title: An endogene-resembling transgene is resistant to DNA methylation and systemic silencing.
abstract: In plants, endogenes are less prone to RNA silencing than transgenes. While both can be efficiently targeted by small RNAs for post-transcriptional gene silencing (PTGS), generally only transgene PTGS is accompanied by transitivity, RNA-directed DNA methylation (RdDM) and systemic silencing. In order to investigate whether a transgene could mimick an endogene and thus be less susceptible to RNA silencing, we generated an intron-containing, endogene-resembling GREEN FLUORESCENT PROTEIN (GFP) transgene (GFP(endo)). Upon agroinfiltration of a hairpin GFP (hpF) construct, transgenic Nicotiana benthamiana plants harboring GFP(endo) (Nb-GFP(endo)) were susceptible to local PTGS. Yet, in the local area, PTGS was not accompanied by RdDM of the GFP(endo) coding region. Importantly, hpF-agroinfiltrated Nb-GFP(endo) plants were resistant to systemic silencing. For reasons of comparison, transgenic N. benthamiana plants (Nb-GFP(cDNA)) carrying a GFP cDNA transgene (GFP(cDNA)) were included in the analysis. HpF-agroinfiltrated Nb-GFP(cDNA) plants exhibited local PTGS and RdDM. In addition, systemic silencing was established in Nb-GFP(cDNA) plants. In agreement with previous reports using grafted scions, in systemically silenced tissue, siRNAs mapping to the 3' of GFP were predominantly detectable by Northern blot analysis. Yet, in contrast to other reports, in systemically silenced leaves, PTGS was also accompanied by dense RdDM comprising the entire GFP(cDNA) coding region. Overall, our analysis indicated that cDNA transgenes are prone to systemic PTGS and RdDM, while endogene-resembling ones are resistant to RNA silencing.
authors.affiliation: RLP AgroScience GmbH; AlPlanta-Institute for Plant Research; Neustadt, Germany; These authors contributed equally to this work.
authors.forename: Elena
authors.initials: E
authors.lastname: Dadami
authors.affiliation: RLP AgroScience GmbH; AlPlanta-Institute for Plant Research; Neustadt, Germany; These authors contributed equally to this work.
authors.forename: Athanasios
authors.initials: A
authors.lastname: Dalakouras
authors.affiliation: RLP AgroScience GmbH; AlPlanta-Institute for Plant Research; Neustadt, Germany.
authors.forename: Michele
authors.initials: M
authors.lastname: Zwiebel
authors.affiliation: RLP AgroScience GmbH; AlPlanta-Institute for Plant Research; Neustadt, Germany.
authors.forename: Gabi
authors.initials: G
authors.lastname: Krczal
authors.affiliation: RLP AgroScience GmbH; AlPlanta-Institute for Plant Research; Neustadt, Germany; Centre for Organismal Studies (COS) Heidelberg; University of Heidelberg; Heidelberg, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Wassenegger
mesh_terms: D019175:DNA Methylation; D020868:Gene Silencing; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007438:Introns; D018515:Plant Leaves / Q000235:genetics / Q000254:growth & development*; D010940:Plant Proteins / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics / Q000254:growth & development; D014026:Nicotiana / Q000235:genetics* / Q000254:growth & development; D019076:Transgenes
chemical_list: D010940:Plant Proteins; D049452:Green Fluorescent Proteins