Optimization of transient Agrobacterium-mediated gene expression system in leaves of Nicotioana benthamiana

Here we report on a simple and reproducible system of Agrobacterium-mediated transient gene expression assay that utilizes infiltration of young Nicotiana benthamiana leaves. Although some of the phenomena described in this paper have been already reported by other researchers, here we have further developed them. The highest level of transient gfp gene expression was detected in the youngest leaves of N. benthamiana infiltrated with A. tumefaciens strains AGL0 and EHA105 precultured in the presence of 450-600 microM acetosyringone. Although the maximum level of transient gfp gene expression was restricted presumably by RNA silencing, it was completely suppressed in the presence of the viral protein HC-Pro. The transient expression system described here can be used to identify new viral suppressors of RNA silencing, for detailed analysis of unidentified genes and for industrial production of proteins in plants as well.


INTROduCTION
Agrobacterium-mediated transient gene expression system in intact plant leaves is a rapid and useful method of analysis of gene expression.By this method many different heterologous proteins can be produced without the need to generate transgenic plants, which is difficult for many plant species (Fischer et al., 1999;2004;Horn et al., 2004).The Agrobacterium-mediated transient gene expression system is often used for identification of new viral suppressors of RNA silencing and for functional analysis of unidentified genes (Qu et al., 2003;Thomas et al., 2003).Transient gene expression system offers a number of advantages over stable expression.One of the most important advantages of the transient gene expression technique as compared to the stable gene expression is its simplicity and easy per-formance.Transient gene expression can be assayed directly post-agroinfiltration (Kapila et al., 1997).
Usually Agrobacterium-mediated transient expression of a transgene achieves the highest level 2-3 days following argoinfiltration, after which the expression level decreases rapidly.This observation can be explained by triggering of local RNA silencing which blocks expression of transgenes (Johansen & Carrington, 2001;Voinnet et al., 2003).RNA silencing is one of the major mechanisms of defense against viruses and other nucleic acid invaders in plants (Dougherty & Parks, 1995;Lindbo et al., 1993;Smith et al., 1994;English et al., 1996;Baulcombe, 1996;2004;Al-Kaff et al., 1998;Tenllado et al., 2004).Specific viral proteins able to suppress the RNA silencing mechanism have been identified in many plant viruses (Roth et al., 2004).The plant viral suppressor proteins are expected to play im-portant roles in the study of the mechanism of RNA silencing and as a tool for industrial production of proteins in plants.The molecular mechanism of suppression of RNA silencing has been fully described only for two viral suppressors: protein p21 encoded by the beet western yellow virus (Reed et al., 2003) and p19 encoded by the tomato bushy stunt virus (TBSV) (Lakatos et al., 2004).Both proteins (p21 and p19) bind to siRNA targeted to viral RNA and block siRNA interaction with its target -the specific viral RNA (Baulcombe & Molnár, 2004).A potyviral protein called helper component proteinase (HC-Pro) was one of the first identified suppressors of RNA silencing (Kasschau & Carrington, 1998;Anandalakshmi et al., 1998).In contrast to the p21 and p19 suppressors, the molecular mechanism of HC-Pro activity is yet to be described.Many transient and stable transformation approaches have been used to explain the HC-Pro suppression of RNA silencing.HC-Pro influences the siRNA metabolism and alters the accumulation of various classes of siRNA (Mallory et al., 2002;Hamilton et al., 2002).
In this paper, we report on a modified Agrobacterium-mediated transient expression analysis based on suppression of gene silencing (Voinnet et al., 2003) by efficient pre-induction of Agrobacterium tumefaciens vir genes.The green fluorescent protein (gfp) gene was used as a sensitive and non-invasive marker to visualize transient gene expression in transformed cells.This transient expression system can be used for identification of new viral suppressors of RNA silencing, for detailed analysis of unidentified genes and for industrial production of proteins in plants as well.

MATERIALs ANd METHOds
Plant material.Wild-type Nicotiana benthamiana plants were grown under standard greenhouse conditions.For all assays 5-to 8-week-old N. benthamiana plants were used.
Bacterial strains and binary plasmids.Agrobacterium tumefaciens strains EHA105, LBA4404, AGL0 and AGL1 harboring binary vectors were used for transient transformation.Plasmid pCAMBIA(gfp)1302 (Center for the Application of Molecular Biology of International Agriculture, Canberra, Australia) contains hygromycin phosphotransferase (hptII) gene and a modified green fluorescent protein (mgfp5) gene.Plasmid pBINmgfp5-ER (Haseloff et al., 1997) was kindly provided by Jacek Augustyniak (Adam Mickiewicz University, Poznań, Poland).All constructs used in this study are listed in Fig. 1.
Construction of suppressor-vectors.Total RNA was extracted with TRIZOL (GibcoBRL) from leaves infected with TuMV or PVY viruses and RT-PCR reactions were carried out.The incubation mixture for the reverse transcription reaction was based on the method given in the GeneAmp RNA PCR kit (Roche).In our routine experiments we used 50 units of reverse transcriptase (M-MLV), 25 µM random primer and 1 µg of total RNA as a template in the incubation mixture to synthesize the first-strand cDNA.After incubation at 99 o C for 5 min, 2.5 units of AmpliTaq DNA polymerase and two primers were added to the reverse transcriptase mixture and then the PCR reaction was performed.To obtain the P1 and HC-Pro genes for preparation of the construct pJR1Ri-P1/HC-Pro(TuMV), the following two primers were used: NPL50 5' AATCGGTAC-CAACAATGGCAGCAGTAACATTCGCA 3' and NPL43ASC 5' ATCATCATCATCGTCGACTCATC-CGACGCGGTAGTGTTTCAAGC 3' with sequences corresponding to the P1 and HC-Pro coding region of TuMV virus.For preparation of the construct pJR1Ri-HC-Pro(TuMV) the following two primers were used: NPL45A 5' ATCGGTACCAACAAT-GAGTGCGGCAGGAGCCAATTTCTGG 3' and Optimization of transient Agrobacterium-mediated gene expression system NPL43ASC 5' ATCATCATCATCGTCGACTCATC-CGACGCGGTAGTGTTTCAAGC 3' with sequences corresponding to HC-Pro of TuMV virus.For preparation of the construct pJR1Ri-HC-Pro(PVY) the following two primers were used: NPL50B 5' ATCGGTACCAACAATGGCCAGCTTGCCAGT-TAGCGATCTG 3' and NPL43BSC 5' ATCATCAT-CATCGTCGACTCATATGCCAGGGGATAGTATT-GATA 3'.All the cloning steps were carried out using standard molecular biology protocols (Sambrook & Russell, 2001) (Kozak, 1984).These constructs (Fig. 1) were used to suppress the silencing during transient expressing of the gfp gene.All constructs were mobilized from Escherichia coli DH5α into Agrobacterium tumefaciens (four strains) containing helper plasmids using direct transformation of Agrobacterium. A. tumefaciens colonies capable of growing in a kanamycin-containing medium were selected for use in these studies.Preparation of Agrobacterium cultures for agroinfiltration.The constructs used in the present study (pBINmgfp5-ER, pCAMBIA(gfp)1302, pJR1Ri-P1/HC-Pro(TuMV), pJR1Ri-HC-Pro(PVY), pJR1Ri-HC-Pro(TuMV)) were introduced into A. tumefaciens strain EHA105 (unless stated otherwise) by direct transformation.Recombinant A. tumefaciens were grown overnight at 28 o C in 100 ml conical flask containing 10 ml of LB medium supplemented with 50 μg kanamycin per ml.Aliquote of 50 μl of this overnight culture was used for inoculation of 10 ml of LB medium supplemented with 10 mM MES buffer, pH 5.7, 50 μg kanamycin per ml and 150 μM acetosyringone (3,5-dimethoxy-4'-hydroxy-acetophenone) (Voinnet et al., 2003).The precultures were grown overnight at 28 o C in a shaker.Cells were harvested by centrifugation and resuspended to a final concentration corresponding to an optical density (OD) of 1.0 (Voinnet et al., 2000) at 600 nm in a solution containing 10 mM MgCl 2 , 10 mM MES pH 5.7, and 150 μM acetosyringone (unless stated otherwise).Cultures were incubated at room temperature for 3 h before infiltration.
Agroinfiltration procedure.For silencing-release assays, two zones on the opposite half-leaves of N. benthamiana plants were infiltrated with a mix of cultures from two recombinant Agrobacterium strains: the first culture carrying a Ti-binary plasmid that contained the gfp gene and the second culture carrying a Ti-binary plasmid in which a transgene encoding a viral suppressor was inserted.A mix two cultures of Agrobacterium carrying the Ti-binary plasmid pJR1Ri (i.e., empty vector) and a Ti-binary plasmid that contained the gfp gene was used as a control.Prior to infiltration a suspension of Agrobacterium harbouring a binary plasmid containing the gfp gene was mixed in a 1:1 ratio with a bacterial suspension carrying one of the three viral silencing suppressors in the binary plasmid.Two top-leaves per plant were infiltrated with a 2-ml syringe without a needle.Leaves were superficially wounded with a needle to improve infiltration.Three plants were agroinfiltrated for each point of a particular chart.The infiltrated leaves were photographed at 4 days after agroinfiltration (unless stated otherwise).
GFP imaging.Visual detection of GFP fluorescence in whole transient transformed leaves was performed using a hand-held long-wavelength ultraviolet lamp.The transiently transformed leaves were photographed with a Digital Camera OLYM-PUS, C-750 Ultra Zoom though a Yellow 8, ES 52 filter (exposure time 16 s).The fluorescence intensity of randomly selected three sites in the agroinfiltrated and nonagroinfiltrated zones (as a background control) was measured in the photographs of Agrobacterium-infected leaves.The fluorescence was quantified using the ImageJ program (National Institutes of Health, USA) and fluorescence intensity was expressed as the mean gray value (Maximova et al., 1998).The data were statistically analyzed by the t-test.

Comparison of levels of transient gfp gene expression in leaves of N. benthamiana mediated by four strains of A. tumefaciens carrying binary plasmids
For comparative purposes, the four strains (EHA105, AGL0, AGL1 and LBA4404) of A. tumefaciens carrying the binary plasmids pCAMBIA(gfp)1302 and pJR1Ri-HC-Pro(TuMV) were infiltrated into leaves of N. benthamiana.Suspensions of particular strains of A. tumefaciens carrying plasmid pCAMBIA(gfp)1302 were infiltrated separately or in combination with a culture carrying plasmid pJR1Ri-HC-Pro into leaves of N. benthamiana.The infiltrated leaves were photographed on the 4th day after agroinfiltration.

2006
M. Wydro and others The highest level of GFP fluorescence without a suppressor was reached with strain AGL0 (about 30 as the mean gray value).The maximal level for strain EHA105 was 14 only.When strain AGL0 carrying pCAMBIA(gfp)1302 was used for agroinfiltration with the suppressor HC-Pro(TuMV) the level of GFP fluorescence was increased twice in relation to that in the absence of this suppressor (from 30 to 60 as the mean gray value).When strain EHA105 was used the GFP fluorescence was increased three times as compared to that without a suppressor (from about 14 to 40) (Fig. 2).We used interchangeably two bacterial strains: AGL0 and EHA105 in our experiments.When strains LBA4404 and AGL1 carrying plasmid pCAMBIA(gfp)1302 were used for agroinfiltration with HC-Pro(TuMV) the increase of the GFP fluorescence in relation to that without a suppressor was very poor (Fig. 2).

Comparison of levels of transient gfp gene expression in leaves of N. benthamiana mediated by Agrobacterium EHA105 carrying the binary plasmids pBINmgfp5-ER or pCAMBIA(gfp)1302
Suspensions of strain EHA105 carrying the binary plasmids: pBINmgfp5-ER, pCAMBIA(gfp)1302 or pJR1Ri-HC-Pro(TuMV) were infiltrated into leaves of N. benthamiana.Our results indicated that a higher level of GFP fluorescence was reached when the binary plasmid pBINmgfp5-ER was used (about 65 as the mean gray value with the suppressor and 35 without).When the plasmid pCAMBIA(gfp)1302 was used these values were 28 and 13, respectively (Fig. 3).Plasmid pBINmgfp5-ER was used in all the experiments in our study with the exception of those when the efficiency of the four strains of A. tumefaciens was being tested.

Agrobacterium-mediated expression of gfp gene in leaves of N. benthamiana
There are numerous controversial data on the effect of acetosyringone concentration on transient gfp gene expression in N. benthamiana.In our study, we monitored the effect of acetosyringone concentration ranging from 50 to 600 μM on the level of GFP fluorescence.Our results indicated that the highest level of GFP fluorescence in the presence of suppressor (HC-Pro), measured as a mean gray value, was reached at 450 μM and remained unchanged up to 600 μM of AS.When agroinfiltration was performed with a control suspension of Agrobacterium carrying the binary plasmid pBINmgfp5-ER only a slight in-  Optimization of transient Agrobacterium-mediated gene expression system crease in the gfp gene expression was observed when the concentration of acetosyringone was increased from 50 to 600 μM (Fig. 4).

Effect of the density of the Agrobacterium suspension on transient gfp gene expression
The studies were performed for four different densities of bacterial suspensions (0.1, 0.5, 1.0 and 1.5 OD 600 ).The infiltrated leaves were photographed on the 4th day after agroinfiltration.The results are presented in Fig. 5.We did not notice any differences in the levels of transient gfp gene expression for the different densities of Agrobacterium suspensions used for infiltration.The co-expression of gfp and HC-Pro genes caused an increase in the level of GFP fluorescence but this enhancement was similar for all the densities of bacterial suspension used (the levels of GFP fluorescence with a suppressor were increased twice in relation to that in the absence of suppressor) (Fig. 5).The density of the bacterial suspension used for infiltration during all the experiments reported in this paper was OD 600 = 1.0.

Changes of transient gfp gene expression levels during days 1-6 after agroinfiltration
In a long term experiment, two cultures of strain EHA105 carrying the binary plasmids pBIN-mgfp5-ER and pJR1Ri-HC-Pro(TuMV) were infiltrated into leaves of N. benthamiana.A combination of two suspensions of A. tumefaciens carrying the binary plasmid pBINmgfp5-ER and the empty vector pJR1Ri was infiltrated into the left half-leaves (controls).A second combination of two cultures of Agrobacterium carrying the binary plasmids pBIN-mgfp5-ER and pJR1Ri-HC-Pro(TuMV) was infiltrated into the right half-leaves.The infiltrated leaves were photographed on each day from the 1st until the 6th day after agroinfiltration.The GFP fluorescence intensity on days 1-6 after agroinfiltration was measured at three random selected sites in non-infiltrated zone (as a background) and three sites in the agroinfiltrated zone.Six data sets were collected over the time period of 6 days (Fig. 6).The GFP fluorescence on the left half-leaves (infiltrated with Agrobacterium carrying the binary plasmids pBINm-gfp5-ER and pJR1Ri) reached about 30 as the mean gray value at day 4 then remained unchanged until day 6.The GFP fluorescence of the N. benthamiana leaves agroinfiltrated with the suspension of strain EHA105 of Agrobacterium carrying the binary plasmids pBINmgfp5-ER and pJR1Ri-HC-Pro at first appeared on the second day post-agroinfiltration and then increased until the end of the experiment on the sixth day (GFP fluorescence was at least about   M. Wydro and others 3-fold higher than that in the leaves infiltrated with pBINmgfp5-ER in the absence of HC-Pro).

Effect of leaf ageing on transient gfp gene expression in leaves of N. benthamiana
For comparative purposes, leaves from three different positions corresponding to different leaf age were selected for agroinfiltration: the top leaf position (youngest leaves), intermediate leaf position and bottom leaf position (oldest leaves) (Fig. 7A).A culture of A. tumefaciens carrying pBINmgfp5-ER and the empty vector-pJR1Ri was infiltrated into the left half-leaves, and the right half-leaves of these plants were infiltrated with a suspension of Agrobacterium carrying the binary plasmids pBINmgfp5-ER and pJR1Ri-HC-Pro(TuMV) at the same time.The inoculated leaves were photographed on the 4th day after agroinfiltration.In the leaves agroinfiltrated with the control suspension, the GFP fluorescence intensity was similar at all leaf positions with the mean gray value ranging from 17 to 24 (Fig. 7B).The co-infiltration with suspension carrying the gfp and HC-Pro genes resulted in strong expression of the gfp gene in the youngest leaves (the top position) and the GFP fluorescence intensity was three times higher in relation to that of the leaves infiltrated with pBINm-gfp5-ER plasmid without HC-Pro.A similar increase was observed in the intermediate position leaves but the levels of GFP fluorescence in the presence and without the suppressor were slightly lower.At the bottom position (oldest leaves) of N. benthamiana the level of GFP fluorescence with the HC-Pro gene was increased twice in relation to that without the HC-Pro gene (Fig. 7B).The GFP fluorescence of N. benthamiana leaves expressed as a mean gray value varied from 11 to 23 in the leaves infiltrated with gfp without the HC-Pro gene, and from 28 to 38 in the leaves infiltrated with the gfp and HC-Pro genes.

Agrobacterium-mediated gfp gene expression in leaves of N. benthamiana
For comparative purposes, the three suppressors (HC-Pro-TuMV, HC-Pro-PVY and P1/HC-Pro TuMV) were used during transient expression in  The green fluorescent protein (gfp) gene was used as a sensitive and non-invasive marker to visualize transient gene expression in transformed cells.When the gfp gene alone was used for transient transformation, the maximum level of expression was rather low.It was much enhanced when a mixture of the gfp and HC-Pro genes at the 1:1 ratio was used for transient transformation.The results of our study provide circumstantial evidence that the transient gfp gene expression was restricted by local RNA silencing and that this silencing was suppressed by viral proteins (HC-Pro) from TuMV or PVY viruses, being well-known suppressors of RNA silencing (Kasschau & Carrington, 1998;Anandalakshmi et al., 1998).
Our data reveal that plasmid pBINmgfp5-ER is more efficient for transient gfp gene expression in leaves of N. benthamiana than pCAMBIA(gfp)1302.Different levels of green fluorescence observed after agroinfiltration with each of the plasmids could

B
A be explained by differences between the variants of the gfp gene contained in those plasmids.Plasmid pCAMBIA1302 contains the mgfp5 version of gfp (Siemering et al., 1996) whereas the gfp gene contained in plasmid pBINmgfp5-ER is a version further modified by the addition of a signal sequence which targets mGFP5-ER to the endoplasmic reticulum (Haseloff et al., 1997).
The analysis of the gfp gene expression level measured as the intensity of GFP fluorescence without a suppressor (HC-Pro) showed that this expression was increasing from days 2 to 4 then it remained unchanged up to day 6.In contrast, when this analysis was performed in the presence of the suppressor (HC-Pro-TuMV) the GFP fluorescence was increasing from the 1st day until the end of the assay (6th day).Our results support the hypothesis put forward by Johansen and Carrington (2001) and Voinnet et al. (2003) that after the transient transformation local RNA silencing is triggered and then suppressed in the presence of some viral suppressor.
Figure 7B shows the GFP fluorescence in N. benthamiana leaves of different ages.The highest GFP fluorescence was found at the top leaf position (youngest leaves), at intermediate and bottom positions it was lower (older leaves).This was true both in the presence and in the absence of a suppressor.Halfhill et al. (2003) suggested that changes in GFP fluorescence were related to changes in the concentration of soluble proteins during leaf ageing.The level of gfp gene expression and concentration of soluble proteins declined at similar times and to similar extents in individual leaves at different positions.The close relationship between these two factors would suggest that the GFP decline was a result of general changes in leaf physiology and not a specific effect on the transgene or its expression controlled by the 35S promoter.
Acetosyringone (AS) is known to induce the vir operon of A. tumefaciens (Hiei et al., 1994).We also monitored the effect of concentration of acetosyringone during incubation of Agrobacterium cultures.The highest level of GFP fluorescence in the presence of a suppressor was reached at 450 μM and remained unchanged up to 600 μM AS.An increase in the concentration of acetosyringone from 50 to 600 μM resulted in a slight increase in the gfp gene expression (GFP fluorescence) when the agroinfiltration was performed without a suppressor.Jeoung et al. (2002) have reported optimization of acetosyringone concentration in the preculture and co-culture medium for transient expression of gfp gene in three inbred lines of sorghum at four different concentrations of AS ranging from 200 to 1000 μM.The concentration of AS in culture medium was found to affect the transient gene expression.Further, the op-timum AS concentration varied for different inbred lines.
Although Voinnet et al. (2003) showed that the p19 protein of tomato bushy stunt virus (TBSV) was an effective suppressor of silencing we decided to use the HC-Pro proteins of turnip mosaic virus (TuMV) and potato virus Y (PVY) in our studies as these proteins are the main objects of our interest.The data presented in this report supports the earlier observations on HC-Pro-TuMV and HC-Pro-PVY (Kasschau & Carrington, 1998;Anandalakshmi et al., 1998).Here they were equally effective since they both resulted in the same level of increase in GFP fluorescence over that detected in the absence of viral suppressors.However, unexpectedly the proteins P1/HC-Pro(TuMV) did not result in an increase in GFP fluorescence.This discrepancy may be explained by the fact that the proteins HC-Pro-TuMV and HC-Pro-PVY encoded in the constructs pJR1Ri-HC-Pro-TuMV and pJR1Ri-HC-Pro-PVY had methionine at the N terminus as a result of addition of the initiation codon ATG to the 5' termini of their genes during construct preparation.When two genes P1 and HC-Pro were used in the construct the initiation codon was at the 5'-terminus of P1 and it was not added to the beginning of HC-Pro, which can imply differences in the three-dimensional structure of the HC-Pro protein in both constructs.In contrast, Pruss et al. (1997) suggested that protein P1 might enhance HC-Pro activity as a suppressor of RNA silencing.
In general, we confirmed that the gfp gene can be used effectively as a reporter for optimizing the conditions of successful expression during transient Agrobacterium-mediated transformation of leaves of N. benthamiana.The results obtained indicate that the transient gfp gene expression was related to leaf aging (the maximum expression was observed in the youngest leaves), depended on acetosyringone concentration during incubation of Agrobacterium (maximum gfp gene expression at 450-600 μM AS) and was independent of the density of the bacterial suspension.Much higher levels of transient expression were observed when the strains AGL0 and EHA105 of A. tumefaciens were used compared to AGL1 and LBA4404.The HC-Pro proteins from TuMV and PVY viruses were equally effective since they both resulted in the same level of increase in GFP fluorescence over that present without of viral suppressors.Voinnet et al. (2003) have demonstrated the use of transient expression in the presence of suppressors in another plant and for other viral genes expressed either individually or in mixtures (Cf-4, Cf-9 and NIa).Vaquero et al. (1999) have described the transient expression of different forms of a tumor-specific chimeric antibody (rAbs) in tobacco leaves.The production of full-size human chimeric antibody was achieved by simultaneous expression of the heavy and light chains.The genes were transferred into tobacco leaf cells by agroinfiltration in two independent Agrobacterium cultures.This result demonstrates that multimeric proteins can be analyzed by using Agrobacterium-mediated transient expression.Several genes encoding proteins can be simultaneously transiently expressed within the same cells.This enables analysis of complex multimeric proteins.In stable transgenic plants such manipulation would require several independent transformations that could take months.
The improvements of the Agrobacterium-mediated transient expression system reported here will be important in the identification of new viral suppressors of RNA silencing, for systematic investigation of unidentified genes and also for industrial production of proteins in plants.In our laboratory, this system will be used also to test expression of constructs before generation of stably transformed plants.

Figure 2 .
Figure 2. Comparison of the levels transient gfp gene expression mediated by four strains A. tumefaciens carrying the binary plasmids in the leaves of N. benthamiana.The cultures of particular strains (EHA105, AGL0, AGL1 and LBA4404) of A. tumefaciens harbouring plasmid pCAMBIA(gfp)1302 were infiltrated singly or in combination with the culture carrying plasmid pJR1Ri-HC-Pro into the leaves of N. benthamiana as described in Materials and Methods section.The infiltrated leaves were photographed at 4 dpi.GFP fluorescence was quantified with ImageJ programme.N. benthamiana leaves had an average fluorescence as a mean gray value.

Figure 3 .
Figure 3.Comparison of the levels transient gfp gene expression mediated by strain EHA105 of Agrobacterium harbouring binary plasmids pBINmgfp5-ER or pCAMBIA(gfp)1302 in the leaves N. benthamiana.The cultures of EHA105 carrying of the binary plasmids pBINmgfp5-ER, pCAMBIA(gfp)1302 and pJR1Ri-HC-Pro(TuMV) were infiltrated into the leaves of N. benthamiana as described in Materials and Methods section.The infiltrated leaves were photographed at 4 dpi.GFP fluorescence was quantified with ImageJ programme.

Figure 4 .
Figure 4. Effect of acetosyringone concentration of on transient expression gfp gene in the leaves of N. benthamiana.Leaves of N. benthamiana were infiltrated as described in the Materials and Methods section with culture of strain EHA105 harbouring the plasmids pBINmgfp5-ER and pJR-1Ri-HC-Pro(TuMV). Photographs were taken at 4 dpi.

Figure 5 .
Figure 5.Effect of density of Agrobacterium suspension on transient gfp gene expression.The four different densities of Agrobacterium suspension: 0.1, 0.5, 1.0 and 1.5 OD at 600 nm carrying of the binary plasmids pBINmgfp5-ER and pJR1Ri-HC-Pro(TuMV) were infiltrated into the leaves of N. benthamiana as described in the Materials and Methods section.The infiltrated leaves were photographed at 4 dpi.

Figure 6 .
Figure 6.Course of transient gfp gene expression within 1-6 days post-agroinfiltration.The two cultures of strain EHA105 carrying of the binary plasmids pBINmgfp5-ER and pJR1Ri-HC-Pro(TuMV) were infiltrated into the leaves of N. benthamiana plants as described in the Materials and Methods section.The infiltrated leaves were photographed on each day from the 1st until the 6th day post-agroinfiltration.

Figure 7 .
Figure 7. (A) Three different leaf positions corresponding to different leaf age were selected for agroinfiltration.(B) Effect of leaf ageing on transient gfp gene expression in the leaves of N. benthamiana.The combination two cultures of A. tumefaciens carrying of the binary plasmids pBINmgfp5-ER and pJR1Ri-HC-Pro(TuMV) was infiltrated into the leaves of N. benthamiana as described in the Materials and Methods section.The infiltrated leaves were photographed at 4 dpi.

Figure 8 .
Figure 8.Effect of the three viral silencing suppressors on the levels of transient gfp gene expression in the leaves of N. benthamiana.(A) Photograph.(B) Chart.The three combinations of two cultures of Agrobacterium carrying the plasmids pBIN-mgfp5-ER and one of the three individual suppressor (HC-Pro-TuMV; HC-Pro-PVY and P1/HC/Pro-TuMV) were infiltrated as described in the Materials and Methods section.Photographs were taken at 4 dpi.