 1000-grain weight (TGW) and one for PYD. qPH1 (a QTL for PH) was found in the same interval as qBBR1 for BB resistance, and qHD11 for HD and qBBR11 for BB resistance also shared a similar interval. Additionally, BB resistance was significantly correlated with PH or HD in the RIL population. This suggests that the resistance genes may have pleiotropic effects on, or close linkage to, genes controlling PH or HD. These results will help deduce the resistance mechanisms of the novel resistance gene(s) and provide the basis for cloning them and using them in marker-assisted breeding.

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.

In this study, high-resolution melting (HRM) analysis was evaluated for gene mapping in rice with sequence-tagged site (STS) and simple sequence repeat (SSR) markers. A total of 103 out of 353 normal STS and SSR markers revealed polymorphic melting curves among the parental genotypes, and 12 of these were successfully used to genotype the F2 mapping population for HRM analysis. Additional electrophoresis findings demonstrated that HRM genotyping matched with traditional electrophoresis results. To optimize the HRM-marker screening efficiency, different HRM reaction conditions were evaluated. A 5-μl touchdown-polymerase chain reaction (PCR) system provided no significant improvement in screening efficiency but in a 10-μl touchdown-PCR system, the marker screening efficiency increased by 75%. Twenty-one markers were obtained for mapping purposes under the optimized reaction conditions. This study indicates that HRM analysis can speed up the gene mapping progress in rice, while saving a lot of manpower.

Artificial microRNA (amiRNA) has become a powerful tool for gene silencing in plants. A new method for easy and rapid construction of rice artificial miRNA vector is described. The procedure involved modification of the pCAMBIA1300-UR vector by insertion of a 'vector modification fragment'. This was prepared from the precursor of Os-amiR528 by eliminating the central miRNA-containing region while simultaneously creating an AfeI restriction site. The fragment was then introduced to the destination vector to produce a multipurpose 'Highly Efficient gene Silencing Compatible vector' (HESC vector). AfeI was used to produce linearized HESC vectors, and a blunt end PCR product that included amiRNA sequence was cloned into this site by a single ligation reaction to create the completed amiRNA vector. Tests showed that the method was highly efficient, and greatly reduced the time needed for vector construction and resulted in a DNA sequence identical to that of the current method, making it particularly suitable for use in a systems biology approach to functional genomic research.

Primer sequences used for quantitative RT-PCR of differentially expressed genes in susceptible rice subject to Xoo inoculation. (DOCX 14.3Â kb)

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.

Primer sequences used for over-expression and RT-PCR of Xoo3654 gene. (DOCX 14.1Â kb)

Primer sequences used for transient expression of Xoo genes in rice protoplast cells. (DOCX 13.8Â kb)

Cajal bodies (CBs) are distinct sub-nuclear structures that are present in eukaryotic living cells and are often associated with the nucleolus. CBs play important roles in RNA metabolism and formation of RNPs involved in transcription, splicing, ribosome biogenesis, and telomere maintenance. Besides these primary roles, CBs appear to be involved in additional functions that may not be directly related to RNA metabolism and RNP biogenesis. In this review, we assess possible roles of plant CBs in RNA regulatory pathways such as nonsense-mediated mRNA decay and RNA silencing. We also summarize recent progress and discuss new non-canonical functions of plant CBs in responses to stress and disease. It is hypothesized that CBs can regulate these responses via their interaction with poly(ADP ribose)polymerase (PARP), which is known to play an important role in various physiological processes including responses to biotic and abiotic stresses. It is suggested that CBs and their components modify PARP activities and functions.

Oryza meyeriana is highly resistant to rice bacterial blight (BB) and this resistance trait has been transferred to cultivated rice (O. sativa) using asymmetric somatic hybridization. However, no resistance genes have yet been cloned. In the present study, a progeny of the somatic hybridization with high BB resistance was crossed with a rice cultivar with high BB susceptibility to develop an F2 population. Using bulked segregant analysis (BSA), 17 polymorphic markers that were linked to rice BB resistance were obtained through scanning a total of 186 simple sequence repeats (SSR) and sequence-tagged site (STS) markers, evenly distributed on 12 chromosomes. A genetic linkage map was then constructed based on the 17 linkage markers and the F2 segregating population, which was followed by mapping for quantitative trait loci (QTLs) for BB resistance. Three QTLs were identified on chromosomes 1, 3 and 5, respectively, and the alleles of the resistant parent at any of the QTLs increased BB resistance. All of the three QTLs had a strong effect on resistance, explaining about 21.5%, 12.3% and 39.2% of the resistance variance, respectively. These QTLs were different from the loci of the BB resistance genes that have been identified in previous studies. The QTLs mapped in this work will facilitate the isolation of novel BB resistance genes and their utilization in rice resistance breeding.

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.

Emaravirus is a recently described genus of negative-strand RNA plant viruses. Emaravirus P4 protein localizes to plasmodesmata, suggesting that it could be a viral movement protein (MP). In the current study, we showed that the P4 protein of raspberry leaf blotch emaravirus (RLBV) rescued the cell-to-cell movement of a defective potato virus X (PVX) that had a deletion mutation in the triple gene block 1 movement-associated protein. This demonstrated that RLBV P4 is a functional MP. Sequence analyses revealed that P4 is a distant member of the 30K superfamily of MPs. All MPs of this family contain two highly conserved regions predicted to form β-strands, namely β1 and β2. We explored by alanine mutagenesis the role of two residues of P4 (Ile106 and Asp127) located in each of these strands. We also made the equivalent substitutions in the 29K MP of tobacco rattle virus, another member of the 30K superfamily. All substitutions abolished the ability to complement PVX movement, except for the I106A substitution in the β1 region of P4. This region has been shown to mediate membrane association of 30K MPs; our results show that it is possible to make non-conservative substitutions of a well-conserved aliphatic residue within β1 without preventing the membrane association or movement function of P4.

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.

Background Techniques that enable high levels of transgene expression in plants are attractive for the commercial production of plant-made recombinant pharmaceutical proteins or other gene transfer related strategies. The conventional way to increase the yield of desired transgenic products is to use strong promoters to control the expression of the transgene. Although many such promoters have been identified and characterized, the increase obtainable from a single promoter is ultimately limited to a certain extent. Results In this study, we report a method to magnify the effect of a single promoter by using a weak promoter-based selection system in transgenic rice. tCUP1, a fragment derived from the tobacco cryptic promoter (tCUP), was tested for its activity in rice by fusion to both a β-glucuronidase (GUS) reporter and a hygromycin phosphotransferase (HPT) selectable marker. The tCUP1 promoter allowed the recovery of transformed rice plants and conferred tissue specific expression of the GUS reporter, but was much weaker than the CaMV 35S promoter in driving a selectable marker for growth of resistant calli. However, in the resistant calli and regenerated transgenic plants selected by the use of tCUP1, the constitutive expression of green fluorescent protein (GFP) was dramatically increased as a result of the additive effect of multiple T-DNA insertions. The correlation between attenuated selection by a weak promoter and elevation of copy number and foreign gene expression was confirmed by using another relatively weak promoter from nopaline synthase (Nos). Conclusions The use of weak promoter derived selectable markers leads to a high T-DNA copy number and then greatly increases the expression of the foreign gene. The method described here provides an effective approach to robustly enhance the expression of heterogenous transgenes through copy number manipulation in rice.