Compared with the transgenic approach, transient assays provide a convenient alternative to analyze gene expression. To analyze the relationship between miRNAs and their target genes, a rice protoplast system to detect target gene activity was established. The MIRNA and GFP-fused target sequence (or GFP-fused mutated sequence as a non-target control) were constructed into the same plasmid, and then delivered into rice protoplasts. The GFP expression level decreased significantly when the protoplasts were transfected with the plasmid containing GFP-fused target compared to that of the plasmid with non-target sequence either by fluorescence microscopy or qRT-PCR method. Two microRNA genes, osaMIR156 and osaMIR397, and their target sequences were used to prove the feasibility of the rice protoplast transient assay system. This method will facilitate large-scale screening of rice miRNA target in vivo, and may be suitable for functional analysis of miRNAs of other monocot plants that might share the evolutionarily conserved small RNA processing system with rice.
Gene Targeting ; Green Fluorescent Proteins ; genetics ; MicroRNAs ; genetics ; Oryza ; genetics ; Plasmids ; Protoplasts ; metabolism ; RNA, Plant ; genetics ; Transfection

Small interfering RNAs (siRNAs) can efficiently inhibit gene expression by sequence-specific RNA interference (RNAi). A common 5' leader sequence exists in the genomic RNA and all subgenomic RNAs of SARS-CoV, and is well conserved among various SARS-CoV strains, thus providing a preferable target for RNAi of SARS-CoV replication. Here efficient depletion of the SARS-CoV mRNAs by either a synthetic siRNA or DNA vector-derived short hairpin RNAs (shRNAs) targeting the leader sequence in mammalian cell lines were reported. The siRNA or shRNAs efficiently suppressed the expression of an EGFP reporter gene which contains the leader sequence at the 5' end. Both the siRNA and shRNAs efficiently knocked down the levels of leader-containing transcripts of three SARS-CoV genes encoding the spike protein, membrane protein and nucleocapsid protein were demonstrated. The results suggest that RNAi targeting the leader sequence is a potential efficient strategy for anti-SARS-CoV therapy.

To gain insights into the function of potential post-translational modifications on the activity of the Cucumber mosaic virus (CMV)-encoded silencing suppressor protein 2b, one predicted phosphorylation site (S40) and two predicted ubiquitination/sumoylation sites (K22 and K39) in CMV-Q2b protein were individually or simultaneously mutated by site-directed mutagenesis methods. These Q2b mutants were inserted into plant expression vectors, expressed in plant leaves, and then analyzed for their silencing suppressor activities. The results showed that S40A mutation greatly impaired both the local and systemic silencing suppressor activity, and the K22R mutation has no significant effect on the suppressor activity, while the K22R/K39R double mutation reduced the systemic silencing suppressor activity. To test if the decrease of suppressor activity were due to protein accumulation changes, western blot were performed to monitor the protein level of Q2b mutants. The results indicated that mutations of both K22 and K39 to R or S40 to A all significantly reduced the accumulation of the Q2b protein in plants, while the single mutation of K22 to R did not alter the accumulation of Q2b protein, suggesting that two potential post-translational modification sites, K39 and S40, contribute to the suppressor activity and stability of 2b protein in plant cells.
Cucumovirus ; genetics ; metabolism ; Gene Expression Regulation, Plant ; Gene Silencing ; Mutagenesis, Site-Directed ; Mutation ; Protein Processing, Post-Translational ; Protein Stability ; Viral Proteins ; genetics

Phospholipase Ds (PLDs) exist in many plants. PLDs catalyse the hydrolysis of phospholipids (e.g. phosphatidylcholine) in cell membrane into phosphatidic acid (PA) and polar free heads (e.g. choline). Two PLD members from rice, OsPLD3 and OsPLD4, were studied by reverse genetics approaches. The results showed that the promoters of OsPLD3 and OsPLD4 could drive the expression of the reporter gene in various tissues of the rice flower organs at different levels. The expression of both genes was induced by wounding and methyl jasmonate (MeJA), but with different intensity at different time intervals. No prominent phenotypes were observed by RNA interference with the gene-specific artificial miRNAs or over-expression of the target genes in rice plants, implying the functional redundancy among different members of the rice PLD family.
Acetates ; pharmacology ; Base Sequence ; Cyclopentanes ; pharmacology ; Flowers ; genetics ; metabolism ; Genes, Plant ; genetics ; Genes, Reporter ; genetics ; Molecular Sequence Data ; Oryza ; enzymology ; genetics ; Oxylipins ; pharmacology ; Phospholipase D ; genetics ; metabolism ; Promoter Regions, Genetic ; genetics

The preliminary role of calcineurin B-like protein-interacting protein kinases (CIPKs) in stress response is defined but the exact function of OsCIPK10 gene in rice stress response and its expression pattern yet unclear. In this study we explored the possible functions of OsCIPK10 gene by reverse genetics approaches and also revealed its expression pattern by GUS staining. From the preliminary study of this gene we presumed its function to assist plant to resist stress but over-expressed OsCIPK10 rice transgenic lines showed no significant phenotypic differences from the wild type either under high salt or low potassium conditions, however the gene knockdown plants using inverted repeat strategy presented meaningful healthy plants compared to wild type under the stress of salt. Further we checked the expression profile under high salt and low potassium conditions in wild type and found that OsCIPK10 decreases under high salt and increases on low potassium conditions. So we speculate that OsCIPK10 is actually going to function in response to high salt and low potassium stress. We also explored the expression pattern of this gene using Gus staining and found that gene expresses in all plant tissues, the only exception observed was its higher expression in the vascular tissues.
Cloning, Molecular ; Genes, Plant ; Oryza ; enzymology ; genetics ; Plant Proteins ; genetics ; metabolism ; Plants, Genetically Modified ; genetics ; metabolism ; Potassium ; pharmacology ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; RNA Interference ; RNA, Small Interfering ; genetics ; Salt-Tolerant Plants ; genetics ; Sodium Chloride ; pharmacology ; Stress, Physiological

Gene expression in rice roots under nutritional stress was studied using micro array techniques. The results showed that when re-supplied with sufficient amounts of nutrition after nutrition stress, the expression of OsPra2 (a small G protein which is homologous with Pea Pra2 protein) decreased in the plants root tissue. The cDNA sequence of the OsPra2 gene and its promoter, which is about 1 kb upstream of the translation origin point, was obtained using RT-PCR and PCR approaches. The OsPra2 protein contains four conserved GTP/GDP binding domains and specific domain of Rab small G protein family. The expression of OsPra2 and GST fusion protein in onion epidermal cells showed that OsPra2 protein was localized in the membrane and nucleus of the cell. The fusion expression of OsPra2 promoter and GUS reporter gene in transgenic rice suggested that the OsPra2 promoter allowed GUS expression in coleoptiles and roots. Compared with wild type rice, OsPra2 over expressed transgenic rice showed an obvious dwarf phenotype which resembles the BR deficient rice.
Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; Gene Expression Regulation, Plant ; Genes, Plant ; Molecular Sequence Data ; Oryza ; genetics ; metabolism ; Plant Proteins ; genetics ; metabolism ; Plants, Genetically Modified ; genetics ; metabolism ; Sequence Homology, Amino Acid ; rab GTP-Binding Proteins ; genetics ; metabolism

Biofilms are complex three-dimensional bacterial assemblages that attach to biotic or abiotic solid surfaces, and frequently embed within a self-produced matrix of extracellular polymeric substances. Biofilm formation is a microbial defense response to biotic and abiotic stresses, and a key factor for survival in adverse environments. A wide variety of microorganisms can colonize distant tissues of higher plants, such as leaves, vascular network and roots, and adhere to the surface of the tissues to form biofilms. The dynamic processes in forming biofilms in response to plant internal environment are key steps required for full virulence of phytopathogenic bacteria. Exploring the mechanisms involved in regulation of bacterial biofilms is important for understanding the plant-pathogens interactions. In this review, we summarized the research progresses related to the biofilms of bacterial phytopathogens, including biofilm characteristics, essential regulatory mechanisms and key signals affecting the transition between a planktonic lifestyle and multicellular behavior.