Download Plasmodesmata 2004. Surfing the Symplasm

Meeting Report
Plasmodesmata 2004. Surfing the Symplasm1
Christine Faulkner, Jeri Brandom, Andy Maule, and Karl Oparka*
Faculty of Science, University of Sydney, New South Wales 2006, Australia (C.F.); Section of Plant Biology,
University of California, Davis, California 95616 (J.B.); Department of Disease and Stress Biology, John Innes
Centre, Norwich NR4 7UH, United Kingdom (A.M.); and Unit of Cell-Cell Communication, Scottish Crop
Research Institute, Dundee DD2 5DA, United Kingdom (K.O.)
Communication between cells is necessary to coordinate plant development and physiology, and is
modulated in response to environmental signals and
pathogen attack. Plasmodesmata (PDs; singular plasmodesma), plasma membrane-lined channels that
cross the cell wall, are key components of this intercellular communication network. Historically, PDs
were largely viewed as little more than channels that
allowed the passive movement of small molecules
such as water and metabolites between cells. Research
over the past few decades has revealed that PDs are
structurally and functionally dynamic and complex. It
is now recognized that PDs are a central hub for
intercellular transportation of a wide range of solutes,
proteins, signaling molecules, and ribonucleoprotein
complexes. Rapid progress has been made in many
facets of plasmodesmal research, as reported at the
recent 5th International Plasmodesmata meeting held
at Asilomar, Pacific Grove, California, in August 2004.
The Asilomar meeting is the first dedicated solely to
PD research in the past 3 years, a period that has
shown an expanding interest in cell-to-cell communication in plants. Although, traditionally, the PD field
has brought together cell biologists and virologists, in
recent years the subject has expanded to include
developmental biologists and experts in gene silencing. Indeed, the topic of PD research has gained
increasing relevance, as an increasing number of plant
proteins and endogenous RNAs have been shown to
act in a non-cell autonomous manner. Of particular
topical interest is the movement of gene silencing
signals. In one of the plenary talks, Olivier Voinnet
(IPMB-CNRS, Strasbourg, France) elegantly described
how the small RNAs (sRNAs) of 21 and 24 nucleotides
associated with RNA silencing were differentially
involved in local (cell-to-cell) transmission of the
silencing signal. Mutant plants defective in the accumulation of the 21-nucleotide but not the 24-nucleotide sRNAs showed no movement of the silencing
phenotype. Many research groups are now tackling
the problem of how these and other macromolecules
are transported between cells and throughout the
1
C.F. and J.B. were recipients of the two awards at the conference
for outstanding postgraduate presentations.
* Corresponding author; e-mail [email protected]; fax 01382–
568575.
www.plantphysiol.org/cgi/doi/10.1104/pp.104.057851.
plant. This resurgence and expansion of interest is
being assisted by a major technological shift toward
the use of ‘‘omics’’-based tools and genetic resources.
MACROMOLECULAR COMPONENTS OF PD
A major constraint in advancing our understanding
of PDs has been a lack of knowledge of their constituent
components. Various indirect approaches have previously implicated components of the cytoskeleton, proteins that interact with viral MPs, and molecular
chaperones in PD function. Until recently, potential
plasmodesmal components have been difficult to identify, in part because the location of PD embedded in the
cell wall makes them intractable to biochemical analysis. At this meeting, several groups reported new, more
direct approaches to identifying PD proteins. In an
exciting development, Pat Zambryski and Insoon Kim
(University of California, Berkeley) reported the use of
a genetic screen for altered intercellular trafficking
patterns in Arabidopsis embryos. One mutant, increased
size exclusion 1 (isel1), was identified as a DEAD-box
RNA helicase. Appropriately, as a green fluorescent
protein (GFP) fusion protein, ISEL1 colocalized with
tobacco mosaic virus (TMV) movement protein (MP) as
punctate spots on the cell wall. Three groups (Bernie
Epel, Tel Aviv University, Israel; Andy Maule, John
Innes Centre, Norwich, UK; and Robyn Overall,
Sydney University, Australia) reported the application
of proteomic technologies for identifying PD proteins
from purified PDs or cell wall fractions enriched for
PDs. Again, these candidates were assessed by their
localization as GFP fusions. One protein that appeared
in all these analyses was a REVERSIBLY GLYCOSYLATED POLYPEPTIDE 2. This Golgi-associated protein appears to be targeted to PDs.
An important milestone in this field will be the
identification of the unique structural components
of PDs. By analogy with the nuclear pore complex,
we might expect there to be several tens of structural components. One intriguing possibility was a
tropomyosin-like protein identified from the cell walls
of Chara corallina (Christine Faulkner and Robyn Overall) that localized along the length of the PD. The
abundance of putative PD-associated proteins identified in these recent studies may mean that the long
drought in the isolation of PD components is over.
Plant Physiology, February 2005, Vol. 137, pp. 607–610, www.plantphysiol.org Ó 2005 American Society of Plant Biologists
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2005 American Society of Plant Biologists. All rights reserved.
607
Faulkner et al.
Particularly encouraging was the appearance in the
lists of candidate proteins of proteins found previously
to associate with the PD and the isolation of the same
proteins from different studies, for example, the identification of a DEAD-box RNA helicase by both proteomic and genetic screens.
A complementary and valuable approach to the
challenge of identifying PD functional components
is to ask which plant proteins interact with viral or
endogenous proteins known to move through PDs.
One of the first successful examples of this approach
was the identification of pectin methyl esterase interacting with TMV MP, and it was comforting to hear
that pectin methyl esterase was identified in the lists of
proteomics-derived candidates. From reports at the
meeting, we can now add the DnaJ-like protein CPIP1
(Daniel Hofius, Institute of Plant Genetics and Crop
Plant Research, Gatersleben, Germany), eIF4E (Andy
Maule), and FICa (Takuji Wada, RIKEN, Yokohama,
Japan) as proteins that interact with the potato virus Y
capsid protein, pea seed-borne mosaic virus VPg, and
the non-cell autonomous protein CAPRICE, respectively.
MODES OF PD TRANSPORT
Two modes of operation have been suggested to
explain patterns of intercellular trafficking through
PD. One is nontargeted trafficking in which the size
exclusion limit of the PD is sufficient to allow trafficking of macromolecules not specifically targeted elsewhere within the cell. This could be considered to be
equivalent to diffusion. Alexis Maizel (Max Planck
Institute for Developmental Biology, Tuebingen, Germany) asked the following question: Is nontargeted
movement from cell to cell the default state for many
macromolecules? Maizel addressed this by creating
nonoverlapping deletion mutants of the transcription
factor LEAFY. When fused to GFP, none of these
deletion mutants lost the ability to move from cell
to cell, suggesting that movement of LEAFY is nontargeted. Further analysis of various plant proteins
expressed as GFP fusions showed that all but those
expected to form large protein complexes could move
from cell to cell, further supporting the idea that
movement is the default state.
A second model of trafficking is targeted, or selective,
trafficking in which interaction of translocated macromolecules with PD components mediates a change in
the size exclusion limit of the pore. Viral MPs are typical
examples of targeted movement. One proposed means
of regulating selective trafficking is phosphorylation.
Jung-Youn Lee (Delaware Biotechnology Institute,
Newark, Delaware) and colleagues have screened for
a potential PD-associated protein kinase by testing
fractionated PD-enriched cell wall preparations from
BY-2 cells for phosphorylation of TMV MP. A putative
PD-associated protein kinase belonging to the CASEIN
KINASE 1 gene family was isolated that phosphory-
lates endogenous proteins and selectively phosphorylates viral movement proteins in vitro. Localization
studies of Arabidopsis CASEIN KINASE 1 isoforms
demonstrate various subcellular localization patterns,
including a group that label punctae at the cell periphery, a pattern consistent with PD localization.
Macromolecules that traffic by a selective or targeted
pathway might be expected to contain sequences
necessary for translocation. Kimberley Gallagher
(Duke University, Durham, NC) was able to show
that, while movement of the transcription factor SHR
is dependent upon a single residue in the protein and is
therefore representative of targeted and active movement, SHR cannot be trafficked from cell to cell when
localized to the nucleus. This allows the speculation
that chaperones that facilitate movement of SHR are
localized in the cytoplasm and that SHR (which contains a nuclear localization signal) needs to interact
with exportins to localize to the cytoplasm. Jae-Yean
Kim (Gyeongsang National University, Jinju, Korea)
used a trichome rescue assay to identify a trafficking motif of the endogenous transcription factor
KNOTTED1 (KN1). Fusion of the KN1 homeodomain
to the cell-autonomous GL1 conferred trichome rescue
in gl1 mutants. Fritz Kragler (University of Vienna,
Austria) demonstrated that MPB2C, previously described as a negative regulator of TMV MP movement,
is able to block movement of KN1. Cell-to-cell movement of KN1 is not impaired simply because of the
presence of a large amount of interacting protein, as the
KN1-interacting protein KNB36 has no effect on KN1
trafficking in microinjection experiments. Furthermore,
interaction of KN1 with MPB2C in yeast two-hybrid
assays was dependent upon the KN1 homeodomain.
One of the questions posed at the meeting related to
the mechanism by which macromolecules traffic from
cell to cell. Recent work by Karl Oparka’s group
(Scottish Crop Research Institute, Dundee, UK) identified a Rab protein that localizes to plasmodesmata
(Medina Escobar et al., 2003). This family of proteins is
involved in the targeting of vesicles to specific locations in the cell, and thus the following question was
raised: Are vesicles targeted to the plasmodesmata?
This theme was continued by Alison Roberts (Scottish
Crop Research Institute), who provided evidence that
two viral MPs of potato mop top virus interact with
components of the endocytic machinery, notably a
protein involved in receptor-mediated endocytosis
(RME-8). The intriguing concept of endocytosis was
raised again in Alexis Maizel’s investigation of the secretion and internalization of homeodomain proteins fused
to GFP in COS-7 and primary neuron co-cell culture. He
was able to demonstrate that, while the full-length
KN1 protein fused to GFP could not move from the
COS-7 cells into the neurons, the KN1 homeodomain
fused to GFP was able to move. If the movement of
this protein from cell to cell in animals is analogous
to the movement from cell to cell in plants, this raises
the following intriguing question: Are homeodomain
proteins, rather than being transported through
608
Plant Physiol. Vol. 137, 2005
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2005 American Society of Plant Biologists. All rights reserved.
Plasmodesmata 2004: Surfing the Symplasm
plasmodesmata, trafficked by a mechanism that more
closely resembles the secretion and internalization
mechanism adopted by animal cells? This question is
likely to be debated hotly in the near future.
A common view of PD trafficking is that molecules
exploit the cytoplasmic sleeve between the plasma
membrane lining of the PD and the central rod-like
appressed ER component, often called the desmotubule. Delivery to this location might involve targeting
motifs on vesicles, while directionality to and through
PDs is provided by components of the cytoskeleton.
Talks by Manfred Heinlein (IPMB-CNRS) and Petra
Boevink (Scottish Crop Research Institute) examined
the role of the microtubule (MT) cytoskeleton in delivering MP-RNA complexes to plasmodesmata. Although both groups agreed that the decoration of MT
later in the infection process is not involved in cellto-cell transport, debate continues as to whether the
targeting of MP-RNA to plasmodesmata requires the
actin or MT cytoskeleton.
Traditionally, we have assumed that the lumen of
the ER is inert with respect to molecular movement
between cells. Both Robyn Overall and Bernie Epel
addressed the question of whether the ER forms a
functional transport pathway through the PD. Epel
presented data that suggest that, in the presence of
TMV MP, the desmotubule dilates and that proteins can
be transported from cell to cell through the ER lumen.
Overall described intricate experiments that showed
that microinjection of a 3-kD dye into the ER lumen of
tobacco trichomes moved into the adjacent cells, first
being seen in the nucleus of the neighboring cells.
However, FRAP analysis of ER-targeted GFP suggested
that ER itself was not moving from cell to cell.
TISSUE CONNECTIVITY AND LONG
DISTANCE TRANSPORT
Plasmodesmata are ubiquitous throughout plant
tissues and, except for the few examples of symplasmically isolated cells (e.g. stomatal guard cells), provide
symplasmic continuity throughout the body of the
plant. Although crucial in establishing the routes for
symplasmic transport, intercellular flow does not
appear to be regulated by the abundance of PDs. In
an exhaustive study of plasmodesmata frequency and
distribution in mature stems, Aart van Bel (JustusLiebig-University, Giessen, Germany) determined that
plasmodesmatal density does not appear to control the
transport capacity of the dye lucifer yellow from cell to
cell. This was similar to the findings of Bob Turgeon
(Cornell University, Ithaca, NY), who, by comparing
phloem loading in plants that translocate raffinosefamily oligosaccharides with those that translocate
Suc, concluded that plasmodesmata frequency in the
minor vein phloem has little to do with phloem
loading. The phloem has emerged as a crucial element
in defining the long-distance routes for signaling
within the plant. The concept of the phloem as
a superhighway for the delivery of systemic informational molecules (Bill Lucas, University of California,
Davis) has gained considerable support from the
study of movement of viruses and endogenous macromolecules. Recent work establishing that non-cell
autonomous macromolecules are able to function at
a supracellular level has, as Lucas stated, raised more
questions than answers about the roles of endogenous
macromolecules translocated in the phloem, and the
cellular components mediating this process. Some of
the issues the Lucas lab is addressing include the
following. Are proteins and transcripts selectively
transported in the phloem? If so, what are the mechanisms that establish selectivity? Also, is the RNA
present in the phloem involved in signaling and/or
transmission of phenotype? RNA-binding proteins
(RBPs) are likely to be one of the components involved
in the transmission of RNA molecules in the phloem.
Eriko Miura of the Lucas lab has identified a pumpkin
phloem-localized protein, orthologous to eIF-5A, that
interacts with exportin 4 (Lipowsky et al., 2000) and
is involved in RNA shuttling (Bevec et al., 1996) in
animal cells. Investigation into the specific roles of
these RBPs will help to elucidate the mechanisms of
long-distance RNA transport in the phloem.
Ultimately, the purpose of local and remote signaling
is to maintain a tightly regulated program for growth
and development, plant defense, and physiological
control. The breadth of impact of these processes
is being addressed by Shmuel Wolf (The Institute of
Plant Sciences in Agriculture, Rehovot, Israel) and colleagues, who are using both genomic and proteomic approaches to study the composition of phloem exudate.
Microarrays will be used to compare gene expression
profiles in flowering and nonflowering melon. Comparison of proteins isolated from phloem exudate of
these plants identified several proteins present only in
flowering plants, including three protein kinases. Interestingly, analysis of 1,200 clones of a cDNA phloem
library returned 81% singletons, confirming the large
population of transcripts present in phloem exudate.
THE WAY FORWARD
Speculation on the application of new technologies
for plasmodesmal research was also considered at the
meeting. Karl Oparka described the many different
approaches to image the trafficking between cells in
his opening address at the meeting. The vast array of
fluorescent technologies available, such as FlAsH,
fluorescent proteins, and Q-Dots, has opened up the
potential for detailed analysis of the transport capacity
of PDs. Further, Wolf Frommer (Carnegie Institution of
Washington, Stanford, CA) presented the development of nanosensors for the imaging of metabolites.
Some of these techniques might answer the following key questions that arose at the meeting. Do
homeodomain proteins really traffic through PDs?
What is the role of vesicle-mediated trafficking with
Plant Physiol. Vol. 137, 2005
609
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2005 American Society of Plant Biologists. All rights reserved.
Faulkner et al.
respect to PD function? What are the roles of the
expanding list of putative PD-associated proteins
identified? What function do RBPs have with respect
to short- and long-distance transport of mRNA? How
are macromolecules targeted to PD?
This is an important and exciting area of research
that will have impacts across the whole of plant
biology, but there are many challenges ahead. Bill
Lucas and David Jackson (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) called for collaboration between researchers to meet these challenges. The
Plasmodesmata meetings are opportunities for the
initial sharing of information and ideas, and Plasmodesmata 2004 was no exception. We regret that, due to
space restrictions, we are unable to detail many of the
interesting presentations at the meeting, and apologize
to colleagues for omissions. The organizers, David
Jackson, Rick Nelson, Pat Zambryski, and Bob Tur-
geon, did a wonderful job of putting together a fascinating scientific program in a beautiful location by the
Pacific.
Received December 8, 2004; returned for revision December 16, 2004; accepted
December 16, 2004.
LITERATURE CITED
Bevec D, Jaksche H, Oft M, Wohl T, Himmelspach M, Pacher A, Schebesta
M, Koettnitz K, Dobrovnik M, Csonga R, et al (1996) Inhibition of
HIV-1 replication in lymphocytes by mutants of the Rev cofactor
eIF-5A. Science 271: 1858–1860
Lipowsky G, Bischoff FR, Schwarzmaier P, Kraft R, Kostka S, Hartmann
E, Kutay U, Gorlich D (2000) Exportin 4: a mediator of a novel nuclear
export pathway in higher eukaryotes. EMBO J 19: 4362–4371
Medina Escobar N, Haupt S, Thow G, Boevink P, Chapman S, Oparka K
(2003) High-throughput viral expression of cDNA-green fluorescent
protein fusions reveals novel subcellular addresses and identifies unique
proteins that interact with plasmodesmata. Plant Cell 15: 1507–1523
610
Plant Physiol. Vol. 137, 2005
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2005 American Society of Plant Biologists. All rights reserved.