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Background  Using plant viruses to produce desirable proteins in plants allows for using non-transgenic plant hosts and if necessary, the ability to make rapid changes in the virus construct for increased or modified protein product yields. The objective of this work was the development of advanced CMV-based protein production systems to produce  Acidothermus cellulolyticus  endo-1, 4-β-glucanase (E1) in non-transgenic plants.    Results  We used two new  Cucumber mosaic virus  (CMV)-based vector systems for producing the green fluorescent protein (GFP) and more importantly, the  Acidothermus cellulolyticus  endo-1, 4-β-glucanase (E1) in non-transgenic  Nicotiana benthamiana  plants. These are the inducible  CMVin  (CMV-based inducible) and the autonomously replicating  CMVar  (CMV-based advanced replicating) systems. We modified a binary plasmid containing the complete CMV RNA 3 cDNA to facilitate insertion of desired sequences, and to give modifications of the subgenomic mRNA 4 leader sequence yielding several variants. Quantitative RT-PCR and immunoblot analysis showed good levels of CMV RNA and coat protein accumulation for some variants of both  CMVin  and  CMVar . When genes for E1 or GFP were inserted in place of the CMV coat protein, both were produced in plants as shown by fluorescence (GFP) and immunoblot analysis. Enzymatic activity assays showed that active E1 was produced in plants with yields up to ~ 11 μg/g fresh weight (FW) for specific variant constructs. We also compared  in vitro  CMV genomic RNA reassortants, and CMV RNA 3 mutants which lacked the C’ terminal 33 amino acids of the 3A movement protein in attempts to further increase E1 yield. Taken together specific variant constructs yielded up to ~21 μg/g FW of E1 in non-transgenic plants.    Conclusions  Intact, active E1 was rapidly produced in non-transgenic plants by using agroinfiltration with the CMV-based systems. This reduces the time and cost compared to that required to generate transgenic plants and still gives the comparable yields of active E1. Our modifications described here, including manipulating cloning sites for foreign gene introduction, enhance the ease of use. Also,  N. benthamiana,  which is particularly suitable for agroinfiltration, is a very good plant for transient protein production.

bmc biotechnology

Bipartite and tripartite Cucumber mosaic virus-based vectors for producing the Acidothermus cellulolyticus endo-1,4-β-glucanase and other proteins in non-transgenic plants