when in contact or in the proximity of MTs and MT organizing
centers [35–38]. The targeting of MP to PD and the spread of
infection depend on myosin motor proteins [39] and can be inhibited by over-expression of actin-binding proteins [40]. Taken
together, these observation led to the model that TMV forms
eVRCs at cortical MT-associated ER sites (cMERs) [36] with the
help of MP, and that myosin motor proteins (class VIII and class XI
myosins) target the VRCs along the ER-actin network to PD and
into adjacent cells to spread infection [21, 41].
The use of fluorescent protein fusions and their imaging with
advanced fluorescent microscopy technologies allow singlemolecule detection with sub-diffraction resolution in vivo
[42]. However, unlike proteins, RNAs cannot be expressed as
fluorescent molecules. Thus, new techniques for the in vivo detection of RNA molecules are under continuous development. RNA
molecules may be imaged upon in vitro labeling with fluorochromes followed by introduction intro cells by transfection or
microinjection [43], or need to be visualized by indirect methods.
Native RNAs can be detected either by hybridization with fluorescent probes delivered into the cell [44] or by using RBPs, such as
the Pumilio family of RBPs, whose modular structure can be modified to recognize and bind virtually any RNA sequence of interest
[45, 46]. More recently, nuclease-inactivated CRISPR-Cas9 systems modified to bind ssRNA have been applied [47]. Other
approaches involve the modification of the studied RNA, for example, by the introduction of structural RNA elements called aptamers. These aptamers are capable of binding small molecules, like
malachite green, or various types of recently developed fluorogens
such as “spinach,” which gain fluorescence upon aptamer binding
[48, 49]. RNAs of interest may also be modified by fusion to
aptamers able to bind proteins. An example is the broadly used
“MS2 system.” This system relies on the fusion of the RNA target
with a tandem repetition of a sequence motif derived from the
origin of assembly of the bacteriophage MS2. Upon transcription,
these motifs adopt a stem-loop structure (MSL) that is specifically
recognized and bound by the co-expressed and fluorescent proteintagged MS2 coat protein (MCP), thus allowing RNA imaging
through binding of an RBP to its cognate RNA-binding motif in
the target RNA [50]. Other analogous aptamer/RBP-dependent
RNA-labeling systems have been developed and most of them have
been comprehensively described [51–53]. Moreover, their application to plant systems has been reviewed [54].
The MS2 system was first developed in yeast and has been
extensively applied for the study of all aspects of RNA life and in a
broad range of organisms, including plants [10, 35, 50, 55–
57]. The successful application of the MS2 method for the study
of a given mRNA molecule depends on several aspects that need to
be considered. Zipcodes (usually located at the 3
0 untranslated
106
Eduardo Jose ´ Pen ˜ a and Manfred Heinlein
centers [35–38]. The targeting of MP to PD and the spread of
infection depend on myosin motor proteins [39] and can be inhibited by over-expression of actin-binding proteins [40]. Taken
together, these observation led to the model that TMV forms
eVRCs at cortical MT-associated ER sites (cMERs) [36] with the
help of MP, and that myosin motor proteins (class VIII and class XI
myosins) target the VRCs along the ER-actin network to PD and
into adjacent cells to spread infection [21, 41].
The use of fluorescent protein fusions and their imaging with
advanced fluorescent microscopy technologies allow singlemolecule detection with sub-diffraction resolution in vivo
[42]. However, unlike proteins, RNAs cannot be expressed as
fluorescent molecules. Thus, new techniques for the in vivo detection of RNA molecules are under continuous development. RNA
molecules may be imaged upon in vitro labeling with fluorochromes followed by introduction intro cells by transfection or
microinjection [43], or need to be visualized by indirect methods.
Native RNAs can be detected either by hybridization with fluorescent probes delivered into the cell [44] or by using RBPs, such as
the Pumilio family of RBPs, whose modular structure can be modified to recognize and bind virtually any RNA sequence of interest
[45, 46]. More recently, nuclease-inactivated CRISPR-Cas9 systems modified to bind ssRNA have been applied [47]. Other
approaches involve the modification of the studied RNA, for example, by the introduction of structural RNA elements called aptamers. These aptamers are capable of binding small molecules, like
malachite green, or various types of recently developed fluorogens
such as “spinach,” which gain fluorescence upon aptamer binding
[48, 49]. RNAs of interest may also be modified by fusion to
aptamers able to bind proteins. An example is the broadly used
“MS2 system.” This system relies on the fusion of the RNA target
with a tandem repetition of a sequence motif derived from the
origin of assembly of the bacteriophage MS2. Upon transcription,
these motifs adopt a stem-loop structure (MSL) that is specifically
recognized and bound by the co-expressed and fluorescent proteintagged MS2 coat protein (MCP), thus allowing RNA imaging
through binding of an RBP to its cognate RNA-binding motif in
the target RNA [50]. Other analogous aptamer/RBP-dependent
RNA-labeling systems have been developed and most of them have
been comprehensively described [51–53]. Moreover, their application to plant systems has been reviewed [54].
The MS2 system was first developed in yeast and has been
extensively applied for the study of all aspects of RNA life and in a
broad range of organisms, including plants [10, 35, 50, 55–
57]. The successful application of the MS2 method for the study
of a given mRNA molecule depends on several aspects that need to
be considered. Zipcodes (usually located at the 3
0 untranslated
106
Eduardo Jose ´ Pen ˜ a and Manfred Heinlein