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

• Main Authors: Hwang Min, Lindenmuth Benjamin E, McDonald Karen A, Falk Bryce W
• Format: Article
• Language: English
• Published: BMC 2012-09-01
• Series: BMC Biotechnology
• Subjects: <it>Cucumber mosaic virus</it>; Protein production; Endoglucanase; <it>Agrobacterium tumefaciences</it>; Viral vector; Transient protein expression; <it>Nicotiana benthamiana</it>
• Online Access: http://www.biomedcentral.com/1472-6750/12/66

<p>Abstract</p> <p>Background</p> <p>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 <it>Acidothermus cellulolyticus</it> endo-1, 4-β-glucanase (E1) in non-transgenic plants.</p> <p>Results</p> <p>We used two new <it>Cucumber mosaic virus</it> (CMV)-based vector systems for producing the green fluorescent protein (GFP) and more importantly, the <it>Acidothermus cellulolyticus</it> endo-1, 4-β-glucanase (E1) in non-transgenic <it>Nicotiana benthamiana</it> plants. These are the inducible <it>CMVin</it> (CMV-based inducible) and the autonomously replicating <it>CMVar</it> (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 <it>CMVin</it> and <it>CMVar</it>. 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 <it>in vitro</it> 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.</p> <p>Conclusions</p> <p>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, <it>N. benthamiana,</it> which is particularly suitable for agroinfiltration, is a very good plant for transient protein production.</p>