The Plant Journal (2023) 115, 299–300
16358
RESEARCH HIGHLIGHT
Local call or long-distance call: studying cell-to-cell transport
by Arabidopsis callus grafting
Gwendolyn K. Kirschner, Research Highlights Editor
 lu et al. To view this article visit https://doi.org/10.1111/tpj.16326.
Linked article: This is a Research Highlight about Hasbiog
Connections between plant cells were discovered more
than a century ago by the Austrian botanist Eduard Tangl.
He proposed that they serve for communication and trans-
port between cells (Tangl, 1880). Today, it is clear that these
connections—termed plasmodesmata—facilitate the move-
ment of molecules such as mRNA and proteins between
cells (Kragler, 2013). Friedrich Kragler, project leader at the
Max Planck Institute of Molecular Plant Physiology in
Potsdam-Golm, became fascinated early in his career by
the finding that plants have intercellular plasmodesmata
pores that transfer viral RNA–protein complexes. At that
time in the 1990s, nothing was known about this transport
mechanism, and it was even disputed whether intercellular
transport of viral proteins and RNAs was regulated at
all (Chasan, 1993). This prompted Kragler to start research
on this intercellular transport. Now, his group mainly
researches the mechanisms for intercellular and long-dis-
tance transport of macromolecules. Recently, they demon-
strated that it is possible to create stable gene-edited,
transgene-free plants by grafting a wild-type shoot stock to
a rootstock transformed with a CRISPR/Cas9 construct
(Yang et al., 2023). Fusions of Cas9 and guide RNA tran-
scripts to tRNA-like sequence motifs move the RNA from
root to shoot and create inheritable mutations in the shoot.
This RNA mobility across graft junctions raised the
question how the RNA transport is achieved between cells
over short distances. However, it is difficult to study sym-
plasmic transport of macromolecules between cells in
plants independent of transport in the vasculature in a
whole plant system. In the highlighted publication, Kragler
and Frank Machin, together with the master students
Yag mur Hasbiog  lu and Stephanie Weber, describe a callus
grafting system to study local macromolecule transport
without the presence of a vascular system (Hasbiog  lu
et al., 2023). For this, they co-cultivated calli to form a chi-
meric tissue. They used YFP as a mobile protein expressed
in the donor callus and Cherry fused to histone H2B as an
immobile protein expressed in the recipient tissue. At the
attachment sites of the calli, they detected cells with both
fluorescent proteins, suggesting that YFP was transported
between the cells (Figure 1a–c). The highest number of
Ó 2023 Society for Experimental Biology and John Wiley & Sons Ltd.
doi: 10.1111/tpj.
cells with a YFP transfer between the calli was achieved by
placing the recipient callus in the centre and the donor cal-
lus around it (Figure 1d,e). To exclude the possibility that
cell fusion during the grafting caused the presence of both
fluorescent proteins in a single cell, the authors used two
immobile fusion proteins and confirmed that there was no
signal in any common cell. To test whether excretion from
the donor callus could be the reason for the protein trans-
fer to the recipient callus, they performed grafts between
calli with secreted RFP and YFP, but never detected any
RFP in the YFP cells.
The authors went on to demonstrate that calli from
two different ecotypes, Columbia and Landsberg erecta, as
well as calli derived from different plant organs, could
form symplasmic connections. They analysed whether the
symplasmic connection at the junction site was formed by
simple or complex plasmodesmata, by grafting calli with
two different plasmodesmata markers. MP17 labels com-
plex/branched PDs and could widely be detected in the cal-
lus tissue, suggesting that plasmodesmata in calli are
mostly complex. Cryo-sections of the connection sites
showed that the plasmodesmata were labelled with MP17
from one side and with PDPL5 as a general marker for
plasmodesmata from the other side, suggesting that plas-
modesmata are formed at the chimeric junction site. They
then tested the mobility of macromolecules other than YFP
through these plasmodesmata, confirming the mobility of
the transcription factor, KNOTTED 1 (KN1), which is known
to move from cell to cell by regulated transport (Lucas
et al., 1995). They did not, however, detect movement of
the MOVEMENT PROTEIN BINDING PROTEIN 2C that
retains KN1 within cells (Winter et al., 2007). They also
tested PHLOEM ASSOCIATED RNA CHAPERONE-LIKE 1
(PARCL1), which moves as RNA–protein complex through
the phloem, but its expression in the companion cells sug-
gests that short distance transport occurs between cells
(Ostendorp et al., 2022), and indeed found the protein
mobile between callus grafts.
The unorganised nature of the calli and their loose
structure limited the number of observed fluorescent pro-
tein mobility at the graft junction, making the authors
299

300 Gwendolyn K. Kirschner
speculate the events were undercounted. Therefore, they
went on to use small interference RNA (siRNA) silencing
spread, which depends on symplasmic transport and is
amplifying, so that only one connection event at the calli
junction site should be enough to silence gene expression
in the whole callus (Voinnet et al., 1998). The authors used
two gRNA-TLS (tRNA-like sequence) expressed in the
recipient callus to target VENUS and a control gRNA tar-
geting another locus. In the negative control, VENUS sig-
nal was still visible in the recipient callus expressing 35S:
H2B-VENUS, but abolished in the experimental setup con-
taining VENUS-targeting siRNA, suggesting that the silenc-
ing spread was established throughout the recipient callus.
A time course showed that the silencing occurred starting
from 7 days after grafting and was delayed in the dark con-
ditions, confirming previous observations that plasmodes-
mata permeability of fluorescent proteins is influenced by
the light conditions (Brunkard & Zambryski, 2019).
As well as proteins, mRNAs are known to be transported
across cells and tissues via the symplasm. The mRNA of PER-
OXIN 11D (PEX11D) is predicted to be mobile, but the protein
localises to the ER and peroxisomal membranes and is
therefore unlikely to move itself (Koch et al., 2010; Thieme
et al., 2015). Nevertheless, when Hasbiog  lu et al. expressed
YFP-PEX11D in the donor callus, the authors were able to
detect the protein reporter in the recipient calli at the grafting
sites, suggesting that the mRNA moved between the calli and
that the method was feasible to detect this movement.
According to Machin, the callus grafting method can there-
fore answer the most pressing question in the field of mobile
mRNA research, ‘which mRNAs can be detected moving
across callus grafts?’ It will give insights into the properties of
specific mRNAs and help to identify sequence motifs that can
indicate if a particular mRNA is cell-to-cell mobile. Currently,
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Figure 1. Callus grafting for analysing symplasmic
transport.
(a–c) YFP is mobile at the grafting sites between a
callus expressing YFP and a callus expressing H2B-
Cherry; arrowheads mark the nuclei that show YFP
and Cherry signal; YFP channel in green (a), Cherry
channel in magenta (b), merge (c).
(d, e) Assembly of calli on callus induction medium;
the recipient callus is placed in the centre [0 days
after germination (DAG)], (d) and the calli are
grown until the cells attach at the junction site (e).
(f, g) Graft between a callus expressing 35S:H2B-
VENUS and a donor callus expressing gRNA target-
ing VENUS, which induces silencing of VENUS in
the recipient callus (dashed line, f); no silencing is
observed when FLOWERING LOCUS F is target by
gRNA from the donor callus, modified from Has-
biog lu et al. (2023).
Kragler’s group works on creating both a tissue- and a nutri-
tion-dependent mRNA mobility map, and on establishing a
single-cell transcriptome map to identify cell-to-cell mobile
mRNAs.
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The Plant Journal, (2023), 115, 299–300