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First published online December 22, 2006; 10.1104/pp.106.091488 Plant Physiology 143:801-811 (2007) © 2007 American Society of Plant Biologists MPB2C, a Microtubule-Associated Plant Factor, Is Required for Microtubular Accumulation of Tobacco Mosaic Virus Movement Protein in Plants1Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, Department of Medical Biochemistry, Medical University of Vienna, A–1030 Vienna, Austria (M.C., K.T., E.W.); and Department of Gene Technology, Tallinn University of Technology, 19086 Tallinn, Estonia (E.-L.O., B.I., E.T.)
Movement protein binding 2C (MPB2C) is a plant endogenous microtubule-associated protein previously identified as an interaction partner of tobacco (Nicotiana tabacum) mosaic virus movement protein (TMV-MP). In this work, the role of MPB2C in cell-to-cell transport of TMV-MP, viral spread of TMV, and subcellular localization of TMV-MP was examined. To this end, plants with reduced MPB2C levels were generated by a gene-silencing strategy. Local and systemic spread of TMV and cell-to-cell movement of TMV-MP were unimpaired in MPB2C-silenced plants as compared to nonsilenced plants, indicating that MPB2C is not required for intercellular transport of TMV-MP itself or spread of TMV. However, a clear change in subcellular distribution of TMV-MP characterized by a nearly complete loss of microtubular localization was observed in MPB2C-silenced plants. This result shows that the MPB2C is a central player in determining the complex subcellular localization of TMV-MP, in particular its microtubular accumulation, a phenomenon that has been frequently observed and whose role is still under discussion. Clearly, MPB2C mediated accumulation of TMV-MP at microtubules is not required for intercellular spread but may be a means to withdraw the TMV-MP from the cell-to-cell transport pathway.
For cell-to-cell spread, plant viruses take advantage of plasmodesmata, cytoplasmic bridges that interconnect neighboring plant cells. However, the size of the viral particle is considerably larger than the size exclusion limit (SEL) of plasmodesmata. To overcome this problem, plant viruses encode one or more movement proteins (MPs; for review, see Tzfira et al., 2000  ; Waigmann et al., 2004; Lucas, 2006). Tobacco (Nicotiana tabacum) mosaic virus movement protein (TMV-MP), one of the most extensively studied viral MPs, is known to cooperatively bind single-stranded nucleic acid (Citovsky et al., 1990) and increase the SEL of plasmodesmata (Wolf et al., 1989; Waigmann et al., 1994). In a model, TMV-MP is thought to form complexes with the single-strand RNA genome of TMV (vRNA-TMV-MP) in the vicinity of the endoplasmic reticulum (ER). These complexes are transported to the cell periphery, potentially via the cytoskeleton. At the cell periphery, TMV-MP facilitates the transport of the vRNA-TMV-MP complexes into adjacent cells by inducing an increase in plasmodesmal SEL (Lazarowitz and Beachy, 1999; Tzfira et al., 2000).
When TMV-MP fusions to autofluorescent proteins were employed, three subcellular localization patterns were revealed. TMV-MP localized to cortical puncta associated with the ER, to filamentous structures identified as microtubules, and to plasmodesmata, both in the context of a viral infection (Heinlein et al., 1995
Knowledge about plant endogenous factors interacting with TMV-MP would be highly desirable to gain insight into the functional relevance of these localizations for viral spread. One such factor, movement protein binding 2C (MPB2C), was identified from tobacco and constitutes a novel microtubule-associated plant protein (Kragler et al., 2003 In this work, virus-induced gene silencing (VIGS) of MPB2C was performed to further elucidate the role of MPB2C in TMV infections. Both cell-to-cell movement of TMV-MP and spread of TMV were unimpaired in silenced plants as compared to nonsilenced plants. However, a clear change in subcellular distribution of TMV-MP characterized by a nearly complete loss of microtubular localization and redistribution toward plasmodesmata was observed. All together, these data support the concept that the MPB2C's role in the viral life cycle is not to support TMV-MP-mediated transport function but to accumulate TMV-MP at microtubules. MPB2C might be a central player in a decision process that determines the subcellular localization of TMV-MP. We discuss the biological significance of the MPB2C-mediated accumulation of TMV-MP at microtubules that may be a means to efficiently withdraw the TMV-MP from the cell-to-cell transport pathway.
VIGS of MPB2C in Nicotiana benthamiana
Our previous series of experiments on the role of MPB2C in viral infections relied on overexpression of MPB2C (Kragler et al., 2003
Because at the beginning of our study only the tobacco MPB2C sequence was available (NtMPB2C), we used this sequence to generate the silencing construct, reasoning that the MPB2C sequences in tobacco and N. benthamiana are likely closely related, so that the NtMPB2C sequence would induce silencing of NbMPB2C expression. In line, an anti-MPB2C antiserum raised against recombinant NtMPB2C (Kragler et al., 2003 ) specifically recognized a protein of the expected size in N. benthamiana protein extracts (Fig. 1B, lane 3). Using a PCR-based strategy, we generated two NtMPB2C constructs, a long construct from nucleotide 1 to 966 encompassing the complete cDNA of NtMPB2C and a short construct containing only nucleotides 1 to 473 of NtMPB2C (Figs. 1A and 2A
). Both constructs were introduced into TRV RNA2 in the context of binary plasmid pTV00 to yield silencing construct TRV RNA2-MPB2C long and TRV RNA2-MPB2C short, respectively (Fig. 1A). Viral infections of N. benthamiana plants were achieved by mixing agrobacteria harboring TRV RNA1 with agrobacteria harboring either of the TRV RNA2-MPB2C constructs. Agroinfiltration was performed as described (Ratcliff et al., 2001; http://www.jic.bbsrc.ac.uk/sainsbury-lab/dcb/Services/ptv00.htm). To evaluate the frequency and efficiency of NbMPB2C silencing induced by the two constructs, 13 plants were agroinfiltrated with TRV-MPB2C long and 18 plants with TRV-MPB2C short. Mild symptoms of TRV infections became apparent after 1 week, characterized by dark and light green patches in systemic young leaves (data not shown). Three weeks after agroinfiltration, when silencing was expected to be established (Ratcliff et al., 2001), the level of NbMPB2C protein was determined by western blotting with the anti-MPB2C antiserum in total protein extracts derived from leaf tissue of mature, noninfiltrated leaves. Various degrees of silencing of the NbMPB2C protein were observed, ranging from a reduction in the NbMPB2C level (Fig. 1B, lane 5) to complete silencing below the detection level (Fig. 1B, lane 4). The frequency and efficiency of NbMPB2C silencing was dependent on the type of TRV-MPB2C construct; TRV-MPB2C long induced complete silencing in only one of the 13 infected plants (7.6% frequency) and partial silencing in 15.4% of the infected plants. In contrast, construct TRV-MPB2C short induced complete silencing in 13 out of 18 plants, equal to 72.2% frequency, and partial silencing in five plants, equal to 27.8% frequency (Table I
). Thus, TRV-MPB2C long was considerably less effective in MPB2C silencing, potentially due to instability of the larger MPB2C insert in the virus vector (Lu et al., 2003). As a consequence, in all subsequent MPB2C-silencing experiments, we used the TRV-MPB2C short silencing construct. Furthermore, because even TRV-MPB2C short induced complete silencing in only 72.2% of plants, we individually verified NbMPB2C silencing in each leaf that was analyzed in subsequent experiments. Because the MPB2C-silenced plants did not show any distinguishing phenotype (data not shown), we relied on western blots for verification of NbMPB2C silencing.
Cloning of MPB2C Homolog from N. benthamiana
By database searches, proteins homologous to tobacco MPB2C were found in mono- and dicotyledonous plants with identity ranging from 37% to 75% (Kragler et al., 2003
It was previously shown that upon transient coexpression of TMV-MP:green fluorescent protein (GFP) and MPB2C:red fluorescent protein, cell-to-cell transport of TMV-MP:GFP was impaired (Kragler et al., 2003
TMV-MP:GFP was expressed in single epidermal cells of mature leaves by biolistic delivery of plasmid pRTL TMV-MP:GFP (McLean et al., 1995 ), and the frequency of cell-to-cell movement was evaluated by confocal microscopy on the first and second day after bombardment. TMV-MP:GFP cell-to-cell movement manifested as small cell wall-associated puncta in cells next to the expressing cell (Crawford and Zambryski, 2001; Kotlizky et al., 2001; Trutnyeva et al., 2005; see also Fig. 5, B and C). For each category of plants, the frequency of TMV-MP:GFP cell-to-cell movement was calculated at day 1 and day 2 after bombardment by analyzing at least 50 cells. Data were assembled into a bar graph (Fig. 3). At day1 after bombardment, in wild-type and TRV-infected control plants, 38% of analyzed cells showed TMV-MP:GFP movement to neighboring cells and 44% in MPB2C-silenced plants (Fig. 3A). At day 2 after bombardment, 40% of cells were movement positive in wild-type plants and 41% in TRV-infected control plants, compared to 47% in MPB2C-silenced plants. These data suggest that the MPB2C protein is not required for cell-to-cell movement of TMV-MP:GFP in N. benthamiana plants. Notably, TRV infection does not affect the frequency of TMV-MP:GFP cell-to-cell movement as compared to wild-type plants. The level of MPB2C expression is similar in wild-type and TRV-infected plants (Fig. 3B, compare lanes wild type and TRV infected). Each leaf analyzed for TMV-MP:GFP cell-to-cell transport was also evaluated for the level of MPB2C protein (Fig. 3B). Only data derived from completely silenced leaves (Fig. 3B, lane MPB2C silenced) were included in the results depicted in Figure 3A.
MPB2C Silencing Does Not Impair Local and Systemic TMV Spread
In a next step, the effect of MPB2C silencing on TMV infection was tested. MPB2C-silenced plants as well as untreated wild-type plants were mechanically inoculated with TMV particles. Inoculated leaves were harvested 3 d post inoculation (dpi), and systemic noninoculated leaves were harvested 9 or 16 dpi. A protein extract was prepared and analyzed for the presence of TMV coat protein, which is produced in sufficiently high amounts during infection to be easily detectable in total protein extracts (Waigmann et al., 2000
Next, systemic spread of TMV was evaluated. TMV coat protein was clearly detectable in systemically infected young leaves of MPB2C-silenced plants at 9 dpi (Fig. 4C, lanes 1 and 2) as well as in systemically infected young leaves of TMV-infected wild-type plants (Fig. 4C, lanes 6 and 7) but was not present in young leaves of noninfected wild-type plants (Fig. 4C, lanes 3 and 5). When the same samples were analyzed by western blotting for their MPB2C levels, MPB2C was not detectable in TMV-infected systemic leaves of MPB2C-silenced plants (Fig. 4D, lanes 1 and 2) but was present in noninfected wild-type plants (Fig. 4D, lanes 3 and 4). Interestingly, in TMV-infected wild-type plants, a much faster migrating species of the MPB2C protein became apparent at 9 dpi, which became the dominant MPB2C species at 16 dpi (marked by asterisk in Fig. 4D, lanes 5 and 6). The faster migrating species likely reflects a degradation product of MPB2C that is produced upon TMV infection. In line, up-regulation of E1, the ubiquitin-activating enzyme, was reported upon TMV infection, indicating that the ubiquitin-proteasome system that leads to degradation of proteins is affected by TMV infection (Takizawa et al., 2005 ). Thus, due to this up-regulation, MPB2C might become a target of ubiquitination during TMV infection, which in turn leads to its degradation. Overall, results indicate that TMV local and systemic spread is not dependent on the presence of the MPB2C protein. In addition, the MPB2C protein is either modified or subjected to degradation upon TMV infection.
Even though the MPB2C protein was not required for the movement function of TMV-MP, involvement of the MPB2C in subcellular distribution of TMV-MP was still possible. Keeping in mind that the MPB2C is a microtubule-associated protein (Kragler et al., 2003
On the first day after bombardment, the two most prominent subcellular localization patterns of the TMV-MP:GFP-expressing cells were the ER pattern (53%), followed by the PD pattern (41%), whereas the MT pattern was hardly represented (3%). Two days after bombardment, reduction of the ER pattern to 34% was observed, which correlated to a significant increase in MT pattern to 16% and a slight increase in PD pattern to 45% (Fig. 5D). Cells expressing TMV-MP:GFP in a PD pattern were highly associated with cell-to-cell movement: at day 1, 93% and at day 2, 87% of those cells showed movement. Cells expressing TMV-MP:GFP in a MT pattern showed no movement at day 1, but 53% of those cells showed movement at day 2 after bombardment. Cells expressing TMV-MP:GFP in an ER pattern were not associated with movement to neighboring cells at day 1 or day 2 (Fig. 5D). Collectively, results indicate that the PD pattern is most productive with regard to cell-to-cell movement, whereas the MT pattern contributes to a much lesser extent, and the ER pattern not at all. Movement-associated MT pattern is most likely dependent on plasmodesmally localized TMV-MP:GFP, because the MT pattern frequently consists of a mixture of microtubular and plasmodesmally localized TMV-MP:GFP (Fig. 5B). Lack of movement associated with the ER pattern may reflect that ER association is an early stage of the TMV-MP's journey toward plasmodesmata, as has been previously suggested (Heinlein, 2002
The movement profile suggested that a significant level of microtubular localization was observable at day 2 after bombardment. Therefore, to examine the potential effect of MPB2C silencing on subcellular distribution, and, in particular, on microtubular accumulation of TMV-MP, a direct side-by-side comparison between MPB2C-silenced plants and untreated N. benthamiana plants as well as plants infected with a TRV vector lacking the MPB2C sequence was performed at day 2 after bombardment. Between 100 and 140 cells were analyzed with regard to TMV-MP:GFP subcellular localization for each type of plant. The percentage of cells expressing TMV-MP:GFP in a microtubular localization pattern constituted a substantial fraction, 19% wild-type and 26% TRV-infected plants, but was strongly reduced to 6% in MPB2C-silenced plants (compare Fig. 6 , A and B, to C). Conversely, the frequency of PD pattern increased from 38% and 31% in wild-type or TRV-infected plants, respectively, to 49% in MPB2C-silenced plants (Fig. 6). In contrast, the percentage of cells with TMV-MP:GFP localized in an ER pattern remained nearly unchanged in all three types of plants: 41% and 40% for wild-type and TRV-infected plants, and 43% for MPB2C-silenced plants.
Thus, silencing of MPB2C significantly influenced the subcellular distribution of TMV-MP:GFP, leading to nearly complete loss of MT pattern and a concomitant increase in PD pattern. The loss of microtubular localization implicates the MPB2C protein in accumulating TMV-MP at microtubules. The observed increase of PD pattern in MPB2C-silenced plants may be a direct consequence of the failure of TMV-MP to accumulate at microtubules; because the MT pattern is frequently a mixture of TMV-MP localized at microtubules and TMV-MP localized at plasmodesmata (Fig. 5B), loss of microtubular accumulation would result in PD pattern, thereby increasing the frequency of this pattern.
MPB2C is a previously uncharacterized, plant-specific microtubule-associated protein isolated from tobacco that biochemically interacts and colocalizes with TMV-MP (Kragler et al., 2003 ). Initial studies based on transient overexpression of MPB2C indicated that enhanced MPB2C levels negatively affect cell-to-cell transport of TMV-MP protein; in host N. benthamiana, the frequency of cell-to-cell transport of TMV-MP was reduced to less than one-half in MPB2C-overexpressing cells. The inhibitory effect of MPB2C was not due to unspecific effects on plasmodesmal transport capacity, because cell-to-cell movement of cucumber (Cucumis sativus) mosaic virus MP, and a point mutant of TMV-MP, MPR3, was unimpaired. In line with this observation, the MPR3 mutant was shown to not accumulate at microtubules and to bind MPB2C with reduced affinity in vitro (Gillespie et al., 2002; Kragler et al., 2003). Coupled to the inhibitory effect, transiently overexpressed MPB2C induced a shift in subcellular localization of TMV-MP from plasmodesmal puncta toward its own localization at microtubules (Kragler et al., 2003). Collectively, these data pointed to a non-movement-related or even inhibitory function of MPB2C for cell-to-cell movement of TMV-MP. However, because these analyses were limited to transient overexpression of MPB2C in single cells, it was not possible to test the impact of MPB2C on TMV infection or to completely rule out adverse effects due to overexpression.
To further elucidate the role of MPB2C in TMV infections, and, in particular, cell-to-cell movement, a TRV-based strategy to silence the MPB2C gene in N. benthamiana was employed. This silencing strategy has been used previously to test the role of other plant endogenous factors in TMV infections, such as the role of microtubules (Gillespie et al., 2002 In this work, efficient MPB2C silencing was achieved when a 473-bp fragment of the MPB2C cDNA was incorporated into the TRV-silencing system. Silencing of the MPB2C protein was routinely confirmed in leaves, the primary target tissue of these analyses, by western blotting with anti-MPB2C antibodies. Silenced plants did not show any particular phenotype, indicating that the MPB2C protein was not essential for mature plants. Studies in silenced plants focused on cell-to-cell movement of TMV-MP protein, local and systemic spread of TMV, and subcellular distribution of TMV-MP. The frequency of TMV-MP cell-to-cell movement, evaluated in single epidermal cells transiently expressing TMV-MP:GFP, was similar in MPB2C-silenced plants as compared to two types of MPB2C-expressing control plants, untreated N. benthamiana plants, and TRV-infected, non-MPB2C-silenced plants. Thus, the presence of the MPB2C protein is clearly not required for TMV-MP cell-to-cell movement.
Because it is possible that MPB2C might perform a function in the context of a viral infection that cannot be detected by analyzing cell-to-cell movement of TMV-MP protein, both local and systemic spread of TMV were evaluated in MPB2C-silenced plants. Results show that TMV was able to spread and replicate in inoculated as well as systemic leaves of MPB2C-silenced N. benthamiana plants, suggesting that MPB2C is not required for establishing either local or systemic infection. In TMV-infected leaves, a change in electrophetic mobility of the MPB2C protein became apparent, which was slight in inoculated leaves but pronounced in systemic leaves (Fig. 4). The small change observed in inoculated leaves may be due to either a small decrease in protein size or a change in posttranslational modification such as phosphorylation status. In systemically infected leaves, the change in electrophoretic mobility is most likely due to protein degradation, a process that has been reported to take place upon TMV infection (Takizawa et al., 2005 Transient expression studies indicated that overexpressed MPB2C causes a change in subcellular localization of TMV-MP, redistributing it from plasmodesmata toward the MPB2C localization at microtubules. In MPB2C-silenced plants, just the opposite was the case; microtubular localization of TMV-MP was strongly reduced and plasmodesmal localization of TMV-MP was correspondingly enhanced when compared to wild-type and TRV-infected plants (compare Fig. 6, A–C). These data clearly show that MPB2C is required to efficiently accumulate TMV-MP at microtubules.
We suggest a modified model of accounting for the various subcellular localizations of TMV-MP (Fig. 7
). After association to the ER, which is considered the first localization after production, TMV-MP can either be targeted to plasmodesmata, thereby entering the cell-to-cell transport route, or it can be accumulated at microtubules via the MPB2C (Fig. 7). While plasmodesmal transport would be the default route, the establishment of microtubular localization would be determined by MPB2C. The MPB2C would thus act as a decisive factor determining the balance between the plasmodesmal and microtubular localization of TMV-MP. Once accumulated at microtubules, TMV-MP is no longer available for plasmodesmal targeting or the intercellular transport process. In line with experimental data, the model would predict that absence of MPB2C would lead to strongly reduced microtubular accumulation (Fig. 6) but would not disturb cell-to-cell transport (Fig. 3). Overall, MPB2-mediated microtubular accumulation could be a means to reduce transportability of TMV-MP through plasmodesmata and thus be an efficient strategy to inactivate transport function of TMV-MP. A similar effect on transportability of proteins has been observed previously when GFP was directed to the cytoskeleton or the ER (Crawford and Zambryski, 2000
This interpretation of MPB2C function can also be extended to the infection process. TMV-MP at the leading edge of infection localizes to plasmodesmata, reflecting its commitment to the cell-to-cell transport pathway, whereas it accumulates at microtubules well behind the leading edge of infection. Thus, MPB2C likely acts at later stages of infection to recruit TMV-MP toward microtubules. Such a model would clearly require that the interaction between the MPB2C and TMV-MP is regulated during infection, because microtubular accumulation of TMV-MP is under strict spatiotemporal control. In that context, the observed change in electrophoretic mobility of MPB2C in inoculated leaves of TMV-infected plants may be relevant (Fig. 4B, compare lanes 1 and 2). The nature of this change is so far unknown but may be due to either a small decrease in protein size or a change in posttranslational modification such as phosphorylation status that might influence the affinity between TMV-MP and MPB2C. Instead, or in addition, the well-known phosphorylation of TMV-MP at various sites might play a role in regulating interaction between TMV-MP and MPB2C (Citovsky et al., 1993 ; Haley et al., 1995; Waigmann et al., 2000; Karger et al., 2003; Trutnyeva et al., 2005). Our results also reflect on the more general question of the biological role of microtubules in TMV spread that has been under discussion for more than a decade and is still unclear.
Originally, it was suggested that microtubular accumulation of TMV-MP might reflect a role of microtubules in guiding viral RNA and/or the TMV-MP toward plasmodesmata. This hypothesis was challenged by drug studies suggesting that intact microtubules are not required for TMV spread (Gillespie et al., 2002 In summary, we present evidence that host factor MPB2C is not required for cell-to-cell movement of TMV-MP or for local or systemic spread of TMV. Instead, MPB2C is required to efficiently accumulate TMV-MP at microtubules and can thus be considered a decisive factor in determining the complex subcellular localization of TMV-MP. Further experiments will address the endogenous function of MPB2C, which has not been elucidated so far.
Construction of TRV-RNA2-MPB2C Long and TRV-RNA2-MPB2C Short
Construct TRV-RNA2-MPB2C long was generated by PCR amplification of full-length MPB2C from pGEX-MPB2C (Kragler et al., 2003
Total RNA from Nicotiana benthamiana leaves was isolated using RNeasy Plant Mini kit according to the manufacturer's instructions (Qiagen). Reverse transcription was performed according to the following conditions: 10 µL of total RNA (1 µg) was incubated at 65°C for 5 min to destroy RNA secondary structures and kept on ice until mixed with 10 µL of reaction mixture containing: 1 mM dNTPs, 2x AMV Reverse Transcription buffer (Promega), 2.5 units of AMV Reverse Transcriptase (Promega), 20 mM dithiothreitol, 40 units RNAsin (Promega), and 5 pmol of primer (5'-GCAGAATTCTTATGTTCTCAGAACAAG-3'). Reaction mixture was incubated for 2 h at 42°C. NbMPB2C cDNA was amplified by PCR using forward primer 5'-GCAATGTATGAGGCCCAG-3' and back primer 5'-GCAGAATTCTTATGTTCTCAGAACAAG-3' with the following PCR conditions: 5 min at 95°C, followed by 40 cycles consisting of 30 s of 95°C, 45 s at 55°C, and 1 min at 68°C, followed by 7 min at 72°C. The PCR product was subcloned into 2.1-TOPO vector (Invitrogen), five independent clones were sequenced, and a consensus sequence of NbMPB2C was generated.
N. benthamiana plants were grown in a controlled environment with a cycle of 16 h of light at 22°C and 8 h of darkness at 20°C. Three- to 4-week-old plants were used for agroinfiltration. Three weeks after agroinfiltration, plants were used for bombardment experiments and TMV infections.
Agroinfiltration was performed as described (Ratcliff et al., 2001
Mature leaves of N. benthamiana were mechanically inoculated by mixture of TMV particles and inoculation buffer (50 mM Na-P buffer, pH = 7, 1% Celite). Virus titer was determined by infecting Nicotiana glutinosa leaves and the number of local lesion was counted. A total of 10 µL of inoculation mixture contained approximately 50 viral particles, and this amount was used to infect each N. benthamiana leaf.
For biolistic delivery, 25 µg of plasmid pRTL-TMV-MP:GFP (McLean et al., 1995
A piece of bombarded leaf was excised at days 1 and 2 after bombardment, and all the TMV-MP:GFP-expressing cells in that area were analyzed by confocal microscopy. Routinely, three to six independent experiments were pooled to obtain information on at least 50 cells/experiment and time point. For direct comparison of TMV-MP:GFP subcellular localization in MPB2C-silenced, wild-type, and TRV-infected plants, between 100 and 140 cells were analyzed. Expressing cells were scanned with a TCS-SP confocal microscope (Leica Microsystems). GFP fluorescence was excited with an ArKr laser at 488 nm and detected at 500 to 550 nm. Serial optical sections were acquired in 1-µm steps with a 40x objective. These sections were then assembled into projections using software supplied by the manufacturer (Leica Microsystems).
Expressing cells were assigned to one of the three localization patterns: PD pattern, ER pattern, and MT pattern. Cells that did not fit into one of the three major subcellular localization patterns (typically <5%) were counted but are not represented on the graphs. Therefore, in some graphs, the total percentage of cells does not add up to 100%. Calculation of the mean and statistical evaluation of data was performed as described (Trutnyeva et al., 2005
N. benthamiana leaves were harvested and immediately frozen in liquid nitrogen. Frozen leaves were ground into a fine powder using mortar and pestle. To extract proteins, 100 µL of powder was mixed with the same volume of 1x Laemmli sample buffer (Ausubel et al., 1987
The NbMPB2C sequence was deposited in GenBank, accession number DQ297413.
Binary plasmids PTV00 and pBINTRA6 and agrobacteria strains C58C1 and GV3101 are a gift of David Baulcombe and Plant Bioscience Limited. Received October 19, 2006; accepted November 22, 2006; published December 22, 2006.
1 This work was supported by the Austrian Science Foundation (Spezialforschungsbereich 17, project part 08 to E.W.), by the Wiener Wissenschafts-, Forschungs- und Technologiefonds (project LS123 to E.W.), and by the Estonian Science Foundation (grant no. 6559 to E.T.). 
2 Present address: Clinical Institute for Medical and Chemical Laboratory Diagnostics, Department of Human Genetics, Medical University of Vienna, Waehringerstr. 10, 1090 Vienna, Austria. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Elisabeth Waigmann (elisabeth.waigmann{at}meduniwien.ac.at). www.plantphysiol.org/cgi/doi/10.1104/pp.106.091488 * Corresponding author; e-mail elisabeth.waigmann{at}meduniwien.ac.at; fax 43–1–4277–9616.
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