title: High rates of virus-induced gene silencing by tobacco rattle virus in Populus.
abstract: Virus-induced gene silencing (VIGS) has been shown to be an effective tool for investigating gene functions in herbaceous plant species, but has rarely been tested in trees. The establishment of a fast and reliable transformation system is especially important for woody plants, many of which are recalcitrant to transformation. In this study, we established a tobacco rattle virus (TRV)-based VIGS system for two Populus species, Populus euphratica and P. × canescens. Here, TRV constructs carrying a 266 bp or a 558 bp fragment of the phytoene desaturase (PDS) gene were Agrobacterium-infiltrated into leaves of the two poplar species. Agrobacterium-mediated delivery of the shorter insert, TRV2-PePDS266, into the host poplars resulted in expected photobleaching in both tree species, but not the longer insert, PePDS558. The efficiency of VIGS was temperature-dependent, increasing by raising the temperature from 18 to 28 °C. The optimized TRV-VIGS system at 28 °C resulted in a high silencing frequency and efficiency up to 65-73 and 83-94%, respectively, in the two tested poplars. Moreover, syringe inoculation of Agrobacterium in 100 mM acetosyringone induced a more efficient silencing in the two poplar species, compared with other agroinfiltration methods, e.g., direct injection, misting and agrodrench. There were plant species-related differences in the response to VIGS because the photobleaching symptoms were more severe in P. × canescens than in P. euphratica. Furthermore, VIGS-treated P. euphratica exhibited a higher recovery rate (50%) after several weeks of the virus infection, compared with TRV-infected P. × canescens plants (20%). Expression stability of reference genes was screened to assess the relative abundance of PePDS mRNA in VIGS-treated P. euphratica and P. × canescens. PeACT7 was stably expressed in P. euphratica and UBQ-L was selected as the most suitable reference gene for P. × canescens using three different statistical approaches, geNorm, NormFinder and BestKeeper. Quantitative real-time PCR showed significant reductions in PDS transcripts (55-64%) in the photobleached leaves of both VIGS-treated poplar species. Our results demonstrate that the TRV-based VIGS provides a practical tool for gene functional analysis in Populus sp., especially in those poplar species which are otherwise recalcitrant to transformation.
authors.affiliation: College of Biological Sciences and Technology (Box 162), Beijing Forestry University, Beijing 100083, P.R. China.
authors.forename: Zedan
authors.initials: Z
authors.lastname: Shen
authors.affiliation: College of Life Science, Jiangsu Normal University, Xuzhou, Jiangsu Province 221116, P.R. China.
authors.forename: Jian
authors.initials: J
authors.lastname: Sun
authors.affiliation: College of Biological Sciences and Technology (Box 162), Beijing Forestry University, Beijing 100083, P.R. China.
authors.forename: Jun
authors.initials: J
authors.lastname: Yao
authors.affiliation: College of Biological Sciences and Technology (Box 162), Beijing Forestry University, Beijing 100083, P.R. China.
authors.forename: Shaojie
authors.initials: S
authors.lastname: Wang
authors.affiliation: College of Biological Sciences and Technology (Box 162), Beijing Forestry University, Beijing 100083, P.R. China.
authors.forename: Mingquan
authors.initials: M
authors.lastname: Ding
authors.affiliation: College of Biological Sciences and Technology (Box 162), Beijing Forestry University, Beijing 100083, P.R. China.
authors.forename: Huilong
authors.initials: H
authors.lastname: Zhang
authors.affiliation: College of Biological Sciences and Technology (Box 162), Beijing Forestry University, Beijing 100083, P.R. China.
authors.forename: Zeyong
authors.initials: Z
authors.lastname: Qian
authors.affiliation: College of Biological Sciences and Technology (Box 162), Beijing Forestry University, Beijing 100083, P.R. China.
authors.forename: Nan
authors.initials: N
authors.lastname: Zhao
authors.affiliation: College of Biological Sciences and Technology (Box 162), Beijing Forestry University, Beijing 100083, P.R. China.
authors.forename: Gang
authors.initials: G
authors.lastname: Sa
authors.affiliation: College of Biological Sciences and Technology (Box 162), Beijing Forestry University, Beijing 100083, P.R. China.
authors.forename: Rui
authors.initials: R
authors.lastname: Zhao
authors.affiliation: College of Biological Sciences and Technology (Box 162), Beijing Forestry University, Beijing 100083, P.R. China.
authors.forename: Xin
authors.initials: X
authors.lastname: Shen
authors.affiliation: Forstbotanik und Baumphysiologie, Büsgen-Institut, Georg-August Universität Göttingen, Göttingen 37077, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Polle
authors.affiliation: College of Biological Sciences and Technology (Box 162), Beijing Forestry University, Beijing 100083, P.R. China lschen@bjfu.edu.cn.
authors.forename: Shaoliang
authors.initials: S
authors.lastname: Chen
mesh_terms: D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing; D017343:Genes, Plant; D005822:Genetic Vectors / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics; D010641:Phenotype; D038761:Photobleaching; D010935:Plant Diseases / Q000821:virology; D010942:Plant Viruses / Q000502:physiology*; D032107:Populus / Q000235:genetics / Q000821:virology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012015:Reference Standards
chemical_list: D012333:RNA, Messenger; D010088:Oxidoreductases; C022398:phytoene dehydrogenase
keywords: Agrobacterium infiltration; Populus euphratica; Populus × canescens; leaf photobleaching; phytoene desaturase; silencing efficiency; silencing frequency; tobacco rattle virus