• The Plant Pathology Journal
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• v.24 no.3
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• pp.296-304
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• 2008

The transient and rapid expression system of a foreign protein in planta is a very useful technique in biotechnology application. We have investigated optimum condition of Agrobacterium-infiltration technique in which expression level of foreign proteins were maximized without detrimental effects on plants using GFP and Cucumber mosaic virus 2b protein, which is known as an enhancer of gene expression and a suppressor of post-transcriptional gene silencing(PTGS). The optimum expression level of both RNA and protein of GFP with minimum leaf impairment was obtained at $OD_{600}$=0.2 of Agrobactrium inocula. The steady-state levels of GFP RNA and protein generally peaked at 3 and 7 days post-infiltration(dpi), respectively. In the presence of 2b, both the magnitude and duration of GFP expression was highly increased and we could detect GFP level until 17 dpi. On the other hands, the 2b-mediated higher accumulation of foreign proteins resulted in the repression of normal leaf growth, possibly due to the limitation of supply of energy or materials required for growth maintenance. Using this Agrobacterium-infiltration system with 2b and GFP, we tested a hypothesis for the threshold model of PTGS initiation. Four GFP transgenic lines of N. benthamiana, which shows different expression level of GFP were tested to determine the threshold level for PTGS initiation. Agrobacterium-infiltration of GFP into those GFP-transgenic plants resulted in the co-silencing of the transgenic GFP. It was found that very low concentration of Agrobacterium with GFP and GFP+2b($OD_{600}$=0.002-0.02) which could not phenotypically induce an additive GFP expression, was enough to trigger PTGS pathway in all GFP transgenic plants. This strongly indicates that each GFP-transgenic plant should be expressing the transgenic GFP at its own pre-determined level and there was no buffer zone of additive GFP-expression to the threshold. In other words, the PTGS seems to be immediately activated as a self-defensive mechanism if an internal balance of gene expression is broken.

• Research in Plant Disease
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• v.21 no.3
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• pp.186-192
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• 2015

Cucumber mosaic virus possesses 2b gene known as a suppressor of post-transcriptional gene silencing (PTGS). To investigate its function and effect in plant, transgenic Nicotiana benethamiana expressing 2b gene was developed and analyzed in phenotypic characteristics and differential gene expression (DEG) comparing with wild-type. Eight lines of transgenic plants ($T_0$) were obtained with difficulty and showed severe deformed phenotypes in leaves, flowers, petioles and etc. Moreover, transgenic plants were hardly able to set seeds, but small amounts of seeds were barely produced in some of transgene-hemizygous plants. DEG analysis showed that transgenic plant ectopically accumulated diverse RNA transcripts at higher levels than wild-type probably due to the disturbance in RNA metabolism, especially of RNA decay, caused by 2b-mediated inhibition of PTGS. These ectopic accumulations of RNAs disrupt protein and RNA homeostasis and then subsequently lead to abnormal phenotypes of transgenic plants.

• Plant Biotechnology Reports
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• v.5 no.3
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• pp.273-281
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• 2011

Diverse epigenetic phenotypes are frequently found during research on transgenic plants. To understand the factors underlying such diversity, hundreds of independent 35S-GFP transgenic N. benthamiana plants were analyzed. The diverse GFP-expression phenotypes of the transgenic plants were classified into three major types based on the GFP expression patterns and their response to 35S-GFP agroinfiltration: steady-green, silenced and non-uniform phenotype. The non-uniform phenotype was further sub-divided into five minor phenotypes: variegated, red-dropped, on-silencing, partitioned and misty, according to the distribution of GFP expression on the leaves. Many of transgenic plants continuously generated diverse phenotypes over several generations despite the transgene identity. Such epigenetic GFP phenotyping was found to be the result of spontaneous transgene silencing mediated by either or both of post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS). This finding was verified by the detection of 21- and 24-nt small interfering RNA (siRNA) molecules, and DNA methylation in the transgenic plants that showed repeated epigenetic variation. Agroinfiltration demonstrated that irregular distribution of GFP on a leaf was the result of erratic transgene silencing, and the technique also proved to be a rapid and effective method for selecting fully silenced plants within 3 days. Furthermore, two novel phenotypes described are potential materials for in-depth investigations into the genes and mechanisms responsible for spontaneous transgene silencing.