methylation (m
5 C) in systemic RNA transport [19], the intracellular mechanisms supporting mRNA transport to the PD are not well
understood. One approach for identifying these mechanisms is by
studying the cell-to-cell dissemination of single-stranded (ss) RNA
virus infections [20, 21]. Most plant viruses are RNA viruses and all
plant viruses disseminate infection by targeted movement through
PD [21, 22]. Tobacco mosaic virus (TMV) has been a pioneer in
this regard [23, 24]. This filamentous virus has a positive-sensed
RNA genome that encodes two polymerase subunits (126 kDa and
183 kDa proteins) as well as a movement protein (30 kDa; MP) and
a coat protein (17.5 kDa; CP) from subgenomic RNAs [25]. The
CP is dispensable for cell-to-cell movement [26], thus indicating
that the virus targets and moves its RNA genome through PD in
the form of an RNP, independent of encapsidation by its CP. This
feature positions TMV as an excellent model to study the intra- and
intercellular transport of RNA complexes in plants.
The MP is essential for viral RNA transport [24]. Consistently,
it binds to RNA in a sequence nonspecific manner [27] and also
targets PD. During infection in Nicotiana benthamiana, the protein is transiently expressed and modifies the size exclusion limit
(SEL) of PD between cells at the spreading infection front [28]. At
later stages, thus in cell behind the front of infection, the protein is
ubiquitinylated and degraded through ER-associated degradation
(ERAD) [29] by the 26S proteasome [30]. Infection foci in
N. benthamiana leaves of TMV derivatives in which the CP gene
was deleted and MP expressed as a fusion to a fluorescent protein
reporter indeed show a fluorescent halo [31–33], indicating accumulation of MP at the infection front, thus during a short period of
time. Microscopic observations in cells of such foci led to the
discoveries that MP localizes to the ER-actin network and interacts
with microtubules (MT) [31–33]. In the foremost line of cells at
the virus front, MP accumulates at PD but is also a constituent of
cortical ER-associated, mobile particles. Using conditional mutations within the MP, the mobile particles were functionally associated with the capacity of MP to support the cell-to-cell movement
of the virus [33, 34]. Although these particles likely reflect the
formation of early viral replication complexes (eVRCs) at cortical
ER-MT junctions, similar particles are also observed upon transient
expression of MP alone [35], thus indicating that the formation of
these particles is a function of MP. Indeed, conditional mutations in
MP that correlated the formation of these MP particles (eVRCs)
and the ability of the virus to move between cells during infection
also affected the formation of the MP particles in the noninfected,
MP-transfected cells [35]. The MP particles in TMV-infected and
MP-transfected cells are visible at cortical planes of the cells, and
while many of them are anchored at ER-MT junctions, other
particles disassociate from such sites and show a stop-and-go movement along the cortical ER-actin network, pausing their trajectories
RNA Imaging in Plants Using MS2
105
5 C) in systemic RNA transport [19], the intracellular mechanisms supporting mRNA transport to the PD are not well
understood. One approach for identifying these mechanisms is by
studying the cell-to-cell dissemination of single-stranded (ss) RNA
virus infections [20, 21]. Most plant viruses are RNA viruses and all
plant viruses disseminate infection by targeted movement through
PD [21, 22]. Tobacco mosaic virus (TMV) has been a pioneer in
this regard [23, 24]. This filamentous virus has a positive-sensed
RNA genome that encodes two polymerase subunits (126 kDa and
183 kDa proteins) as well as a movement protein (30 kDa; MP) and
a coat protein (17.5 kDa; CP) from subgenomic RNAs [25]. The
CP is dispensable for cell-to-cell movement [26], thus indicating
that the virus targets and moves its RNA genome through PD in
the form of an RNP, independent of encapsidation by its CP. This
feature positions TMV as an excellent model to study the intra- and
intercellular transport of RNA complexes in plants.
The MP is essential for viral RNA transport [24]. Consistently,
it binds to RNA in a sequence nonspecific manner [27] and also
targets PD. During infection in Nicotiana benthamiana, the protein is transiently expressed and modifies the size exclusion limit
(SEL) of PD between cells at the spreading infection front [28]. At
later stages, thus in cell behind the front of infection, the protein is
ubiquitinylated and degraded through ER-associated degradation
(ERAD) [29] by the 26S proteasome [30]. Infection foci in
N. benthamiana leaves of TMV derivatives in which the CP gene
was deleted and MP expressed as a fusion to a fluorescent protein
reporter indeed show a fluorescent halo [31–33], indicating accumulation of MP at the infection front, thus during a short period of
time. Microscopic observations in cells of such foci led to the
discoveries that MP localizes to the ER-actin network and interacts
with microtubules (MT) [31–33]. In the foremost line of cells at
the virus front, MP accumulates at PD but is also a constituent of
cortical ER-associated, mobile particles. Using conditional mutations within the MP, the mobile particles were functionally associated with the capacity of MP to support the cell-to-cell movement
of the virus [33, 34]. Although these particles likely reflect the
formation of early viral replication complexes (eVRCs) at cortical
ER-MT junctions, similar particles are also observed upon transient
expression of MP alone [35], thus indicating that the formation of
these particles is a function of MP. Indeed, conditional mutations in
MP that correlated the formation of these MP particles (eVRCs)
and the ability of the virus to move between cells during infection
also affected the formation of the MP particles in the noninfected,
MP-transfected cells [35]. The MP particles in TMV-infected and
MP-transfected cells are visible at cortical planes of the cells, and
while many of them are anchored at ER-MT junctions, other
particles disassociate from such sites and show a stop-and-go movement along the cortical ER-actin network, pausing their trajectories
RNA Imaging in Plants Using MS2
105