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Nicotiana Benthamiana Leaves (nicotiana + benthamiana_leaf)
Selected AbstractsThe Arabidopsis class VIII myosin ATM2 is involved in endocytosisCYTOSKELETON, Issue 6 2008Amirali Sattarzadeh Abstract Members of the class XI of the myosin superfamily comprising higher plant, actin-based molecular motors have been shown to be involved in peroxisome and Golgi vesicle trafficking comparable to yeast and animal class V myosins. The tasks of the second class of myosins of higher plants, class VIII, are unclear. In this study the class VIII myosin ATM2 from the model plant Arabidopsis thaliana was selected for the examination of cargo specificity in vivo. Fluorescent protein-fusion plasmid constructs with fragments of the ATM2 cDNA were generated and used for Agrobacterium tumefaciens -based transient transformation of Nicotiana benthamiana leaves. The resulting subcellular localization patterns were recorded by live imaging with confocal laser scanning microscopy (CLSM) in epidermal leaf cells. Expression of a nearly full-length construct displayed labeling of filaments and vesicles, a head + neck fragment led to decoration of filaments only. However, expression of fluorescent protein-tagged C-terminal tail domain constructs labeled vesicular structures of different appearance. Most importantly, coexpression of different RFP/YFP-ATM2 tail fusion proteins showed colocalization and, hence, binding to the same type of vesicular target. Further coexpression experiments of RFP/YFP-ATM2 tail fusion proteins with the endosomal marker FYVE and the endosomal tracer FM4-64 demonstrated colocalization with endosomes. Colocalization was also detected by expression of the CFP-tagged membrane receptor BRI1 as marker, which is constantly recycled via endosomes. Occasionally the ATM2 tail targeted to sites at the plasma membrane closely resembling the pattern obtained upon expression of the YFP-ATM1 C-terminal tail. ATM1 is known for its localization at the plasma membrane at sites of plasmodesmata. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source] Rice cellulose synthase-like D4 is essential for normal cell-wall biosynthesis and plant growthTHE PLANT JOURNAL, Issue 6 2009Ming Li Summary Cellulose synthase-like (CSL) proteins of glycosyltransferase family 2 (GT2) are believed to be involved in the biosynthesis of cell-wall polymers. The CSL D sub-family (CSLD) is common to all plants, but the functions of CSLDs remain to be elucidated. We report here an in-depth characterization of a narrow leaf and dwarf1 (nd1) rice mutant that shows significant reduction in plant growth due to retarded cell division. Map-based cloning revealed that ND1 encodes OsCSLD4, one of five members of the CSLD sub-family in rice. OsCSLD4 is mainly expressed in tissues undergoing rapid growth. Expression of OsCSLD4 fluorescently tagged at the C- or N-terminus in rice protoplast cells or Nicotiana benthamiana leaves showed that the protein is located in the endoplasmic reticulum or Golgi vesicles. Golgi localization was verified using phenotype-rescued transgenic plants expressing OsCSLD4,GUS under the control of its own promoter. Two phenotype-altered tissues, culms and root tips, were used to investigate the specific wall defects. Immunological studies and monosaccharide compositional and glycosyl linkage analyses explored several wall compositional effects caused by disruption of OsCSLD4, including alterations in the structure of arabinoxylan and the content of cellulose and homogalacturonan, which are distinct in the monocot grass species Oryza sativa (rice). The inconsistent alterations in the two tissues and the observable structural defects in primary walls indicate that OsCSLD4 plays important roles in cell-wall formation and plant growth. [source] The conserved Xanthomonas campestris pv. vesicatoria effector protein XopX is a virulence factor and suppresses host defense in Nicotiana benthamianaTHE PLANT JOURNAL, Issue 6 2005Matthew Metz Summary Nicotiana benthamiana leaves display a visible plant cell death response when infiltrated with a high titer inoculum of the non-host pathogen, Xanthomonas campestris pv. vesicatoria (Xcv). This visual phenotype was used to identify overlapping cosmid clones from a genomic cosmid library constructed from the Xcv strain, GM98-38. Individual cosmid clones from the Xcv library were conjugated into X. campestris pv. campestris (Xcc) and exconjugants were scored for an altered visual high titer inoculation response in N. benthamiana. The molecular characterization of the cosmid clones revealed that they contained a novel gene, xopX, that encodes a 74-kDa type III secretion system (TTSS) effector protein. Agrobacterium -mediated transient expression of XopX in N. benthamiana did not elicit the plant cell death response although detectable XopX protein was produced. Interestingly, the plant cell death response occurred when the xopX Agrobacterium -mediated transient expression construct was co-inoculated with strains of either Xcv,xopX or Xcc, both lacking xopX. The co-inoculation complementation of the plant cell death response also depends on whether the Xanthomonas strains contain an active TTSS. Transgenic 35S- xopX -expressing N. benthamiana plants also have the visible plant cell death response when inoculated with the non- xopX -expressing strains Xcv,xopX and Xcc. Unexpectedly, transgenic 35S- xopX N. benthamiana plants displayed enhanced susceptibility to bacterial growth of Xcc as well as other non- xopX -expressing Xanthomonas and Pseudomonas strains. This result is also consistent with the increase in bacterial growth on wild type N. benthamiana plants observed for Xcc when XopX is expressed in trans. Furthermore, XopX contributes to the virulence of Xcv on host pepper (Capsicum annuum) and tomato (Lycopersicum esculentum) plants. We propose that the XopX bacterial effector protein targets basic innate immunity in plants, resulting in enhanced plant disease susceptibility. [source] A DNA replicon system for rapid high-level production of virus-like particles in plantsBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2009Zhong Huang Abstract Recombinant virus-like particles (VLPs) represent a safe and effective vaccine strategy. We previously described a stable transgenic plant system for inexpensive production and oral delivery of VLP vaccines. However, the relatively low-level antigen accumulation and long-time frame to produce transgenic plants are the two major roadblocks in the practical development of plant-based VLP production. In this article, we describe the optimization of geminivirus-derived DNA replicon vectors for rapid, high-yield plant-based production of VLPs. Co-delivery of bean yellow dwarf virus (BeYDV)-derived vector and Rep/RepA-supplying vector by agroinfiltration of Nicotiana benthamiana leaves resulted in efficient replicon amplification and robust protein production within 5 days. Co-expression of the P19 protein of tomato bush stunt virus, a gene silencing inhibitor, further enhanced VLP accumulation by stabilizing the mRNA. With this system, hepatitis B core antigen (HBc) and Norwalk virus capsid protein (NVCP) were produced at 0.80 and 0.34 mg/g leaf fresh weight, respectively. Sedimentation analysis and electron microscopy of transiently expressed antigens verified the efficient assembly of VLPs. Furthermore, a single replicon vector containing a built-in Rep/RepA cassette without P19 drove protein expression at similar levels as the three-component system. These results demonstrate the advantages of fast and high-level production of VLP-based vaccines using the BeYDV-derived DNA replicon system for transient expression in plants. Biotechnol. Bioeng. 2009;103: 706,714. © 2009 Wiley Periodicals, Inc. [source] Optimization of the bioprocessing conditions for scale-up of transient production of a heterologous protein in plants using a chemically inducible viral amplicon expression systemBIOTECHNOLOGY PROGRESS, Issue 3 2009Michael A. Plesha Abstract Use of transient expression for the rapid, large-scale production of recombinant proteins in plants requires optimization of existing methods to facilitate scale-up of the process. We have demonstrated that the techniques used for agroinfiltration and induction greatly impact transient production levels of heterologous protein. A Cucumber mosaic virus inducible viral amplicon (CMViva) expression system was used to transiently produce recombinant alpha-1-antitrypsin (rAAT) by co-infiltrating harvested Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains, one containing the CMViva expression cassette carrying the AAT gene and the other containing a binary vector carrying the gene silencing suppressor p19. Harvested leaves were both infiltrated and induced by either pressure or vacuum infiltration. Using the vacuum technique for both processes, maximum levels of functional and total rAAT were elevated by (190 ± 8.7)% and (290 ± 7.5)%, respectively, over levels achieved when using the pressure technique for both processes. The bioprocessing conditions for vacuum infiltration and induction were optimized and resulted in maximum rAAT production when using an A. tumefaciens concentration at OD600 of 0.5 and a 0.25-min vacuum infiltration, and multiple 1-min vacuum inductions further increased production 25% and resulted in maximum levels of functional and total rAAT at (2.6 ± 0.09)% and (4.1 ± 0.29)% of the total soluble protein, respectively, or (90 ± 1.7) and (140 ± 10) mg per kg fresh weight leaf tissue at 6 days post-induction. Use of harvested plant tissue with vacuum infiltration and induction demonstrates a bioprocessing route that is fully amenable to scale-up. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] High-Level Transient Production of a Heterologous Protein in Plants by Optimizing Induction of a Chemically Inducible Viral Amplicon Expression SystemBIOTECHNOLOGY PROGRESS, Issue 6 2007Michael A. Plesha We have demonstrated that the method of chemical induction using a chemically inducible viral amplicon expression system can be optimized to increase expression of a heterologous protein in plants. A cucumber mosaic virus inducible viral amplicon (CMViva) expression system was used to transiently produce a recombinant human blood protein, ,-1-antitrypsin (AAT), by co-infiltrating intact and detached Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains, one containing the CMViva expression cassette carrying the AAT gene and the other containing a binary vector carrying the gene silencing suppressor p19. Infiltrated plants were induced by either topical applications or pressure injections and inducer was applied at either a single or multiple time points. Applying induction solution every 2 days via topical application resulted in increasing maximum levels of biologically functional rAAT from 0.71% to 1.3% of the total soluble protein (TSP) in detached plant leaves, a 1.8-fold improvement. Multiple applications of induction solution via pressure injection into intact leaves resulted in maximum levels of biologically functional rAAT being elevated 3-fold up to 2.4% of TSP compared to 0.8% of TSP when using the conventional method of a single topical application, and expression levels remained high 6 days post-induction. Overall production of rAAT in intact leaves was found to have a maximum level of 5.8% of TSP or 390 mg rAAT per kg leaf tissue when applying multiple injections of chemical induction solution. [source] | ||||||||||