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First published online February 13, 2009; 10.1104/pp.108.133827

Plant Physiology 149:1810-1823 (2009)
© 2009 American Society of Plant Biologists

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PLANTS INTERACTING WITH OTHER ORGANISMS

Inhibition of Tobacco Mosaic Virus Movement by Expression of an Actin-Binding Protein1,[W],[OA]

Christina Hofmann, Annette Niehl, Adrian Sambade2, André Steinmetz and Manfred Heinlein*

Institut de Biologie Moléculaire des Plantes du CNRS, Université de Strasbourg, 67084 Strasbourg cedex, France (C.H., A.N., A. Sambade, M.H.); and Laboratory of Plant Molecular Biology, CRP-Santé, L–1526 Luxembourg (A. Steinmetz)


   ABSTRACT
TOP
 ABSTRACT
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 LITERATURE CITED
 
The tobacco mosaic virus (TMV) movement protein (MP) required for the cell-to-cell spread of viral RNA interacts with the endoplasmic reticulum (ER) as well as with the cytoskeleton during infection. Whereas associations of MP with ER and microtubules have been intensely investigated, research on the role of actin has been rather scarce. We demonstrate that Nicotiana benthamiana plants transgenic for the actin-binding domain 2 of Arabidopsis (Arabidopsis thaliana) fimbrin (AtFIM1) fused to green fluorescent protein (ABD2:GFP) exhibit a dynamic ABD2:GFP-labeled actin cytoskeleton and myosin-dependent Golgi trafficking. These plants also support the movement of TMV. In contrast, both myosin-dependent Golgi trafficking and TMV movement are dominantly inhibited when ABD2:GFP is expressed transiently. Inhibition is mediated through binding of ABD2:GFP to actin filaments, since TMV movement is restored upon disruption of the ABD2:GFP-labeled actin network with latrunculin B. Latrunculin B shows no significant effect on the spread of TMV infection in either wild-type plants or ABD2:GFP transgenic plants under our treatment conditions. We did not observe any binding of MP along the length of actin filaments. Collectively, these observations demonstrate that TMV movement does not require an intact actomyosin system. Nevertheless, actin-binding proteins appear to have the potential to exert control over TMV movement through the inhibition of myosin-associated protein trafficking along the ER membrane.

The mechanism by which plant viruses move from cell to cell and systemically to cause systemic infection has been the subject of intense studies (for review, see Carrington et al., 1996Go; Citovsky, 1999Go; Lazarowitz and Beachy, 1999Go; Tzfira et al., 2000Go; Heinlein and Epel, 2004Go; Waigmann et al., 2004Go; Lucas, 2006Go; Verchot-Lubicz et al., 2007Go). Plant viruses encode one or more movement proteins (MPs; Lucas, 2006Go) that facilitate the spread of the viral genome through plasmodesmata (PD), dynamic cell wall channels that mediate and control intercellular communication (Heinlein, 2002Go; Heinlein and Epel, 2004Go; Lucas and Lee, 2004Go). The most extensively studied MP is that of Tobacco mosaic virus (TMV). This protein localizes to PD and modifies the size-exclusion limit of the pores (Tomenius et al., 1987Go; Wolf et al., 1989Go; Atkins et al., 1991Go; Ding et al., 1992aGo; Moore et al., 1992Go; Oparka et al., 1997Go). The MP also has the capacity to bind single-stranded nucleic acids in vitro (Citovsky et al., 1990Go), and since complexes of MP and viral RNA (vRNA) were isolated from TMV-infected plants (Dorokhov et al., 1983Go, 1984Go), vRNA movement likely occurs in the form of a ribonucleoprotein complex. Indeed, the viral capsid protein is dispensable for cell-to-cell movement (Siegel et al., 1978Go; Dawson et al., 1988Go; Saito et al., 1990Go; Hilf and Dawson, 1993Go), indicating that the virus moves between cells in a nonencapsidated form. During infection, the protein interacts with endoplasmic reticulum (ER) membranes and microtubules, suggesting a role of these cellular components in vRNA movement (Heinlein et al., 1995Go, 1998Go). The intercellular transport of vRNA involves the occurrence of mobile, microtubule-proximal, MP-containing particles in cells undergoing the spread of infection (Boyko et al., 2007Go). Transient expression experiments provided evidence that the MP-containing particles are associated with RNA and that they move in association with ER membranes, thereby continuously undergoing transient interactions with microtubules (Sambade et al., 2008Go). The movement of the particles involves microtubule polymerization activity (Sambade et al., 2008Go), consistent with the ability of MP to interact with GFP-tagged microtubule end-binding protein 1 (AtEB1a; Brandner et al., 2008Go) and {gamma}-tubulin (Sambade et al., 2008Go).

The observed ER-mediated transport of MP-containing particles is consistent with the general perception that TMV movement involves the ER network. ER membranes are continuous between cells through PD (Ding et al., 1992bGo; Heinlein and Epel, 2004Go) and represent a potential pathway for the movement of membrane-associated replication complexes from the infected cells into adjacent cells. This model of TMV movement is supported by observations indicating that ER membrane proteins can laterally diffuse within the membrane (Runions et al., 2006Go; Guenoune-Gelbart et al., 2008Go) and move from cell to cell (Guenoune-Gelbart et al., 2008Go).

Since the ER is tightly associated with the actin network (Boevink et al., 1998Go), microfilaments could play an important role in supporting the ER-mediated targeting of MP and/or vRNA to PD. Consistent with this hypothesis, the treatment of plants with actin polymerization inhibitors reduces both MP particle trafficking (Sambade et al., 2008Go) and the efficiency by which MP accumulates in PD (Wright et al., 2007Go). Moreover, inhibition of the actin cytoskeleton for several days by either actin silencing (Liu et al., 2005Go) or inhibitor treatment (Kawakami et al., 2004Go) reduced the efficiency of TMV movement. The role of actin in supporting the spread of infection may be indirect, however. Although an interaction of MP with actin filaments has been reported (McLean et al., 1995Go), actin depolymerization reduces but does not fully inhibit PD targeting of MP (Wright et al., 2007Go). In addition, when actin inhibitor was present for longer than 18 h, no effect on the accumulation of MP in PD was observed (Prokhnevsky et al., 2005Go). These findings are consistent with the proposal that the targeting of MP to PD occurs by diffusion in the ER membrane (Wright et al., 2007Go) and that the actin cytoskeleton is not required for this mechanism.

To further clarify the role of the actin cytoskeleton in TMV movement, we applied latrunculin B (LatB) treatments to cells in which actin filaments were labeled by expression of the actin-binding domain 2 (ABD2) of Arabidopsis fimbrin (AtFIM1) fused to GFP (ABD2:GFP; Sheahan et al., 2004Go). Our observations demonstrate that TMV cell-to-cell movement can continue in the absence of an intact actin cytoskeleton and indicate that TMV movement is primarily ER membrane mediated. Nonetheless, ER-associated actin filaments and actin-binding proteins may play an important role in controlling TMV movement efficiency.


   RESULTS
TOP
 ABSTRACT
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 LITERATURE CITED
 

Labeling of Actin Filaments with ABD:GFP

To visualize actin filaments in vivo, we expressed ABD2:GFP from a binary vector under the control of the cauliflower mosaic virus 35S promoter. Transient expression of ABD2:GFP in tobacco (Nicotiana tabacum) BY-2 cells resulted in the green fluorescent labeling of a dense actin network (Fig. 1, A and B ). The effective labeling of actin filaments with ABD2:GFP was confirmed by costaining the cells with rhodamine-phalloidin (Fig. 1, C–E). Actin filaments were also effectively visualized in agroinfiltrated Nicotiana benthamiana leaves, where the coexpression of ABD2:GFP with ABD2 fused to red fluorescent protein (ABD2:RFP) resulted in exactly coinciding patterns of green- and red-labeled filaments (Fig. 1, F–H). The pattern of actin filaments in agroinfiltrated leaf tissues was similar to the pattern detected in ABD2:GFP transgenic plants (Fig. 1, I–M); however, the fluorescence emitted from ABD2:GFP in the agrotransfected tissues increased over time. Immunoblot analysis (Fig. 1N) confirmed that the average steady-state levels of transiently expressed ABD2:GFP and derived products in agroinfiltrated tissues started to exceed the level of ABD2:GFP expressed in transgenic plants at about 3 d postagroinfiltration (3 dpa; lane 6). Consistent with relatively low ABD2:GFP expression levels, the ABD2:GFP transgenic plants developed normally and were indistinguishable from nontransgenic wild-type plants (Fig. 1O). To avoid potential adverse effects produced by accumulating ABD2:GFP in agroinfiltrated plant tissues, subsequent experiments involving transient expression of the protein were performed at 1.5 dpa (if not otherwise noted).


Figure 1
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  		Figure 1. ABD2:GFP illuminates the actin cytoskeleton. A and B, Tobacco BY-2 suspension cell transiently expressing ABD2:GFP at 5 d after transfection by microparticle bombardment. The confocal images show labeled microfilaments in cortical (A) and central (B) optical sections. N, Nucleus. Bars = 10 µm. C to E, Tobacco BY-2 suspension cell transiently expressing ABD2:GFP and stained with rhodamine-phalloidin at 6 d after transfection. The patterns of ABD2:GFP fluorescence (C) and rhodamine-phalloidin staining (D) coincide (E), indicating that ABD2:GFP fluorescence specifically labels actin microfilaments. Bars = 10 µm. F to H, Pattern of ABD2:GFP (F) and ABD2:RFP (G) fluorescence in an epidermal cell of N. benthamiana leaf tissue at 1.5 dpa. ABD2:GFP and ABD2:RFP label the same actin microfilaments as seen in the merged image (H). Bars = 10 µm. I to M, Expression of ABD2:GFP in transgenic N. benthamiana plants. I, Epidermal tissue at low magnification showing strong ABD2:GFP fluorescence in stomata. Bar = 50 µm. J, Magnification of an epidermal cell showing a dense network of ABD2:GFP-labeled microfilaments. Bar = 10 µm. K to M, A trichome (K) with ABD2:GFP fluorescence in cortical (L) and central (M) views. Bars = 10 µm. N, Western-blot analysis. Shown are total protein extracts from leaf tissues of four different ABD2:GFP transgenic N. benthamiana lines (lanes 1–4), of agroinfiltrated plant tissues transiently expressing ABD2:GFP at 1.5 dpa (lane 5), 3 dpa (lane 6), and 6 dpa (lane 7) or GFP at 3 dpa (lane 8), and of mock-infiltrated tissue (lane 9) probed with GFP antibody. The molecular masses of marker proteins are indicated on the left. The prominent protein band of 70 kD (arrow) corresponds to the expected size of ABD2:GFP. Additional proteins of lower molecular mass (lanes 1–7) may represent free GFP and degradation products. The bottom panel shows a section of the Coomassie Brilliant Blue-stained membrane. O, Similar development of wild-type (left) and ABD2:GFP transgenic (right) plants.

 

Transient, But Not Transgenic, ABD2:GFP Expression Interferes with Myosin-Based Motility

The actin cytoskeleton is dynamic and undergoes continuous rearrangements (Staiger and Blanchoin, 2006Go). We used time-lapse microscopy to examine the effect of ABD2:GFP expression on actin filament dynamics in leaf epidermal cells of agroinfiltrated (at 1.5 dpi) or transgenic plants. Whereas the ABD2:GFP-labeled actin cytoskeleton in ABD2:GFP transgenic plants (Fig. 2A ) showed dynamic movements of individual filaments (Supplemental Movie S1), the ABD2:GFP-labeled actin cytoskeleton in agroinfiltrated ABD2:GFP-expressing tissues (Fig. 2B) was static (Supplemental Movie S2). Thus, transient expression of ABD2:GFP causes stabilization of an otherwise dynamic actin cytoskeleton.


Figure 2
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  		Figure 2. Structure and dynamic behavior of actin and ER networks in tissues expressing ABD2:GFP under transient or stable conditions. A, ABD2:GFP-labeled microfilaments in an epidermal cell of an ABD2:GFP transgenic plant. The filaments exert dynamic movements (Supplemental Movie S1). B, ABD2:GFP-labeled microfilaments in an epidermal cell of transiently ABD2:GFP-expressing tissue at 1.5 dpa. The filaments are static (Supplemental Movie S2). C, GFP-tagged ER network of an epidermal cell of N. benthamiana line 16c. D and E, Transgenically expressed ABD2:GFP does not cause detectable alterations in the structure and dynamic behavior of the ER network. D, ABD2:GFP-labeled actin microfilaments. E, ER network of the same cell as shown in D labeled with transiently expressed RFP:HDEL at 1.5 dpa. This is a frame of a time-lapse movie (Supplemental Movie S3). F to M, Transient expression of ABD2:RFP causes varying effects on the structure and dynamic behavior of the ER network at 1.5 dpa. F and G, An epidermal cell of N. benthamiana line 16c showing no obvious effects of ABD2:RFP on the structure of the ER network. However, the dynamic ER-remodeling activity is inhibited (Supplemental Movie S5). F, ABD2:RFP-labeled actin microfilaments. G, Frame of the movie showing dynamic inhibition of the GFP-tagged ER network. H and I, An epidermal cell of N. benthamiana line 16c showing gaps in the ER network. H, ABD2:RFP-labeled actin microfilaments. I, GFP-tagged ER network. J and K, An epidermal cell of wild-type N. benthamiana tissue coexpressing ABD2:RFP and TM17:GFP at 1.5 dpa. The TM17:GFP-labeled ER network is partially coalesced into large aberrant sheets. J, ABD2:RFP-labeled actin microfilaments. K, TM17:GFP-tagged ER network. L and M, Examples of N. benthamiana line 16c epidermal cells showing the partial (L) or complete (M) contraction of the ER network to the vertices. Such contracted ER structures are dynamically inhibited (Supplemental Movie S4). All bars = 10 µm.

 
Since a functional actin-myosin system mediates ER and Golgi motility in plants (Boevink et al., 1998Go; Lichtscheidl and Baluska, 2000Go), we investigated the effect of ABD2 expression on ER and Golgi structure and motility by coexpression of specific, fluorescent protein-tagged markers. The structure and dynamic behavior of the ER is conveniently revealed by confocal microscopy of epidermal cells of N. benthamiana line 16c, which is transgenic for GFP carrying an HDEL ER retention sequence (Ruiz et al., 1998Go). The ER network in these plants consists of the typical polygonal pattern of membrane sheets and tubules (Fig. 2C) and is highly dynamic. A dynamic ER network was also revealed by time-lapse analysis of wild-type plants in which the ER was labeled by transient expression of GFP:HDEL, RFP:HDEL, or TM17:GFP, an ER transmembrane peptide fused to GFP (Brandizzi et al., 2002Go; data not shown). Transient expression of RFP:HDEL in ABD2:GFP transgenic plants revealed that transgenic ABD2:GFP expression does not interfere with the structure and dynamic movements of the ER (Fig. 2, D and E; Supplemental Movie S3). However, upon transient expression of ABD2:RFP in epidermal cells of 16c plants, varying effects on ER structure were observed at 1.5 dpa. Some cells exhibited no obvious defects in the structure of the ER network (Fig. 2, F and G), whereas other cells showed clear defects such as gaps in the ER network (Fig. 2, H and I), the fusion of ER membranes into large sheets (Fig. 2, J and K), or the partial (Fig. 2L) to almost complete (Fig. 2M) contraction of the ER to the vertices. Transient ABD2:GFP expression also caused a partial or complete inhibition of ER membrane movements; this was seen irrespective of whether the ER structure was altered (Fig. 2M; Supplemental Movie S4) or not (Fig. 2G; Supplemental Movie S5). Similar observations were made when ABD2:GFP was coexpressed with the ER marker RFP:HDEL in wild-type plants (data not shown), thus confirming that the transient ABD2:GFP expression affects the structure and dynamic behavior of ER membranes under the conditions applied.

The motility of Golgi stacks depends on specific class XI myosins (Avisar et al., 2008bGo; Peremyslov et al., 2008Go; Prokhnevsky et al., 2008Go; Sparkes et al., 2008Go), requires an intact and dynamic actin cytoskeleton (Nebenführ et al., 1999Go; Brandizzi et al., 2002Go), and appears to occur with the membranes of the ER (Runions et al., 2006Go). To investigate whether expression of ABD2:GFP affects actin-myosin-based Golgi motility, we monitored the motility of Golgi stacks upon transient expression of the red fluorescent, tdTomato-tagged, cis-Golgi marker GmMan1 (GmMan1:tdTomato; Nebenführ et al., 1999Go; Fig. 3, A and B ). Projections of pixel intensities between frames in time-lapse movies display the "paths" of translocating Golgi stacks through the cell (Fig. 3, C and D). In time-lapse movies taken for the duration of 20 s, the velocity of observed Golgi stack movements varied between 0 and 4.1 µm s–1. These values are within the range of velocities reported for Golgi stacks in different plant tissues (Boevink et al., 1998Go; Nebenführ et al., 1999Go; Avisar et al., 2008bGo; Prokhnevsky et al., 2008Go; Sparkes et al., 2008Go). Thus, Golgi trafficking appears not to be affected by transient expression of Golgi marker GmMan1:tdTomato. Expression of this marker in transgenic ABD2:GFP plants revealed dynamic Golgi trafficking comparable to that in wild-type plants (Fig. 3, E–H; Supplemental Movie S6). In contrast, Golgi stacks were immobile in cells transiently coexpressing ABD2:GFP (Fig. 3, I–L; Supplemental Movie S7).


Figure 3
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  		Figure 3. Golgi stack movements in the absence and presence of ABD2:GFP as visualized by transient expression of GmMan1:tdTomato at 1.5 dpa. A to D, Golgi stack movements in N. benthamiana epidermal leaf cells. Bars = 10 µm. A, Single movie frame showing the presence of several GmMan1:tdTomato-labeled Golgi stacks in an epidermal cell. B, Projection of dynamic movie pixels showing that the Golgi stacks are highly mobile. C and D, The paths (red lines) of two individual Golgi stacks (circled) during 20 s. E to H, Time-lapse movie (Supplemental Movie S6) showing Golgi stack movements in ABD2:GFP transgenic N. benthamiana. Bars = 10 µm. E, Single movie frame showing the presence of several red fluorescent GmMan1:tdTomato-labeled Golgi stacks as well as green fluorescent ABD2:GFP-labeled actin filaments in an epidermal cell. F, Projection of dynamic movie pixels showing that the Golgi stacks are highly mobile. G and H, The paths (red lines) of two individual Golgi stacks (circled) during 20 s. I to L, Time-lapse movie (Supplemental Movie S7) showing Golgi stack movements in an epidermal cell of N. benthamiana leaf tissue transiently expressing GmMan1:tdTomato and ABD2:GFP at 1.5 dpa. Bars = 10 µm. I, Single movie frame showing the presence of several red fluorescent GmMan1:tdTomato-labeled Golgi stacks as well as green fluorescent ABD2:GFP-labeled actin filaments. J, Projection of dynamic movie pixels showing that the Golgi stacks are immobile, except for locally wiggling motions at their fixed positions. K and L, The immobile state of two individual Golgi stacks (circled) during 20 s.

 
To investigate whether ABD2:GFP expression may also affect microtubule dynamics, we expressed the Arabidopsis AtEB1a fused to GFP (AtEB1a:GFP; Chan et al., 2003Go). Expression of AtEB1a:GFP has been reported to reveal the dynamic behavior of microtubules upon transgenic or transient expression in Arabidopsis, tobacco, and N. benthamiana cells (Chan et al., 2003Go; Mathur et al., 2003Go; Van Damme et al., 2004Go; Dixit et al., 2006Go; Brandner et al., 2008Go). However, transient coexpression of ABD2:GFP had no significant effect on microtubule polymerization (data not shown), indicating that microtubules are not affected by the inhibition of membrane movements induced by transient ABD2:GFP expression.


TMV Movement in the Presence of an Actin-Disrupting Agent

To investigate the role of actin filaments in TMV movement, we used TMV-MP:RFP, a TMV derivative that encodes full-length MP in fusion to RFP under the control of the MP subgenomic promoter (Ashby et al., 2006Go). Following inoculation of N. benthamiana plants, leaf tissues carrying infection sites were infiltrated at 3.5 d postinfection (dpi) with buffer only or with buffer containing 10 µM or 100 µM LatB. Images of individual infection sites were acquired at 3.5 and 4.5 dpi (Fig. 4B ), and the fold increase in size of each site over the intervening 24 h was determined. The fold increase reflects the extent of cell-to-cell progression of TMV infection within the assay period. Multiple infection sites were measured in three independent experiments. As shown in Figure 4, A and B, TMV-MP:RFP infection sites in leaves of wild-type plants as well as in leaves of ABD2:GFP transgenic plants continued to expand efficiently irrespective of the presence of LatB. Microscopic analysis of infection sites in leaves of ABD2:GFP transgenic plants revealed that the actin cytoskeleton in mock-treated tissues was intact (Fig. 4C), whereas the actin network was disrupted in the cells of leaves treated with LatB (Fig. 4D). Furthermore, after 24 h of LatB treatment, the structure of the ER network was also affected. Consistent with a previous report (Wright et al., 2007Go), we observed that the ER was contracted to the vertices (Fig. 4F).


Figure 4
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  		Figure 4. Movement of TMV-MP:RFP is not inhibited in tissues treated for 24 h with LatB. A, Expansion of TMV-MP:RFP infection sites in the inoculated leaf of wild-type (wt) and ABD2:GFP-transgenic N. benthamiana plants. The fold increase in the size of the infection sites reflects the extent of virus spread during the assay period. LatB treatment does not cause a significant reduction in the spread of infection during this time. Error bars show SD. The number of individually analyzed infection sites is shown in parentheses. B, Examples of TMV-MP:RFP infection sites at 3.5 and 4.5 dpi in leaves treated or not treated with LatB. Bars = 200 µm. C and D, Degradation of microfilaments by LatB treatment in ABD2:GFP transgenic plants. Bars = 10 µm. C, Intact ABD2:GFP-labeled microfilaments in tissue treated for 24 h with mock solution. D, ABD2:GFP-labeled microfilaments are degraded in tissue treated for 24 h with 10 µM LatB. E and F, Structure of the ER network in epidermal cells of N. benthamiana line 16c plants before and after LatB treatment for 24 h. Bars = 10 µm. E, Native structure of the ER network before treatment. F, In tissues treated with LatB, the ER network shows extensive contraction to the vertices.

 

TMV Movement in the Presence of Transiently Expressed ABD2:GFP

As described above, transient expression of ABD2:GFP leads to inhibition of the dynamic myosin-dependent movements of Golgi stacks without affecting the integrity of the actin filaments. To test whether the apparent inhibition of myosin-dependent transport activity has an effect on the efficiency of TMV movement, leaves carrying TMV-MP:RFP infection sites were agroinfiltrated at 3 dpi. After 1.5 dpa, the expression of ABD2:GFP was verified by fluorescence microscopy and images of individual infection sites were acquired. Subsequently, the leaf samples were either mock treated or treated with LatB as described for wild-type and ABD2:GFP transgenic plants, and the individual infection sites were again photographed 24 h later (thus at 2.5 dpa). As a control for the agroinfiltration experiment, the inoculated leaves of control plants were not infiltrated, infiltrated with water, infiltrated with agrobacteria carrying an empty binary vector, or infiltrated with agrobacteria containing a binary vector that encodes free GFP. As summarized in Figure 5A , TMV-MP:RFP infection sites expanded efficiently in these control leaves. However, the spread of infection was significantly inhibited in leaves transiently expressing ABD2:GFP. Importantly, the spread of infection in the ABD2:GFP-expressing tissue was not inhibited if the ABD2:GFP-expressing leaves were treated with LatB, indicating that transiently expressed ABD2:GFP interferes with the spread of TMV-MP:RFP infection through a mechanism requiring actin filaments. Microscopic analysis of ABD2:GFP revealed that the actin cytoskeleton in mock-treated tissues was intact (Fig. 5B), whereas it was disrupted in LatB-treated tissues (Fig. 5, C and D). Consistent with the LatB treatment results obtained with wild-type and ABD2:GFP transgenic plants, LatB had no effect on TMV-MP:RFP movement in control leaves that were not infiltrated or were infiltrated either with water or with agrobacteria carrying either an empty plasmid or a GFP-encoding binary plasmid. We also performed a reverse experiment in which the leaves were first agroinfiltrated and then infected with virus 1.5 d later. As displayed in Table I , TMV-MP:RFP infection sites failed to develop in the transiently ABD2:GFP-expressing leaves, whereas infection sites readily developed in control leaves. Collectively, these observations indicate that TMV movement continues in the absence of an intact actin cytoskeleton and, thus, of actomyosin activity. However, in the presence of the actin cytoskeleton, the progression of TMV infection and myosin-mediated Golgi trafficking are inhibited by transient expression of ABD2:GFP.


Figure 5
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  		Figure 5. Movement of TMV-MP:RFP during 24 h in transiently ABD2:GFP-expressing tissue (1.5–2.5 dpa) treated or not treated with LatB. A, Expansion of TMV-MP:RFP infection sites (fold increase in the size of infection sites) in LatB-treated (gray columns) and nontreated (white columns) tissues during the assay period. In the absence of LatB, the efficiency of TMV movement is significantly reduced in agroinfiltrated tissues transiently expressing ABD2:GFP but not in control infiltrated cells. LatB treatment has no inhibitory effect on the extent of TMV-MP:RFP movement during 24 h. However, LatB treatment abolishes the inhibitory effect of transient ABD2:GFP expression on TMV-MP:RFP movement. Error bars show SD. The number of individually analyzed infection sites is shown in parentheses. no inf., No infiltration. B to D, Degradation of microfilaments by LatB treatment in tissues transiently expressing ABD2:GFP. Bars = 10 µm. B, Intact ABD2:GFP-labeled microfilaments in tissue treated for 24 h with mock solution. C, ABD2:GFP-labeled microfilaments are degraded in tissue treated for 24 h with 10 µM LatB. D, Image shown in C is merged with a differential interference contrast image of the same cell.

 

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  		Table I. Established ABD2:GFP expression interferes with infection

Values shown are numbers of TMV-MP:RFP infection sites in 10 N. benthamiana leaves that were first (agro)infiltrated and then, after 36 h, infected with the virus.

 

Subcellular Localization of MP:RFP with Respect to Actin Filaments

In TMV-MP:RFP infection sites, the subcellular localization patterns of MP:RFP are similar to those described for the MP:GFP of TMV-MP:GFP (Heinlein et al., 1998Go; Supplemental Fig. S1) and include the association with microtubules (Heinlein et al., 1998Go; Boyko et al., 2000bGo; Ashby et al., 2006Go). Because immunostaining procedures applied to fixed protoplasts have indicated that MP may also have the capacity to align along phalloidin-rhodamine-labeled microfilaments (McLean et al., 1995Go), we investigated whether some of the in vivo MP:RFP-decorated filaments may represent actin filaments. However, confocal microscopy of infection sites in ABD2:GFP transgenic plants revealed that MP:RFP-associated filaments are clearly distinct from the ABD2:GFP-labeled microfilaments (Fig. 6, A–F ). Nevertheless, some MP:RFP-associated aggregate structures (ER-associated viral replication sites) occurred in the vicinity of ABD2:GFP-labeled filaments (Fig. 6, A–C and G–I), which is consistent with the actin-dependent development and trafficking of these structures (Heinlein et al., 1998Go; Kawakami et al., 2004Go; Liu et al., 2005Go).


Figure 6
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  		Figure 6. Subcellular localization of MP:RFP within TMV-MP:RFP infection sites in relation to the actin cytoskeleton in ABD2:GFP transgenic plants. Bars = 10 µm. A to C, ABD2:GFP-labeled actin cytoskeleton (A) in a cell in which MP:RFP localizes to filaments and irregular structures (B). The merged image (C) demonstrates that the MP:RFP-associated filaments are distinct from ABD2:GFP-labeled actin filaments. Moreover, the irregular structures seem to occur in the vicinity of the two filament systems. A magnification of the area highlighted by the white square is shown in the inset. D to F, ABD2:GFP-labeled actin cytoskeleton (D) in a cell in which MP:RFP localizes to filaments (E). The merged image (F) demonstrates that the MP:RFP-associated filaments are distinct from ABD2:GFP-labeled actin filaments. G to I, Confocal sectioning through an image volume in which red and green channels have been merged. The red fluorescent MP:RFP-associated irregular structures are associated with green fluorescent ABD2:GFP-labeled actin filaments. However, sectioning through the volume of the large structures is required to reveal this association.

 

   DISCUSSION
TOP
 ABSTRACT
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 LITERATURE CITED
 
In this study, we investigated the role of the actin cytoskeleton as well as the potential involvement of interactions between MP and actin filaments in TMV movement. To address the in vivo localization of MP with respect to actin filaments and to verify the effect of treating plant tissue with the actin polymerization inhibitor LatB, we labeled the actin filaments by expression of the actin-binding protein ABD2:GFP. Interestingly, we found that transient, unlike stable, expression of this marker interferes with actomyosin-dependent motility, as indicated by the lack of Golgi stack movements. Thus, in our analysis of the role of the actin cytoskeleton in TMV movement, ABD2:GFP had a dual role: in addition to serving as a marker to visualize the actin network, its transient expression enabled us to apply this protein as an inhibitor of myosin-based motility in the presence of an intact actin cytoskeleton. Our results demonstrate that TMV movement is inhibited upon inhibition of myosin-based motility by transient expression of ABD2:GFP. This inhibition is dependent on the actin cytoskeleton and eliminated upon disruption by LatB. Thus, whereas the inhibition of TMV movement with transiently expressed ABD2:GFP is actin dependent, the mechanism that supports TMV movement is actin independent. Collectively, these findings are consistent with the proposal that the intracellular transport of TMV occurs via the ER and that the adjacent actin cytoskeleton may exert control over ER-mediated transport via myosin bridges.


Inhibition of TMV Movement by ABD2:GFP through Interference with Macromolecular Trafficking in the ER Membrane

The observation that transient ABD2:GFP expression interfered with myosin-mediated motility is in agreement with several other reports indicating that the expression of GFP fusions with actin-binding proteins can interfere with actin dynamics and organization, even though they are valuable tools for evaluating cytoskeletal functions (Kost et al., 1998Go; Mathur et al., 1999Go; Fu et al., 2001Go; Brandizzi et al., 2002Go; Ilgenfritz et al., 2003Go; Holweg et al., 2004Go; Ketelaar et al., 2004Go; Weerasinghe et al., 2005Go; Xu and Scheres, 2005Go; Holweg, 2007Go; Wang et al., 2008Go). The inhibition of myosin-based dynamics upon transient expression of ABD2:GFP in our N. benthamiana system is probably related to the ABD2:GFP expression level, which during the time of the experiment (1.5 and 2.5 dpa) was in excess of the level of ABD2:GFP in transgenic plants. The mechanism that inhibits myosin-mediated motility and TMV movement upon transient ABD2:GFP expression is LatB sensitive and, therefore, actin dependent. Based on several reports describing the inhibition of kinesin-based movements by microtubule-associated proteins (Lopez and Sheetz, 1993Go; Ebneth et al., 1998Go; Ashby et al., 2006Go; Dixit et al., 2008Go; Vershinin et al., 2008Go), it seems probable that excess binding of ABD2:GFP to the actin cytoskeleton interferes with the translocation of myosin motor proteins along the filaments. The observed restriction of Golgi and TMV trafficking likely involves the inhibition of class XI myosins, which move along actin filaments (Tominaga et al., 2003Go) and are responsible for the dynamic movements of Golgi complexes and other organelles (Avisar et al., 2008bGo; Peremyslov et al., 2008Go; Prokhnevsky et al., 2008Go; Sparkes et al., 2008Go) as well as for those of the ER (Kashiyama et al., 2000Go; Morimatsu et al., 2000Go; Kimura et al., 2003Go; Yokota et al., 2009Go). The myosin-driven Golgi movements occur along actin filaments that align the ER (Boevink et al., 1998Go) and have been proposed to reflect myosin-supported movements of ER membrane proteins (Runions et al., 2006Go). The inhibition of Golgi trafficking upon transient ABD2:GFP expression may thus reflect inhibition of the trafficking of protein complexes in the ER membrane. Given that TMV movement occurs via diffusion in the ER membrane (Brill et al., 2000Go; Wright et al., 2007Go; Guenoune-Gelbart et al., 2008Go; Sambade et al., 2008Go), we propose that the ABD2:GFP-mediated restriction in TMV movement occurs through obstruction of the ER membrane by immobilized protein complexes (Fig. 7 ). We speculate that the inhibitory mechanism involves interference with class XI myosins; nevertheless, class VIII myosins may also have a role, since they are associated with PD (Reichelt et al., 1999Go; Volkmann et al., 2003Go; Golomb et al., 2008Go). Although specific inhibition of class VIII myosin cargo binding was reported to have no effect on the targeting of the TMV MP to PD (Avisar et al., 2008aGo), inhibiting the translocation of this myosin class along actin filaments could still lead to hindrance in the cellular pathway that supports the spread of infection. We also consider the possibility that ABD2:GFP expression may trigger inhibition of TMV movement already during the formation or functioning of ER-associated TMV replication complexes. Nonetheless, a significant influence of transient ABD2:GFP expression on virus replication efficiency appears unlikely, since the fluorescence intensity of TMV-MP:RFP infection sites was not decreased irrespective of the presence of this protein.


Figure 7
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  		Figure 7. Model of inhibition of TMV movement by transient overexpression of ABD2:GFP. A, Actin filaments and associated myosin motors permit the movements of Golgi complexes. MP and associated vRNA as well as complexes that link myosin motors to the actin filaments move laterally within the ER membrane. B, Upon disruption of the actin filaments, the Golgi movements are inhibited. However, MP and associated vRNA are still transported by lateral diffusion in the ER. C, Low-level expression of ABD2:GFP in transgenic plants does not interfere with Golgi movements or with the ER-associated diffusion of MP and vRNA. D, Disruption of actin filaments in transgenic ABD2:GFP-expressing tissues interferes with Golgi motility but not with the diffusion of MP and vRNA in the ER membrane. E, High amounts of ABD2:GFP produced upon transient expression accumulate on actin filaments and block the processing of myosin motors. Inhibition of the motile system leads to the immobilization of protein complexes in the ER membrane. This interferes with the targeting of MP and vRNA to PD. F, Disruption of actin filaments interferes with the formation of the inhibitory ABD2:GFP complex. Thus, MP and associated vRNA can target PD by diffusion in the ER membrane even in the presence of high amounts of ABD2:GFP. T.m., TMV movement.

 

TMV Movement Is Independent of Actin

The ability of LatB to eliminate the inhibition of TMV movement caused by transiently expressed ABD2:GFP as well as the absence of any significant effect of LatB on TMV movement efficiency in wild-type or ABD2:GFP transgenic plants demonstrate that an intact actin cytoskeleton is dispensable for TMV movement. Since MP traffics in the ER membrane (Brill et al., 2000Go; Wright et al., 2007Go; Guenoune-Gelbart et al., 2008Go; Sambade et al., 2008Go), this finding is in agreement with the reported ability of ER membrane proteins to continue their trafficking in the membrane upon disruption of actin filaments with LatB (Runions et al., 2006Go). Nevertheless, although an effect of LatB treatment on the spread of infection was not observed, the absence of intact actin filaments has important effects with respect to the efficiency of PD targeting. Thus, disruption of the actin cytoskeleton slowed down the trafficking of ER membrane-associated MP (Sambade et al., 2008Go) and of other ER proteins (Runions et al., 2006Go). LatB treatment also reduced the accumulation of MP in PD (Wright et al., 2007Go). These effects of LatB on ER-mediated transport have the potential to reduce the number of virus genomes that spread between cells over time. However, because the number of transported genomes is usually not a limiting factor in the spread of infection (Sacristan et al., 2003Go; Li and Roossinck, 2004Go), the effects of LatB do not necessarily translate into an obvious effect at the level of infection sites. Thus, even though we did not observe an effect of LatB treatment on the spread of infection in N. benthamiana leaf tissues under our conditions, we do not exclude the possibility that a potential reduction in the number of transported virus genomes may represent a critical factor for infection in other hosts or tissues. Moreover, an inhibition of virus movement in LatB-treated cells may depend on the experimental conditions. As concluded here, TMV movement occurs undisturbed upon disruption of the actin cytoskeleton for 24 h; however, according to previous reports, TMV movement can be inhibited if the disruption of the actin cytoskeleton continues for several days (Kawakami et al., 2004Go; Liu et al., 2005Go). We note that our conclusions are based on the analysis of over 150 individual infection sites treated with LatB (Figs. 4 and 5), whereas the previous reports were based on smaller sample sizes. Nevertheless, inhibition of TMV movement following prolonged LatB treatment may be expected, since the absence of an intact actin network gradually induces the contraction of the ER (Wright et al., 2007Go). The contraction of the ER is not observed if the drug is applied during shorter time periods (Boevink et al., 1998Go; Sambade et al., 2008Go). Thus, the general structure and function of the ER is independent of actin and persists over a certain time in the presence of LatB. It might be considered that the alterations in ER structure observed in ABD2:GFP-overexpressing cells contributed to the ABD2:GFP-induced inhibition of TMV movement. However, this possibility appears unlikely, since the inhibition by ABD2:GFP was abolished upon LatB treatment; rather, LatB should enhance inhibition if mediated by an aberrant ER structure. We also observed the contraction of ER following the application of LatB for 24 h, thus at the end of the time window during which our TMV movement assay was performed and TMV efficiently moved from cell to cell. Since LatB-induced ER contraction occurs gradually, we assume that the changes in ER structure did not reach inhibitory levels within the 24-h observation period of our assay. However, longer LatB treatments may affect the structure of the ER to an extent that interferes with TMV movement.


MP Does Not Align to Actin Filaments

Neither in cells infected with TMV-MP:RFP nor in cells ectopically expressing MP:RFP did we observe any alignment of MP:RFP to ABD2:GFP-labeled actin filaments. Although MP:RFP-associated filaments were observed, these filaments were not labeled with ABD2:GFP. We cannot exclude the possibility that ABD2:GFP binding to actin filaments interferes with MP:RFP binding. However, since other studies have demonstrated that the filaments to which MP binds in plant and mammalian cells are microtubules (Heinlein et al., 1995Go, 1998Go; Boyko et al., 2000aGo, 2000bGo; Ashby et al., 2006Go; Ferralli et al., 2006Go), this possibility appears unlikely. Our observation is consistent with the finding that MP does not bind to actin filaments in mammalian cells (Ferralli et al., 2006Go), and actin is highly conserved between plants and mammals. The lack of alignment of MP:RFP to ABD2:GFP-tagged actin filaments in living cells does not confirm the previous observations by McLean et al. (1995)Go, who showed the alignment of antibody-stained MP to phalloidin-rhodamine-labeled filaments in chemically fixed TMV-infected protoplasts. It is conceivable that MP has the potential to bind actin in protoplasts or in the presence of a fixative. It is also possible that the binding to plant actin is a property of nonfused MP, although nonfused MP did not show interactions with actin filaments when expressed in mammalian cells (Ferralli et al., 2006Go). Nevertheless, our findings are consistent with the model that MP and MP/vRNA particle trafficking is primarily ER mediated and does not involve direct interactions with actin filaments.

Although MP:RFP binding along ABD2:GFP-labeled actin filaments was not observed, we noticed that MP:RFP-containing bodies occurred in the vicinity of the filaments. This finding presumably reflects the previously reported association of viral replication complexes and fluorescent protein-tagged 126-kD replicase protein with microfilaments labeled with fluorescent protein-tagged talin or ABD2 markers (Liu et al., 2005Go).

Collectively, our results suggest that the PD-targeted transport of MP and associated viral RNA occurs by diffusion in the ER membrane and that TMV movement can be inhibited by conditions that dominantly block this pathway (Fig. 7). The actomyosin system, including its actin-binding factors, may control this pathway by supporting or slowing down the transport of membrane-embedded protein complexes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
LITERATURE CITED
 

DNA Constructs

The plasmid encoding the cDNA of TMV-MP:RFP has been described previously (Ashby et al., 2006Go). Binary plasmids pK7.ABD2:GFP and pK7.ABD2:RFP were generated by Gateway cloning. Here, the open reading frame of ABD2 was PCR amplified from pRSAT.GFP:ABD2 (Sheahan et al., 2004Go) and cloned into Gateway donor vector pDONRzeo (Invitrogen). Subsequently, the open reading frame was recombined into destination plasmids pK7FWG2.0 (Karimi et al., 2002Go) and pK7RWG2.0 (kindly provided by R. Tsien) for expression in C-terminal fusion to GFP and RFP, respectively. Binary plasmids encoding GmMan1:tdTomato, GFP:HDEL, and RFP:HDEL were provided by Prof. A. Nebenführ. The membrane marker TM17:GFP was provided by N. Paris.


BY-2 Cell Suspension Culture, Microparticle Bombardment, and Actin Staining

Tobacco (Nicotiana tabacum) BY-2 suspension cells were maintained at 28°C under constant agitation at 120 rpm in BY-2 medium (Murashige and Skoog; Duchefa), 1 mg L–1 thiamine, 200 mg L–1 KH2PO4, 0.2 mg L–1 2,4-dichlorophenoxyacetic acid, 100 mg L–1 myoinositol, and 3% Suc, pH 5.8). Cells were transfected with plasmid pK7.ABD2:GFP by microprojectile bombardment as described previously (Vetter et al., 2004Go).

For visualization of actin filaments, the cells were briefly stained for 2 min with 5 µg mL–1 rhodamine-phalloidin in staining buffer (50 mM PIPES, 5 mM EGTA, 2 mM MgCl2, 0.05% Triton, and 5% dimethyl sulfoxide) and immediately observed by fluorescence microscopy.


Plant Material

Nicotiana benthamiana wild-type plants as well as plants of transgenic plant line 16c (Ruiz et al., 1998Go) and of a plant line transgenic for ABD2:GFP (see below) were grown and maintained under greenhouse conditions (50% humidity, 18°C–25°C). Three- to 4-week-old plants were used for infiltration and inoculation experiments.


Agroinfiltration

Transformed agrobacteria (Agrobacterium tumefaciens, line LBA4404; Life Technologies) were grown at 28°C in 2 mL of Luria-Bertani medium containing antibiotics. Upon harvest by centrifugation and resuspension in water at an optical density at 600 nm of 0.05, bacteria were syringe infiltrated into the abaxial side of the leaf.


Plant Transformation

ABD2:GFP transgenic N. benthamiana plants were generated by Agrobacterium-mediated leaf disc transformation (Horsch et al., 1985Go) using plasmid pK7.ABD2:GFP. Following recovery of whole plants on selective medium, plants were screened for expression of ABD2:GFP using fluorescence microscopy. Four different lines showing similar ABD2:GFP localization patterns to filaments were selected for further experiments.


LatB Treatment

LatB solutions (10 or 100 µM in BY-2 cell medium) were freshly prepared for each experiment and syringe infiltrated into the abaxial leaf side. Control infiltrations were performed with the same buffer but without inhibitor.


Western-Blot Analysis

Total proteins were isolated from leaf discs (diameter, 0.5 cm) in extraction buffer (50 mM Tris-HCl, 100 mM dithiothreitol, 10% glycerol, 2% SDS, and 0.1% bromphenol blue). Following denaturation at 95°C for 5 min and subsequent sonication for 20 min in a Branson 2200 sonicator, the proteins were size fractionated by SDS gel electrophoresis, blotted onto membranes, and probed with a 1:2,000 dilution of mouse monoclonal GFP antibody (Clontech) and anti-mouse IgG horseradish peroxidase-linked secondary antibody (Molecular Probes). The antibody-decorated membranes were then treated with the Lumi-Light PLUS Western Blotting Kit (Roche) for chemiluminescence signal detection autoradiography.


Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy was performed using a Zeiss LSM510 laser scanning confocal microscope with a C-Apo-chromat (63x/1.2-W Korr) water objective lens in multitrack mode. Excitation/emission wavelengths were 488 nm/505 to 545 nm for GFP and 543 nm/585 to 615 nm for RFP. Images were acquired using LSM510 version 2.8 software (Zeiss).


Time-Lapse Fluorescence Microscopy

Time-lapse fluorescence microscopy was performed using a Nikon TE2000 inverted microscope equipped for real-time imaging with a Roper CoolSnap digital CCD camera, piezo-driven Z-focus, and a 60x, 1.45 numerical aperture total internal reflection fluorescence objective. Excitation/emission wavelengths were 460 to 500 nm/510 to 560 nm for GFP and 550 to 600 nm/615 to 665 nm for RFP. For simultaneous dual-color acquisitions, a Dual-View beam splitter (Optical Insights) was used. The beam splitter was equipped with GFP-mRFP1 exciter and mirror as well as with emission filters HQ510/30m for GFP and HQ650/75m for RFP. To reduce red background emission fluorescence caused by chlorophyll, an e640sp short-pass filter (Chroma) was inserted between the beam splitter and the CCD camera. Images were acquired using Metamorph (6.2r6) software.


Image Processing

Following acquisition, images were processed using Metamorph (6.2r6), ImageJ (1.38u), and Adobe Photoshop (version 7.0) software. Specific algorithms (http://rsb.info.nih.gov/ij/macros/) implemented in the software ImageJ (1.38u) were used for dynamic pixel analysis and display ("slice-to-slice-difference"), the tracking of individual Golgi stacks and display of paths ("trace"), and the measurement of the sizes of TMV-MP:RFP infection sites ("threshold" to define regions of interest, "particle analyzer" to count the number of pixels within the regions of interest).


Analysis of TMV-MP:RFP Infection Sites

Leaf discs carrying TMV-MP:RFP infection sites were excised at 3.5 dpi and images were taken. The leaf discs were then incubated individually for 24 h in wells of a 24-well plate containing BY-2 cell medium. After the incubation period, images of the same infection sites, applying strictly constant acquisition conditions, were again taken. The fluorescent area of the individual infection sites before and after the 24-h incubation period was measured using ImageJ software. To compare the sizes of many infection sites with different initial sizes, for each infection site the initial size value was set to 1 and the relative fold size increase was calculated. Measurements were performed with multiple infection sites in three individually performed experiments.


Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Subcellular localization of MP:RFP within TMV-MP:RFP infection sites at 5 dpi.
Supplemental Movie S1. Dynamic movements of actin filaments in epidermal cells transgenic for ABD2:GFP; time is in seconds.
Supplemental Movie S2. Absence of dynamic actin filament movements in epidermal cells upon transient expression of ABD2:GFP; time is in seconds.
Supplemental Movie S3. Dynamic ER movements in epidermal cells transgenic for ABD2:GFP; time is in seconds.
Supplemental Movie S4. Aberrant ER structure and absence of dynamic ER movements in an epidermal cell transiently expressing ABD2:RFP; time is in seconds.
Supplemental Movie S5. Absence of dynamic ER movements but presence of an otherwise normal ER network structure in an epidermal cell transiently expressing ABD2:RFP; time is in seconds.
Supplemental Movie S6. Dynamic Golgi movements in epidermal cells transgenic for ABD2:GFP; the Golgi complexes are labeled with GmMan1:tdTomato; time is in seconds.
Supplemental Movie S7. Absence of dynamic Golgi movements in epidermal cells upon transient expression of ABD2:GFP; time is in seconds.


   ACKNOWLEDGMENTS
 
We thank D. McCurdy (University of Newcastle, Australia) for providing plasmid pRSAT.GFP:ABD2. We are also grateful to R. Tsien (University of California, San Diego) for providing plasmids pK7FWG2.0 and pK7RWG2.0, A. Nebenführ (University of Tennessee, Knoxville) for binary plasmids encoding GmMan1:tdTomato, GFP:HDEL, and RFP:HDEL, and N. Paris (Université de Rouen, France) for providing the membrane marker TM17:GFP. We also thank Pascal Cobanov (RLP AgroScience, AlPlanta, Institute for Plant Research, Neustadt/Weinstraße, Germany) for assistance in N. benthamiana transformation and Jerome Mutterer (Institut de Biologie Moléculaire des Plantes [IBMP] CNRS 2357, Strasbourg, France) for support in microscopic imaging and image analysis. We are grateful to Christophe Ritzenthaler (IBMP CNRS 2357) for discussion and provision of cellular markers. We thank Mark Seemanpillai (IBMP CNRS 2357) and Jamie Ashby (John Innes Centre, Norwich, UK) for support in preparing the manuscript.

Received December 5, 2008; accepted February 9, 2009; published February 13, 2009.


   FOOTNOTES
 
1 This work was supported by the Ministere de la Culture, de L'Enseignement Superieur et de la Recherche, Luxembourg (doctoral fellowship no. BFR04/068 to C.H.); by the Generalidad Valenciana, Spain (postdoctoral fellowship grant nos. CTBPDC/2204/015 and BPOSTDOC06/072 to A. Sambade); and by the Le Ministère Délégué à la Recherche et aux Nouvelles Technologies, France, and the Human Frontier Science Program Organization (grant nos. ACI BCMS187 and HFSP 22/2006, respectively, to M.H.). Back

2 Present address: Department of Cell and Developmental Biology, John Innes Centre, Norwich NR4 7UH, United Kingdom. Back

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: Manfred Heinlein (manfred.heinlein{at}ibmp-ulp.u-strasbg.fr).

[W] The online version of this article contains Web-only data. Back

[OA] Open Access articles can be viewed online without a subscription. Back

www.plantphysiol.org/cgi/doi/10.1104/pp.108.133827

* Corresponding author; e-mail manfred.heinlein{at}ibmp-ulp.u-strasbg.fr.


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