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 (GU5) reporter gene in sugarcane,a major sugar and biomass producing polyploid.Functionality of these suppressors was first verified in Nicotiono benthomiono 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 Borley stripe mosoic 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 GU5 and P19 suppressor showed the highest accumulation of GUS levels with an average of 2.7-fold more than when GU5 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.
San-Ji Gao Veria Y. Alvarado Keerti S. Rathore Herman B. Scholthof T. Erik Mirkov Mona B. Damaj Jong-Won Park Getu Beyene Marco T. Buenrostro-Nava Joe Molina Xiaofeng Wang Jessica J. Ciomperlik Shuga A. Manabayeva
Key Laboratory of Sugarcane Biology and Genetic Breeding,Ministry of Agriculture,Fujian Agriculture Stoller Enterprises,Inc.,Norman E.Borlaug Center for Southern Crop Improvement,Texas A&M University, Laboratory for Crop Transformation,Institute for Plant Genomics and Biotechnology,Norman E.Borlaug C Department of Plant Pathology and Microbiology,Texas A&M University,College Station,Texas,United Sta Department of Plant Pathology and Microbiology,Texas A&M AgriLife Research,Weslaco,Texas,United Stat Institute for International Crop Improvement,Donald Danforth Plant Science Center,Saint Louis,Missou FCBA-Laboratorio de Biotecnologia,Universidad de Colima,Tecoman,Colima,Mexico Department of Plant Pathology,Physiology and Weed Science,Virginia Tech University,Blacksburg,Virgin National Center for Biotechnology of the Republic of Kazakhstan,Astana,Republic of Kazakhstan