Characterization of resistance mechanism in transgenic Nicotiana benthamiana containing Turnip crinkle virus coat protein.
Two transgenic lines, of Nicotiana benthamiana expressing Turnip crinkle virus (TCV)-coat protein (CP) gene with contrasting phenotype, the highest (#3) and the lowest (#18) CP expressers, were selected and challenged with the homologous TCV. The former, the highest expresser, showed nearly five times more CP expression than the latter. Progenies of #3 and #18 lines showed 30 and 100% infection rates, respectively. The infected progenies of #3 line showed mild and delayed symptom with TCV. This is a coat protein-mediated resistance (CP-MR), and its resistance level is directly proportional to CP transgene expression. However, CP-MR of the transgenic plants was specific only for TCV but not for heterologous viruses. Newly growing leaves of those infected progenies of #3 line did not show any visible symptoms at 4-week post-inoculation (wpi) with TCV, suggesting a reversal from infection. This was confirmed by RT-PCR analysis with the disappearance of the target at 4 wpi. This is a case of RNA-mediated resistance, and a threshold level of transgene expression may be needed to achieve the silent state. To confirm the RNA silencing, we infiltrated Agrobacterium carrying TCV-CP into leaves of progenies of #3 and performed RT-PCR analysis. The results indicate that TCV-CP's suppressor activity against RNA silencing itself can be silenced by the homologous expression of TCV-CP in the transgenic plants. The transgenic plants containing TCV-CP seem to be a model system to study viral protection mediated by a combination of protein and RNA silencing.
Vasudevan
Ayyappan
A
Department of Biology and Medicinal Science, Pai Chai University, Daejeon, South Korea.
Oh
Tae-Kyun
TK
Park
Jae Sung
JS
Lakshmi
Sumitra Vijayachandran
SV
Choi
Bong Kum
BK
Kim
Sue Hoon
SH
Lee
Hyun Ju
HJ
Ji
Jing
J
Kim
Joo-Hwan
JH
Ganapathi
Andy
A
Kim
Sei Chang
SC
Choi
Chang Won
CW
D001483:Base Sequence; D015153:Blotting, Western; D036022:Capsid Proteins / Q000235:genetics*; D019184:Carmovirus / Q000235:genetics*; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014026:Nicotiana / Q000235:genetics* / Q000821:virology; D014158:Transcription, Genetic
D036022:Capsid Proteins; D017931:DNA Primers