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Transient co-expression of post-transcriptional gene silencing suppressors for increased in planta expression of a recombinant anthrax receptor fusion protein.

ABSTRACT: Potential epidemics of infectious diseases and the constant threat of bioterrorism demand rapid, scalable, and cost-efficient manufacturing of therapeutic proteins. Molecular farming of tobacco plants provides an alternative for the recombinant production of therapeutics. We have developed a transient production platform that uses Agrobacterium infiltration of Nicotiana benthamiana plants to express a novel anthrax receptor decoy protein (immunoadhesin), CMG2-Fc. This chimeric fusion protein, designed to protect against the deadly anthrax toxins, is composed of the von Willebrand factor A (VWA) domain of human capillary morphogenesis 2 (CMG2), an effective anthrax toxin receptor, and the Fc region of human immunoglobulin G (IgG). We evaluated, in N. benthamiana intact plants and detached leaves, the expression of CMG2-Fc under the control of the constitutive CaMV 35S promoter, and the co-expression of CMG2-Fc with nine different viral suppressors of post-transcriptional gene silencing (PTGS): p1, p10, p19, p21, p24, p25, p38, 2b, and HCPro. Overall, transient CMG2-Fc expression was higher on intact plants than detached leaves. Maximum expression was observed with p1 co-expression at 3.5 days post-infiltration (DPI), with a level of 0.56 g CMG2-Fc per kg of leaf fresh weight and 1.5% of the total soluble protein, a ten-fold increase in expression when compared to absence of suppression. Co-expression with the p25 PTGS suppressor also significantly increased the CMG2-Fc expression level after just 3.5 DPI.

SUBMITTER: Arzola L

PROVIDER: S-EPMC3179146 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Transient co-expression of post-transcriptional gene silencing suppressors for increased in planta expression of a recombinant anthrax receptor fusion protein.

Arzola Lucas L Chen Junxing J Rattanaporn Kittipong K Maclean James M JM McDonald Karen A KA

International journal of molecular sciences 20110805 8

Potential epidemics of infectious diseases and the constant threat of bioterrorism demand rapid, scalable, and cost-efficient manufacturing of therapeutic proteins. Molecular farming of tobacco plants provides an alternative for the recombinant production of therapeutics. We have developed a transient production platform that uses Agrobacterium infiltration of Nicotiana benthamiana plants to express a novel anthrax receptor decoy protein (immunoadhesin), CMG2-Fc. This chimeric fusion protein, de ...[more]

PMID: 21954339

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Project description:BackgroundLocalized introduction and transient expression of T-DNA constructs mediated by agro-infiltration of leaf tissues has been largely used in dicot plants for analyzing the transitivity and the cell-to cell movement of the RNAi signal. In cereals, however, the morphology of the leaf and particularly the structure of the leaf epidermis, prevent infiltration of a bacterial suspension in cells by simple pressure, a method otherwise successful in dicots leaves. This study aimed at establishing a rapid method for the functional analysis of rice genes based on the triggering of RNA interference (RNAi) following Agrobacterium-mediated transient transformation of leaves.ResultsUsing an agro-infection protocol combining a wound treatment and a surfactant, we were able to obtain in a reliable manner transient expression of a T-DNA-borne uidA gene in leaf cells of japonica and indica rice cultivars. Using this protocol to transiently inhibit gene expression in leaf cells, we introduced hairpin RNA (hpRNA) T-DNA constructs containing gene specific tags of the phytoene desaturase (OsPDS) and of the SLENDER 1 (OsSLR1) genes previously proven to trigger RNAi of target genes in stable transformants. SiRNA accumulation was observed in the agro-infected leaf area for both constructs indicating successful triggering of the silencing signal. Accumulation of secondary siRNA was observed in both stably and transiently transformed leaf tissues expressing the HpRNA OsSLR1 construct. Gene silencing signalling was investigated in monitoring the parallel time course of OsPDS-derived mRNA and siRNA accumulation in the agro-infiltrated leaf area and adjacent systemic sectors. The sensitive RT-Q-PCR method evidenced a consistent, parallel decrease of OsPDS transcripts in both the agroinfiltred and adjacent tissues, with a time lag for the latter.ConclusionsThese results indicate that the method is efficient at inducing gene silencing in the agro-infected leaf area. The transfer of low amounts of siRNA, probably occurring passively through the symplastic pathway from the agro-infected area, seemed sufficient to trigger degradation of target transcripts in the adjacent tissues. This method is therefore well suited to study the cell-to-cell movement of the silencing signal in a monocot plant and further test the functionality of natural and artificial miRNA expression constructs.

Project description:BackgroundMg chelatase is a multi-subunit enzyme that catalyses the first committed step of chlorophyll biosynthesis. Studies in higher plants and algae indicate that the Mg chelatase reaction product, Mg-protoporphyrin IX plays an essential role in nuclear-plastid interactions. A number of Mg chelatase mutants have been isolated from higher plants, including semi-dominant alleles of ChlI, the gene encoding the I subunit of the enzyme. To investigate the function of higher plant CHLI, bacterial orthologues have been engineered to carry analogous amino acid substitutions to the higher plant mutations and the phenotypes examined through in vitro characterization of heterologously produced proteins. Here, we demonstrate the utility of a transient expression system in Nicotiana benthamiana for rapidly assaying mutant variants of the maize CHLI protein in vivo.ResultsTransient expression of mutant maize ChlI alleles in N. benthamiana resulted in the formation of chlorotic lesions within 4 d of inoculation. Immunoblot analyses confirmed the accumulation of maize CHLI protein suggesting that the chlorosis observed resulted from an interaction between maize CHLI and endogenous components of the N. benthamiana chlorophyll biosynthetic pathway. On the basis of this assay, PCR-based cloning techniques were used to rapidly recombine polymorphisms present in the alleles studied allowing confirmation of causative lesions. A PCR-based mutagenesis was conducted and clones assayed by transient expression. A number of novel allelic variants of maize ZmChlI were generated and analyzed using this assay, demonstrating the utility of this technique for fine mapping.ConclusionTransient expression provides a convenient, high-throughput, qualitative assay for functional variation in the CHLI protein. Furthermore, we suggest that the approach used here would be applicable to the analysis of other plastid-localized proteins where gain-of-function mutations will result in readily observable mutant phenotypes.

Dataset Information

Transient co-expression of post-transcriptional gene silencing suppressors for increased in planta expression of a recombinant anthrax receptor fusion protein.

Publications

Transient co-expression of post-transcriptional gene silencing suppressors for increased in planta expression of a recombinant anthrax receptor fusion protein.

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