Enhanced Transgene Expression in Sugarcane by Co-Expression of Virus-Encoded RNA Silencing Suppressors

Abstract

Post-transcriptional gene silencing is commonly observed in polyploid species and often poses a major limitation to plant improvement via biotechnology. Five plant viral suppressors of RNA silencing were evaluated for their ability to counteract gene silencing and enhance the expression of the Enhanced Yellow Fluorescent Protein (EYFP) or the β-glucuronidase (GUS) reporter gene in sugarcane, a major sugar and biomass producing polyploid. Functionality of these suppressors was first verified in Nicotiana benthamiana and onion epidermal cells, and later tested by transient expression in sugarcane young leaf segments and protoplasts. In young leaf segments co-expressing a suppressor, EYFP reached its maximum expression at 48–96 h post-DNA introduction and maintained its peak expression for a longer time compared with that in the absence of a suppressor. Among the five suppressors, Tomato bushy stunt virus-encoded P19 and Barley stripe mosaic virus-encoded γb were the most efficient. Co-expression with P19 and γb enhanced EYFP expression 4.6-fold and 3.6-fold in young leaf segments, and GUS activity 2.3-fold and 2.4-fold in protoplasts compared with those in the absence of a suppressor, respectively. In transgenic sugarcane, co-expression of GUS and P19 suppressor showed the highest accumulation of GUS levels with an average of 2.7-fold more than when GUS was expressed alone, with no detrimental phenotypic effects. The two established transient expression assays, based on young leaf segments and protoplasts, and confirmed by stable transgene expression, offer a rapid versatile system to verify the efficiency of RNA silencing suppressors that proved to be valuable in enhancing and stabilizing transgene expression in sugarcane.

Multiplexed Gene Editing and Protein Overexpression Using a Tobacco mosaic virus Viral Vector

“…benthamiana 16c plants or leaves of TRBO-G infiltrated plants were visualized under a handheld UV mercury lamp as previously described (Odokonyero et al, 2015, Everett et al, 2010. TRBO-gGFP-and RM-gGFPtreated leaf images were visualized using an Olympus SZX7 stereomicroscope with a GFP filter essentially as described previously (Gao et al, 2013).…”

A biolistic-based genetic transformation system applicable to a broad-range of sugarcane and energycane varieties

“…We have successfully used it to engineer several cane lines with improved biotic and abiotic stress tolerance, as well as to produce high-value bioproducts. [10][11][12][13][16][17][18]26 Furthermore, given the cane slow maturation and conventional labor-intensive nodal propagation method, the bioreactor-based multiplication offers a promising alternative to reduce overall time and cost of transformation.…”

“…This approach is highly amenable for transforming sugarcane, with greater reproducibility, and with lower genotype-dependent recalcitrance, when compared to the other methods. [10][11][12][13][14][15] Over the past two decades, technical advances in in vitro regeneration and plant tissue culture have allowed for further optimization of the transformation systems. [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] Our group is among the first research groups in sugarcane biolistic transformation, 22 and our transformation protocols were improved with time through their application in a variety of research efforts.…”

“…[10][11][12][13][14][15] Over the past two decades, technical advances in in vitro regeneration and plant tissue culture have allowed for further optimization of the transformation systems. [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] Our group is among the first research groups in sugarcane biolistic transformation, 22 and our transformation protocols were improved with time through their application in a variety of research efforts. [10][11][12][13][16][17][18]26 Here, we provide a comprehensive, step-by-step, biolistic-based cane transformation and micro-propagation system valuable for the cane community and applicable to a broad-range of commercial sugarcane and energycane varieties.…”

Sugarcane biotechnology: tapping unlimited potential

“…In addition to research on gene promoters in sugarcane, molecular techniques exploiting enhancers of gene expression (Kinkema et al 2014b, Rathus et al 1993, codon optimization of transgenes (Kinkema et al, 2014b), transgene silencing suppression (Gao et al 2013), and RNAi interference (Gan et al 2010, Gao et al 2013, Jung et al 2012 are additional valuable tools that may assist in improving transgene expression in sugarcane.…”