Transgene silencing in grapevines transformed with GFLV resistance genes: analysis of variable expression of transgene, siRNAs production and cytosine methylation.
Eight transgenic grapevine lines transformed with the coat protein gene of Grapevine fanleaf virus (GFLV-CP) were analyzed for a correlation between transgene expression, siRNAs production and DNA methylation. Bisulphite genome sequencing was used for a comprehensive analysis of DNA methylation. Methylated cytosine residues of CpG and CpNpG sites were detected in the GFLV-CP transgene, in the T7 terminator and in the 35S promoter of three grapevines without transgene expression, but no detectable level of siRNAs was recorded in these lines. The detailed analysis of 8 lines revealed the complex arrangements of T-DNA and integrated binary vector sequences as crucial factors that influence transgene expression. After inoculation with GFLV, no change in the levels of cytosine methylation was observed, but transgenic and untransformed plants produced short siRNAs (21-22 nt) indicating that the grapevine plants responded to GFLV infection by activating a post-transcriptional gene silencing mechanism.
Gambino
Giorgio
G
Plant Virology Institute CNR (IVV), Grugliasco Unit., 10095 Grugliasco-TO, Italy. g.gambino@ivv.cnr.it
Perrone
Irene
I
Carra
Andrea
A
Chitarra
Walter
W
Boccacci
Paolo
P
Torello Marinoni
Daniela
D
Barberis
Marco
M
Maghuly
Fatemeh
F
Laimer
Margit
M
Gribaudo
Ivana
I
D003596:Cytosine / Q000378:metabolism*; D019175:DNA Methylation / Q000235:genetics; D015257:DNA-Cytosine Methylases / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing / Q000502:physiology*; D007113:Immunity, Innate / Q000235:genetics*; D010935:Plant Diseases / Q000235:genetics / Q000276:immunology / Q000821:virology; D010942:Plant Viruses / Q000276:immunology*; D030821:Plants, Genetically Modified; D034741:RNA, Small Interfering / Q000096:biosynthesis*; D014170:Transformation, Genetic / Q000502:physiology; D019076:Transgenes / Q000235:genetics / Q000502:physiology*; D027843:Vitis / Q000235:genetics* / Q000276:immunology / Q000378:metabolism / Q000821:virology
D034741:RNA, Small Interfering; D003596:Cytosine; D015257:DNA-Cytosine Methylases