of 21
Plant biomacromolecule delivery
methods in the 21st century
Sachin Rustgi
1
*, Salman Naveed
1
, Jonathan Windham
1
,
Huan Zhang
2
and Gözde S. Demirer
3
*
1
Department of Plant and Environmental Sciences, School of Health Research, Clemson University Pee
Dee Research and Education Center, Florence, SC, United States,
2
School of Agriculture and Biology,
Shanghai Jiao Tong University, Shanghai, China,
3
Department of Chemical Engineering, California
Institute of Technology, Pasadena, CA, United States
The 21st century witnessed a boom in plant genomics and gene
characterization studies through RNA interference and site-directed
mutagenesis. Speci
fi
cally, the last 15 years marked a rapid increase in
discovering and implementing different genome editing techniques.
Methods to deliver gene editing reagents have also attempted to keep pace
with the discovery and implementation of gene editing tools in plants. As a
result, various transient/stable, quick/lengthy, expensive (requiring specialized
equipment)/inexpensive, and versatile/speci
fi
c (species, developmental stage,
or tissue) methods were developed. A brief account of these methods with
emphasis on recent developments is provided in this review article. Additionally,
the strengths and limitations of each method are listed to allow the reader to
select the most appropriate method for their speci
fi
c studies. Finally, a
perspective for future developments and needs in this research area is
presented.
KEYWORDS
biomacromolecule delivery methods, genome editing, CRISPR, RNA interference,
genetic transformation, site-directed mutagenesis, nanoparticles, plants
Introduction
There has been an exponential increase in the availability of genomic information for
plant species in the last two decades, from the complete genomic sequence of
Arabidopsis
thaliana
(
The Arabidopsis Genome Initiative, 2000
) to the pangenomes of maize
(
Hufford et al., 2021
) and wheat (
Walkowiak et al., 2020
). However, the
understanding of gene function has not caught up with the pace of gene discovery.
The main reasons for this hindrance are the ability to transform only a limited number of
plant species (and only a few selected genotypes within a species) and the time and effort
required to transform a selected genotype. This lag between the mounting genomic
information and gene characterization further highlights the need to develop or improve
upon existing biomolecule delivery methods and, if possible, eliminate the need for trans-
differentiation, tissue culture, and genotype-dependence. The US National Science
Foundation acknowledged this need with the creation of the Plant Transformation
Challenge Grants (TRANSFORM-PGR) under the Plant Genome Research Program
(PGRP) in 2016.
OPEN ACCESS
EDITED BY
Matthew R. Willmann,
Pairwise, United States
REVIEWED BY
Alfred (Heqiang) Huo,
University of Florida, United States
Kangquan Yin,
Beijing Forestry University, China
*CORRESPONDENCE
Sachin Rustgi,
srustgi@clemson.edu
Gözde S. Demirer,
gdemirer@caltech.edu
SPECIALTY SECTION
This article was submitted to Genome
Editing in Plants,
a section of the journal
Frontiers in Genome Editing
RECEIVED
04 August 2022
ACCEPTED
03 October 2022
PUBLISHED
14 October 2022
CITATION
Rustgi S, Naveed S, Windham J, Zhang H
and Demirer GS (2022), Plant
biomacromolecule delivery methods in
the 21st century.
Front.GenomeEd.
4:1011934.
doi: 10.3389/fgeed.2022.1011934
COPYRIGHT
© 2022 Rustgi, Naveed, Windham,
Zhang and Demirer. This is an open-
access article distributed under the
terms of the
Creative Commons
Attribution License (CC BY)
. The use,
distribution or reproduction in other
forums is permitted, provided the
original author(s) and the copyright
owner(s) are credited and that the
original publication in this journal is
cited, in accordance with accepted
academic practice. No use, distribution
or reproduction is permitted which does
not comply with these terms.
Frontiers in
Genome Editing
frontiersin.org
01
TYPE
Review
PUBLISHED
14 October 2022
DOI
10.3389/fgeed.2022.1011934
This review article covers the established and newer
biomacromolecule delivery methods and recently investigated
variations of conventional methods. Substantial progress has also
been made on the use of morphogenic genes (developmental
regulators) to avoid genotype-dependence and tissue culture via
inducing trans-differentiation of explants, supernumerary, or
quiescent meristems and by promoting clonal propagation via
seeds (parthenogenesis) by converting meiosis to mitosis (MeMi)
and altering genes that induce haploidy. These topics, however,
are out of the scope of this article, and readers interested in them
are referred to the reviews by
Khanday and Sundaresan, (2021)
,
Kuo et al. (2021)
, and
Chan, (2010)
.
The macromolecular cargo delivery methods discussed in
this review article could be classi
fi
ed in various ways. For
instance, they could be divided into conventional and novel
methods depending on their usage. Furthermore, based on the
mode of delivery, these methods could be classi
fi
ed into either
direct or vector (biological/physical)-mediated cargo delivery
methods. Based on the mode of delivery, these gene delivery
methods could also be grouped under physical, chemical, or
biological methods. Lastly, these biomacromolecule delivery
methods could be classi
fi
ed as either transient/permanent or
non-integrative/integrative. For ease of presentation, different
delivery methods are discussed below based on a hybrid
classi
fi
cation system that emphasizes both the conventionality
of a method and mode of delivery.
Conventional biomacromolecule
delivery methods
In this section that focuses on conventional
biomacromolecule delivery methods, the intent is not to
provide a comprehensive historical account of their
discoveries, as memoirs are available from the discoverers and
can be consulted by interested readers (
Paszkowski et al., 1984
;
Sanford, 2000
;
Chilton, 2001
;
Klein, 2011
;
van Montagu, 2011
).
Furthermore, this section will not document all research done
using these conventional gene delivery methods, as there are
speci
fi
c books dedicated to these topics (
Wang, 2015
;
Rustgi and
Luo 2020
). Instead, an effort is made to list certain modi
fi
cations/
updates to these procedures.
Biolistic approach and its modi
fi
cations
The discovery of biolistic (a portmanteau of
biological
and
ballistics
) biomolecule delivery was one of the
fi
nest
agricultural innovations of the 20th century. It was one of two
gene delivery methods available to researchers to genetically
modify plants (
Rustgi and Luo, 2020
), the other being
Agrobacterium
-mediated gene delivery.
Agrobacterium
-
mediated gene delivery slightly predated the biolistic method
and has its limitations, such as genotype dependence, as it relies
heavily on the gene interactions among the host genome, the
Agrobacterium
genome, and the Ti (tumor-inducer) or Ri (hairy
root-inducing) plasmids (
Christou, 1994
,
1996
). On the contrary,
the major advantage offered by the biolistic method is its
genotype independence, as it relies on physical force rather
than biological interactions for biomolecule delivery. Some
other advantages include the delivery of large DNA fragments
(even some the size of whole bacterial arti
fi
cial chromosome),
which could be linear or circular (
Yuan et al., 2012
). The
possibility of delivering linear DNA offers another advantage
by eliminating the integration of the plasmid backbone (
Yuan
et al., 2012
). Further, this method allows for a vast scope of
alterations, in particular delivery of proteins, nucleoprotein
complexes, and
fl
uorescent dyes (technique dubbed
DiOlistics
)(
Sherazee and Alvarez, 2013
;
Sudowe and Reske-
Kunz, 2013
;
Rustgi and Luo, 2020
). Other alterations involve
research on the microprojectile (particle) size, type, and distance
between the explant and the microprojectile accelerator or nozzle
(
Sanford, 2000
). Some research also went into the development
of the Hepta
adaptor, which branches the acceleration tube in
seven sections over seven macrocarriers to widen the
fi
eld of
particle delivery. Hence, it is supposed to uniformly spread the
DNA-coated particles over a larger area and maximize the
number of cells transformed during one bombardment. The
Hepta
adaptor for the PDS-1000/He biolistic system was
claimed to have transformed 7
10 times more cells than the
standard adaptor (
https://www.bio-rad.com/
). Since the gas is
partitioned into seven sections, pressure and particle velocities
are reduced, making it an ideal system for explant cultures and
cell cultures requiring less forceful penetration. Research on
microprojectiles and coating methods is ongoing (
Ismagul
et al., 2018
;
Cunningham et al., 2020
). Recently, coating gold
microcarriers with polyethylene glycol (PEG) and magnesium
salt solutions, instead of spermidine and calcium chloride,
substantially improved transformation frequency in common
wheat (
Ismagul et al., 2018
).
The biolistic method remains one of the most used gene
delivery methods in plants and was the source of most
commercially released transgenics developed in the 1990s
(
Christou, 1996
). The method is still evolving and has been
modi
fi
ed to deliver nano-sized particles coated with nucleic
acids/nucleoprotein complexes; a procedure termed
nanobiolistics
(
O
Brien and Lummis, 2011
; for detailed
examination of this topic, see
Cunningham et al., 2018
,
2020
).
Another modi
fi
cation known as
Agrolistic transformation
combined the bene
fi
ts of
Agrobacterium
-mediated gene
delivery with the biolistic method. In this approach, the
Agrobacterium
virulence genes, virD1 and virD2 which are
needed to liberate T-strands from the Ti plasmid in bacteria,
were placed under the control of a constitutive cauli
fl
ower
mosaic virus (CaMV)
35S
promoter and co-delivered via the
biolistic approach with a target plasmid containing border
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sequences
fl
anking the gene of interest. Hence, this approach
offers the genotype independence of the biolistic method
combined with the single-copy gene integration bene
fi
t of the
Agrobacterium
-mediated transformation method (
Hansen and
Chilton, 1996
). This method was speci
fi
cally used to modify
dif
fi
cult-to-transform crops, such as cotton and soybean (for
details and later modi
fi
cations of the method, see
Basso et al.,
2020
).
Other modi
fi
cations to the biolistic method include
delivering proteins to plant and animal cells. This method,
named
proteolistics,
requires the deposition of proteins
along with microcarriers onto the macrocarrier surface. This
does not require protein precipitation onto the micro-projectile
surface, it does not involve any chemical modi
fi
cation of the
microcarriers, and there is no chemical interaction between the
protein and the microcarriers; hence, this method is not limited
by the protein that can be delivered (
Martin-Ortigosa and Wang,
2014
,
2020
). Despite the given advantages, this method did not
gain much traction at the time of discovery until recently with the
increased demand to deliver genome-editing reagents, i.e., guide
RNAs complexed with Cas9 protein. There are several
advantages
of
directly
delivering
CRISPR/
Cas9 ribonucleoprotein complexes (RNPs) or in vitro
transcripts (IVTs) of gRNA and Cas9 into plant cells. One
such advantage is reducing off-target mutations via avoiding
insertional mutagenesis or the integration of foreign DNA
fragments in the genome. To deliver the RNP complex via
microprojectile bombardment, the same concentrations of
gRNA and Cas9 are mixed in a 4-5:1 proportion in order to
develop the RNP complex and are then subsequently mixed with
gold particles, spread onto macrocarriers, and air-dried. This
method was successfully used in common wheat for inducing
mutations in the desired genes and is expected to function equally
well in other crop plants (
Liang et al., 2018
,
2019
;
Zhang Y. et al.,
2021
).
Agrobacterium
-Mediated gene delivery
method and its modi
fi
cations
Agrobacterium
-mediated gene delivery is one of the
predominant plant genetic transformation methods. One of
the primary reasons for its popularity is the ease with which it
can be adopted and implemented in laboratories familiar with
plant tissue culture and molecular cloning procedures, without
signi
fi
cant resources (
Hiei et al., 1997
). Bacterial species other
than
Agrobacterium
/
Rhizobium
were later identi
fi
ed to be
capable of delivering DNA to plant cells (
Hooykaas et al.,
1977
). However, so far,
Agrobacterium
remained the primary
vector for DNA transfer.
Two species,
Agrobacterium tumefaciens
and
A. rhizogenes
(
Rhizobium rhizogenes
), and several
A. tumefaciens
strains are
used to transform a wide variety of plant species. Collectively, the
availability of these strains increased the host range of
Agrobacterium
spp. However, using different
Agrobacterium
strains with different host plants needs lots of optimization, as
the co-culture duration and subsequent elimination afterwards is
species/genotype-speci
fi
c.
Agrobacterium tumefaciens
and
R.
rhizogenes
also differ in their modes of genetic transformation
and use different proteins to mobilize DNA into plant cells
(
Ream, 2009
).
Agrobacterium tumefaciens
-mediated gene
delivery yields plants that express transgenes throughout or
completely transformed plants (
Fernández-Piñán et al., 2019
)
whereas
R. rhizogenes
produces transgenic hairy roots on wild-
type shoots resulting in plants that are a composite of a wild-type
shoot with transformed hairy roots (
Fernández-Piñán et al.,
2019
). Therefore, the choice of the
Agrobacterium
species for
transformation depends on the experiment
s objective; for
instance,
R. rhizogenes
is the vector of choice when the
function of the root-speci
fi
c gene needs to be studied promptly.
In an earlier study,
Hooykaas et al. (1977)
introduced the
Agrobacterium
Ti plasmid into
Rhizobium trifolii
and found that
R. trifolii
infected Kalanchoe leaves produced tumors, which
suggested DNA transfer. Similarly, van Veen et al. transformed
Phyllobacterium myrsinacearum
with the
A. tumefaciens
Ti
plasmid and observed that it also induced tumorigenesis in
Kalanchoe (
van Veen et al., 1988
). Later,
Broothaerts et al.
(2005)
demonstrated three other bacteria,
Rhizobium
sp.
strain NGR234,
Sinorhizobium meliloti
, and
Mesorhizobium
loti
(collectively known as Transbacter
) modi
fi
ed with
A.
tumefaciens
Ti plasmid to genetically transform
Arabidopsis
thaliana
,
Nicotiana tabacum
, and
Oryza sativa
(
Broothaerts
et al., 2005
). However, these bacteria exhibited low
transformation ef
fi
ciencies relative to
A. tumefaciens
, hence
they were not used widely in plant transformations. Similarly,
but more recently, Rathore and Mullins demonstrated
Ensifer
adhaerens
OV14 modi
fi
ed with
A. tumefaciens
Ti plasmid to
transform potato, tobacco,
Arabidopsis
, rice, and cassava (for a
comprehensive review on this topic, see
Rathore and Mullins,
2018
). However,
Ensifer
was also shown to be less virulent than
Agrobacterium
. In addition, Cho et al. demonstrated that a
phytopathogenic bacteria,
Ochrobactrum haywardense
H1,
modi
fi
ed to express the
A. tumefaciens
Ti plasmid,
successfully transformed soybean, which otherwise remained
challenging to transform using
Agrobacterium
spp. (
Cho et al.,
2022
).
It is apparent from these studies that the so-called
transforming principle,
the Ti/Ri plasmid, was only
identi
fi
ed from
Agrobacterium
/
Rhizobium
species and was
used to transform different bacterial species to deliver the
T-DNA to a wide range of host plants, which was otherwise
impossible using
Agrobacterium
due to its speci
fi
c host range. It
will be interesting to see if, in the future, the
transforming
principle
will be identi
fi
ed for more bacterial species and if they
could be modi
fi
ed similar to
Agrobacterium
to deliver DNA or
nucleoprotein complexes to plant, fungal, and animal cells. The
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common feature among these bacteria is the type IV secretion
system that allows delivery of the nucleoprotein complex to the
plant cells, followed by its integration in the plant genome. More
recently,
Agrobacterium
transformation ef
fi
ciency and host range
was improved by modifying its genome to express
Pseudomonas
syringae
type III secretion system (T3SS). Using the engineered
Agrobacterium
, a 250%
400% increase in wheat, alfalfa, and
switchgrass transformation is observed (
Raman et al., 2022
).
Moreover, bacteria and fungi that can deliver proteins to plant
cells have been identi
fi
ed, and their use as vectors is discussed in
the later sections of this review.
Polyethylene glycol-mediated gene
delivery method
Inspired by the successful demo
nstration of fungal and animal
cell transfections via chemical tre
atment, polyethylene glycol (PEG),
poly-L-ornithine, and polyethylenimine have been used in similar
experiments performed on plant protoplasts (
Altmann et al., 1992
;
Webb and Morris, 1992
;
Bilang et al., 1994
;
Kuwano, Shirataki and
Itoh, 2008
;
Kawai, Hashimoto and Murata, 2010
). Polyethylene
glycol is a petroleum-derived po
lyether polymer that exists in
various molecular weights. The polymer is hydrophilic and
exhibits low biotoxicity; hence, its use for various biological
applications. One of its primary uses is as a transfection agent to
increase the permeability of the plasma membrane and to improve
the transmissibility of charged macromolecules (
Altmann et al.,
1992
). Indeed, PEG in the presence of divalent cations at high
pH was demonstrated to deliver na
ked or liposome-encapsulated
DNA into plant protoplasts as early as the 1980s (
Altmann et al.,
1992
). The PEG-mediated delivery of DNA has since been
improved signi
fi
cantly (
Webb and Morris, 1992
;
Bilang et al.,
1994
). Several alterations of the method were tested to improve
PEG
s transformation ef
fi
ciency and frequency and are discussed in
the reviews by
Altmann et al. (1992)
,and
Webb and Morris, (1992)
.
There are several advantages associated with the PEG-
mediated transformation. This method is technically simple;
hence, it allows simultaneous processing of many samples. It
utilizes inexpensive supplies and does not have specialized
equipment requirements. PEG-mediated transformation is
versatile; hence, it does not exhibit the host range limitations
of
Agrobacterium
-mediated transformation and could be readily
adapted to various plant species and tissue sources with little
optimization (
Bilang et al., 1994
). Additionally, this method is
suitable for transient expression of a transgene leading to the
production of transgene-free genome-edited mutants. However,
the major bottleneck of this method is the regeneration of plant
protoplasts into complete plants. Given these advantages, this
method has witnessed resurging interest, speci
fi
cally with the
advent of genome-editing procedures, both to test the genome-
editing reagents and to regenerate plants with desired mutations
after genome editing (
Yue et al., 2021
). Also, this method has
been used to deliver the triplex-forming oligonucleotides (TFOs)
or Gene Repair OligoNucleotide (GRON) to induce mutations in
plant cells (
Gocal, 2014
;
Gocal et al., 2015
;
Sauer et al., 2016
).
Protoplast isolation and transformation with PEG were
performed in many crop plants (
Bilang et al., 1994
) including
but not limited to wheat (
Brandt et al., 2020
), rice (
Lin et al., 2020
;
Bes et al., 2021
), maize (
Svitashev et al., 2016
), potato (
Carlsen
et al., 2022
), and soybean (
Patil et al., 2022
). Additionally, in
recent years, the PEG-mediated protoplast transfection of
Cas9 protein and Cas9 complexed with in vitro synthesized
guide RNA (ribonucleoprotein, RNP) was successfully used in
major row crop and horticultural crops in a quest to establish a
DNA-free genome editing platform (
Sant
Ana et al., 2020
;
Subburaj et al., 2022
;
Liu et al., 2022
).
With the rapid and competitive pace of development in the
fi
eld
of plant genome editing, several new breeding technologies have
arisen, which aim improved targeting ef
fi
ciency and lower costs.
Among those are the Rapid Trait Development System (RTDS
)by
Cibus. The RTDS
is a transgene-free, precision gene editing
platform that utilizes Oligonuc
leotide-Directed Mutagenesis
(ODM) by targeting a speci
fi
ed gene sequence with a Gene
Repair Oligonucleotide (GRON). The engineered GRON
(typically around 40 bp) shares homology with the target DNA
sequence except for one or a few mismatched base pairs (
Gocal,
2014
;
Gocal et al., 2015
). The RTDS
then relies on the plant cell
s
endogenous DNA repair machinery
to recognize the mismatch and
repair the DNA using the GRON as a template (
Gocal, 2014
). After
ODM, the GRON is degraded by the plant cell, reducing the
opportunities for off-target muta
tions. This system is in contrast
to the nuclease-based gene editing systems mentioned above, as
supplying a nuclease to induce cleavage is not necessary.
Furthermore, GRONs do not require a delivery vector, avoiding
integration of foreign DNA into the host genome. Similar to gene
editing RNPs, however, GRONs can be delivered into protoplasts via
PEG-mediated delivery, electro
poration (see below) or particle
bombardment (see above) (
Gocal et al., 2015
). According to
Gocal et al. (2015)
, the use of ODM has been demonstrated in
Arabidopsis
, canola, corn, rice, tobacco, and wheat. The simplicity,
accuracy, and avoidance of transgene integration renders ODM, and
subsequent systems such as the RTDS
, attractive choices for rapid
gene editing. Despite these advantages, however, the major
bottleneck in implementing this system is a lack of ef
fi
cient
protoplast production, transform
ation, and regeneration protocols
for most crops along with the genotype-dependence of the
protoplast regeneration system.
Unconventional gene delivery
methods
Other than the conventionally-used gene delivery methods,
there are other methods developed and used by the research
community. Some of these methods are elaborated on below.
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Electroporation
Electroporation, a physical, genetic-transformation method,
is used to deliver DNA constructs through plasma membranes by
producing transient, unstable pores, allowing the transportation
of macromolecules such as DNA, RNA, and proteins into cells
(
Ozyigit, 2020
). In plants, protoplasts (
Fromm et al., 1985
) are
often used for electroporation due to the ease of uptake of
plasmids for stable or transient genetic transformation. After
optimizing electroporation parameters, the method was used
successfully to transform embryonic cells (
Xu and Li, 1994
),
zygotes (
Li and Yang, 2000
), mitochondria (
Farre and Araya,
2001
), microspores (
Brew-Appiah et al., 2013
;
Bhowmik et al.,
2018
), and shoot apices (
de GarcÃ-a and Villarroel, 2007
)in
several plant species, such as tobacco, rice, wheat, and maize (for
a review, see
Barampuram et al., 2011
). However, after
electroporation-mediated gene delivery, the explant needs to
be regenerated to obtain transformed progeny, making the
method labor-intensive and genotype-dependent.
More recently, Furuhata et al. demonstrated that proteins
(Cre recombinase) could be delivered to cultured
Arabidopsis
thaliana
cells with intact cell walls with up to 83% ef
fi
ciency,
which is a step forward for electroporation-mediated plant
genetic transformation (
Furuhata et al., 2019
). In summary,
electroporation-mediated plant gene delivery is fast and
inexpensive; however, it may need optimization of some
parameters, such as
fi
eld strength, pulse duration, cargo
concentration, and explant type to obtain satisfactory
transformation ef
fi
ciencies with minimal damage.
Magnetofection
Magnetofection has proven to be a simple and ef
fi
cient
method of transforming target animal cells by applying an
external magnetic
fi
eld (
Scherer et al., 2002
;
Plank et al.,
2003
). The most commonly used magnetofection system
comprises superparamagnetic iron oxide nanoparticles coated
with the cationic polymer polyethylenimine (PEI), which can
bind negatively charged nucleic acid molecules through
electrostatic interaction (
Zuvin et al., 2019
). Magnetic
nanoparticle (MNP) mediated delivery can be achieved via
associating viral or non-viral vectors with MNPs. The major
bene
fi
t of magnetofection lies in the rapid and ef
fi
cient
transfection using a relatively low vector dose and the
possibility of targeting the vector to a speci
fi
c explant area
under a magnetic
fi
eld (
Plank et al., 2011
).
Although magnetofection has been broadly applied in the
animal system, only two published articles described the
successful application of magnetofection in plants. Speci
fi
cally,
in 2017, Zhao et al. demonstrated the use of magnetofection to
introduce plasmid DNA-coated PEI functionalized iron oxide
nanoparticles (168 nm in diameter) into pollen grains of cotton,
pumpkin, zucchini,
capsicum
, and lily (
Zhao et al., 2017
). They
speculated that the DNA-loaded nanoparticles entered pollens
via the apertures (~5
μ
m) in the pollen wall. Through pollen
magnetofection, both transient transformation and direct
production of transgenic seeds without regeneration can be
achieved; hence the authors claimed the system is tissue
culture- and genotype-independent. However, in 2020,
Vejlupkova et al. published an article claiming that the results
of the cotton study were irreproducible in monocots, speci
fi
cally
in maize, sorghum, and lily (
Vejlupkova et al., 2020
). Recently,
Wang et al. established a maize pollen transfection system using
MNPs for a large-scale, fast, and ef
fi
cient maize transfection
(
Wang Z. et al., 2022
). Importantly, they pointed out that
opening the pollen aperture via pretreatment with the
transfection buffer for 10 min and transfection at 8
°
C (to
protect maize pollen viability) is essential for exogenous gene
delivery.
Although magnetofection has not yet been accepted as a
mainstream genetic transformation method, it possesses some
desirable features, such as genotype-independence and low
toxicity. If optimized for more crops, it has potential to be
used widely.
Sonication
Sonication-mediated gene delivery employs an ultrasound
that can produce a variety of nonthermal bioeffects such as
acoustic cavitation and disrupting the cell membrane,
permeabilizing it and facilitating the uptake of genetic
materials (
Joersbo and Brunstedt, 1992
;
Miller et al., 2002
).
This technique provides an attractive alternative to other
physical methods due to its low cost. It has been reported to
allow gene delivery in plant protoplast (
Joersbo and Brunstedt,
1990
), suspension cells (
Liu et al., 2006
;
Zolghadrnasab et al.,
2021
), and even intact leaf segments (
Zhang et al., 1991
). Due to
the cavitation, sonication was also combined with other gene
delivery methods to reach higher ef
fi
ciencies, such as sonication-
assisted
Agrobacterium
-mediated transformation (SAAT) (
Trick
and Finer, 1997
;
Dutta et al., 2013
) and sonication-mediated
pollen-transformation (
Yang et al., 2017
). Sonication, being a
mechanical method, is less dependent on the explant type. It
could be an effective means of delivering DNA to plant cells/
tissues, given sonication conditions are optimized to minimize
any damage to cells and tissues while providing effective cargo
delivery (
Liu et al., 2006
).
Silicon carbide whiskers
Silicon carbide (SiC) whisker-mediated transformation in
plants was
fi
rst reported by Kaeppler et al. where small needle-
like silicon carbide whiskers are mixed in the liquid medium,
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usually with a vortex, in the presence of plasmid DNA and target
plant cells (
Kaeppler et al., 1990
;
Kaeppler et al., 1992
).
Transgenic plants such as rice (
Matsushita et al., 1999
), maize
(
Frame et al., 1994
), and cotton (
Asad et al., 2008
) were produced
using this method. Despite the method being simple, requiring
less resources, and being cost-effective, only a handful of papers
report stable genetic transformation using this method. SiC
whiskers are hazardous to humans (
Svensson et al., 1997
;
Chen et al., 2018
); therefore, safer alternatives, such as the
aluminum borate whiskers (ABW), were later explored for
producing transgenic rice plants (
Mizuno et al., 2004
).
Moreover, ABW was also shown to signi
fi
cantly increase
Agrobacterium
infection ef
fi
ciency (
Nanasato et al., 2011
)
during the adventitious shoot organogenesis in kabocha
squash (
Cucurbita moschata
Duch). Several factors need to be
considered when using SiC-mediated transformation: the type of
the whiskers, the type of plant cell/tissue, and the potential health
hazard the material imposes.
Use of fungi and bacteria to deliver native
or recombinant proteins into plant cells
These methods were initially developed to characterize
fungal effector proteins and understand host-pathogen
interactions. However, these methods have much potential for
delivering other recombinant proteins, such as TAL effector
nucleases or zinc-
fi
nger nucleases, to edit genomes at the
desired site or to transiently express a transcription factor or a
regulatory protein. For instance, van der Linde et al. reported a
unique
Trojan horse approach
to deliver recombinant proteins
to maize using the secretory capabilities of the smut fungus
Ustilago maydis
(
van der Linde et al., 2018
). This strategy allowed
authors to deliver recombinant proteins into individual corn cells
at certain cell layers and at a precise time point. The method
utilized host-pathogen interactions to transport recombinant
proteins to host cells and tissues and, for the
fi
rst time,
demonstrated the potential of
fi
lamentous fungi as plant gene
delivery vectors.
On the other hand, bacteria with a type III secretion system
(T3SS) were used more broadly for direct protein delivery,
particularly in mammalian cells for biomedical applications
(
Ittig et al., 2015
;
Bai et al., 2018
). However, the utility of
bacteria with T3SS in plant research is somewhat limited,
largely due to the hypersensitive response (HR) induced in
the host plants by these vectors. Given this limitation,
recombinant strains of HR-inducing and non-HR-inducing
pathogens were identi
fi
ed to serve as delivery vehicles. These
bacterial vectors are used in eudicots and monocots, and some
examples are included here. A variant strain of
Pseudomonas
syringae
with a deletion of multiple effectors with reduced
hypersensitive response was used in wheat. Also,
Pseudomonas
fl
uorescens
with an engineered T3SS and no HR were used to
characterize bacterial and fungal effectors in wheat (
Yin and
Hulbert, 2011
). Similarly, Sharma et al. reported using the
effector delivery system of the rice pathogen
Burkholderia
glumae
to characterize the AVR-Pik and AVR-Pii effectors of
the fungal rice pathogen
Magnaporthe oryzae
(
Sharma et al.,
2013
). In line with this study, Upadhyaya et al. demonstrated the
use of the wheat pathogen
Xanthomonas translucens
to deliver
fusion proteins containing T3SS signals from
P. syringae
(AvrRpm1) and
X. campestris
(AvrBs2) avirulence (Avr)
proteins into wheat leaf cells (
Upadhyaya et al., 2014
). This
area of research is evolving, and in due course of time, we may
witness more pests/pathogens with similar capabilities will be
identi
fi
ed and used in gene product characterization or genome
editing.
Nanoparticle-based gene delivery
methods
Several nanoparticle-based technologies have been developed
for plant gene delivery in the past 3 years and exhibited many
advantages over conventional methods. First, nanoparticles
enable plant species-independent delivery of cargoes as the
cell entry is hypothesized to be mechanically-driven (
Lew
et al., 2020a
). Second, nanoparticles protect or at least delay
the genetic cargo degradation, increasing the active life-time of
cargoes in plant tissues and cells, which is especially important
for fragile cargoes such as RNA (
Shidore et al., 2021
). Third,
nanoparticle delivery enables the possibility of transient
expression without gene insertion into plant host genomes
(
Demirer et al., 2019a
;
Wang et al., 2019
). This is not only
desirable for many research applications where gene function is
rapidly studied in planta, but also is a transformative technology
for transgene- and GMO-free CRISPR/Cas9 crop engineering
(
Demirer et al., 2021
). Lastly, a wider range of cargo types,
including nucleic acids, proteins, small molecules, and
agrochemicals, can be delivered with nanoparticles (
Ng et al.,
2016
). There are, however, some limitations of nanoparticles
such as lower ef
fi
ciency, their inability of systemic travel in plant
tissues, and limited studies on their environmental safety and
accumulation.
Below, we highlight the recent developments in nanoparticle-
mediated delivery
fi
eld covering only the last 3 years. For a
comprehensive review of technologies before 2019, readers are
encouraged to refer to these cited reviews (
Wang et al., 2016
;
Cunningham et al., 2018
;
Sanzari, Leone and Ambrosone, 2019
;
Wang et al., 2021
).
One of the well-studied nanomaterial types for plant gene
delivery is single-walled carbon nanotubes (SWCNTs). When
covalently-functionalized with positively-charged polymers, such
as PEI and chitosan (CHI), these nanomaterials were able to
deliver electrostatically-attached DNA plasmids into plant nuclei
and chloroplasts, respectively (
Figure 1A
)(
Demirer et al., 2019b
;
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Kwak et al., 2019
). These
fi
rst-generation studies with carbon
nanomaterials established a transient and plant species-
independent delivery in various eudicot and monocot species,
in which the reporter gene expression lasted 7
10 days in
tobacco, arugula, spinach, wheat, watercress, and cotton. More
recently, second-generation studies were carried out to optimize
the nanoparticle physical parameters and cargo binding mode to
increase the cargo delivery ef
fi
ciency (
Hu et al., 2020
;
Ali et al.,
2022
;
Sharma and Lew, 2022
).
Another hollow nanotube platform called rosette nanotubes
(RNTs) were composed via the self-assembly of complementary
guanine
cytosine motifs, and these nanoparticles were
complexed with plasmid DNA for plant delivery (
Cho et al.,
2020
). RNTs entered wheat microspores and did not affect
health, division, or regeneration abilities of microspores.
Separate labeling of both the DNA and RNTs showed that
while RNTs reached the microspore nucleus, DNA was mostly
present in the cell cytosol, which caused low ef
fi
ciency of gene
expression from delivered DNA. However, this study is
promising to enable the discovery of nanomaterial
formulations that can enter crop microspores.
Numerous nanomaterial platforms have been developed for
the delivery of single- and double-stranded RNA cargoes for gene
silencing, in addition to the DNA plasmids for gene expression
(
Figure 1C
). For instance, oligo-wrapped SWCNTs were
generated to deliver small interfering RNA (siRNA) cargoes to
plant leaves to silence endogenous disease susceptibility genes
with high ef
fi
ciency (
Demirer et al., 2020
). Moreover, DNA
nanostructures with programmable size, shape, and stiffness
features were established to deliver siRNA into the leaves of
tobacco, arugula, and watercress (
Zhang et al., 2019a
). Carbon
and DNA-based nanomaterials are not the only formulations
used for plant RNA delivery. Recently, several studies performed
siRNA delivery into plant leaves via spherical and rod-shaped
gold nanoparticles (
Zhang et al., 2022
), and with gold
nanoclusters (
Zhang Y. et al., 2021
). It is noteworthy to
mention that these studies discovered that nanoparticle
cellular internalization was not needed for siRNA-mediated
FIGURE 1
Nanoparticle-mediated delivery to plants.
(A)
Polymer-functionalized single-walled carbon nanotubes (SWCNTs) deliver plasmid DNA into
plant nucleus and chloroplast for gene expression.
(B)
Cell penetrating peptides deliver multiple DNA, RNA, and protein cargoes to plant leaves.
(C)
SWCNTs, DNA nanostructures, and gold nanoparticles are used to deliver siRNA and dsRNA to plants via leaf in
fi
ltration.
(D)
BioClay and carbon
nanodots are topically sprayed on leaves and pollen for dsRNA and siRNA delivery. Figure created with
BioRender.com
.
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gene silencing and that while rod-shaped gold nanoparticles had
higher ef
fi
ciency of cellular uptake, siRNA delivered via spherical
gold nanoparticles induced stronger gene silencing (
Zhang et al.,
2022
).
Nanoparticle-cargo conjugates can be delivered through leaf
in
fi
ltration using a needless syringe in a lab research context, and
for scalable
fi
eld applications, there are some studies
demonstrating the feasibility of topical application of double-
stranded RNA (dsRNA) and siRNA cargoes with layered double
hydroxide (LDH) nanoparticles (
Mitter et al., 2017
) and carbon
dots (
Schwartz et al., 2020
)(
Figure 1D
), which eliminates the
issues of the instability of naked RNA sprayed on plants. The
LDH platform, which is called BioClay, enabled virus/fungus
protection for 21 days when sprayed on virus/fungus-challenged
leaves (
Mitter et al., 2017
;
Niño-Sánchez et al., 2022
). More
recently, the BioClay technology has been topically applied for
dsRNA delivery into tomato pollen (
Yong et al., 2021
), extending
the plant tissue types for various applications. In addition to the
BioClay, carbon dots (CDs) were used to topically deliver siRNA
molecules and generate highly ef
fi
cient gene silencing in tobacco
and tomato leaves (
Schwartz et al., 2020
).
There has been a substantial amount of progress in the
fi
eld
of nanomaterial-mediated biomolecule delivery to plants in the
last three to 4 years. The choice of nanoparticles to use typically
depends on the cargo of interest, target plant tissue, and
application type. For instance, while SWCNTs are effective in
plasmid delivery for transient expression applications in somatic
cells, RNTs may be a better option for microspore delivery, and
BioClay is highly advantageous for topical RNA delivery. Yet,
there is even much more to achieve in the areas of plant CRISPR
gene editing using nanoparticle-mediated delivery and stable
crop transformations.
Cell-penetrating peptides
In addition to nanoparticles, cell-penetrating peptides
(CPPs), which are short peptides composed of 5
30 amino
acids, have shown remarkable abilities to deliver diverse
biomolecules, such as DNA, RNA, proteins, and RNP
complexes into many plant species (
Figure 1B
)(
Thagun et al.,
2020
;
Zhang S. et al., 2021
).
Cationic CPPs are the most commonly used type of peptides,
and they can be loaded with negatively charged DNA/nucleic
acids through electrostatic interaction and yield transient
expression of proteins from DNA cargoes. Similarly, RNA
molecules were delivered to plant cells using CPPs, which had
increased half-life compared to free RNA. Lastly, CPPs were not
only used to deliver nucleic acids, but also proteins and multiple
biomolecules to plant cells simultaneously (
Zhang S. et al., 2021
).
Similar to nucleic acids, negatively charged proteins, such as BSA,
can be electrostatically grafted onto cationic CPPs. To
demonstrate simultaneous multiple cargo delivery, Thagun
et al. used a superfolder GFP-based complementation assay
with a cytosolic homodimer of the
Coffee arabica
7-
methylxanthine methyltransferase 1 protein. The codelivery of
multiple plasmids or proteins with CPPs resulted in the creation
of complemented GFP
fl
uorescence in plant leaf cells (
Thagun
et al., 2020
). Similarly, Wang et al. developed a
fl
uorescent
complementation-based assay to quantify CPP-mediated
protein delivery to plant cells (
Wang J. W. et al., 2022
).
Compared to nanoparticles, they are more biodegradable,
hence potentially have better suitability for
fi
eld studies.
However, most studies are limited to cationic CPPs limiting
delivery to only negatively charged cargoes.
Virus mediated delivery
The use of viruses to deliver DNA to bacterial cells has its roots
in the early years of molecular biology. However, the use of viruses
to deliver gene expression constructs to eukaryotic cells did not
start until much later. The use of disarmed viruses and, more
recently, virus-like particles (VLPs) to deliver cargos such as DNA,
mRNA, and nucleoprotein complexes is more common in medical
research than in plants (for a review, see
Zhang S. et al., 2021
).
However, this method is gaining traction in plant research to
determine gene function by expressing/silencing genes, popularly
known as virus-induced gene silencing (VIGS) and virus-mediated
overexpression (VOX). It is also being used in site-directed
mutagenesis using CRISPR-associated nucleases, the technology
being dubbed virus-enabled gene editing (VEdGE) or virus-
induced genome editing (VIGE) (for a detailed examination of
these topics, see
Rössner et al., 2022
;
Gentzel et al., 2022
). There are
well-established viral gene delivery systems that have been
developed and utilized in VIGS, VOX, and VIGE (discussed in
Gentzel et al., 2022
;
Rössner et al., 2022
). These include systems
based on the cabbage leaf curl virus (CaLCuV, a DNA virus;
Baltes
et al., 2014
), the foxtail mosaic virus (FoMV, a DNA virus;
Mei
et al., 2019
), the broad bean wilt virus2 (BBWV2, a RNA virus;
Choi et al., 2019
), and a system based on the tobacco rattle virus
(TRV, a RNA virus;
Khakhar et al., 2021
) dubbed
VipariNama.
Emphasis is given to lesser expounded upon viral systems in the
following paragraphs.
DNA virus-based delivery system
Many DNA viruses are used to deliver gene expression
cassettes or gene silencing reagents to plants (
Peretz et al.,
2007
;
Mei et al., 2019
;
Rössner et al., 2022
), in addition to
their application in virus-induced gene silencing. The use of
DNA viruses in VIGS is elaborated on in a separate paragraph;
also, it has been reviewed recently in
Rössner et al. (2022)
.
Therefore, in the following paragraphs, we focused discussion
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on a unique tomato yellow leaf curl virus (TYLCV)-based
delivery system, TraitUP
, to avoid repetition.
Peretz et al. (2007)
developed a universal vector system, IL-
60, for silencing and exogenous gene expression from TYLCV, a
Begomovirus of Geminiviridae. This vector was successfully
tested on tomato plants, where it was mechanically injected
into the stem. The recombinant virus spreads systemically, but
the plants remain asymptomatic. Moreover, the vector did not
integrate into the genome, produced no ssDNA progeny, and did
not spread to other plants by
B. tabaci
, making it a desirable gene
delivery system.
Later, to further improve th
e cargo capacity of the TYLCV
vector system (TraitUP
),thesameresearchgroupdeleted
different components of the viral genome and identi
fi
ed a
314-bp intergenic region (IR) as the only viral element
required for viral dsDNA replication, and two sense-
oriented viral genes (V1 and V2) for its expression and
movement (
Gover et al., 2014
). The authors named this
minimal viral construct p1470 and demonstrated its ability
todelivermarkergenes.
So far, the IL-60 vector system has been successfully used in
over 40 plant species belonging to 14 different families including
perennial woody plants, such as orange, apple, and grape (
Peretz
et al., 2007
;
Cusin, Revers and Maraschin, 2017
;
Malabarba et al.,
2018
). The vector was introduced into the plants via root
in
fi
ltration or by syringe inoculation. The former method was
used to express a 6.3 kb bacterial operon that allowed the
introduction of a complete metabolic route in tomato plants
and the production of an antifungal metabolite, pyrrolnitrine
(PRN), rendering them resistant to
Rhizoctonia solani
(
Mozes-
Koch et al., 2012
). This study has demonstrated the ability of this
vector system to deliver and express large gene constructs,
without a negative impact on its movement and replication,
which is an advantage over other viral vectors.
In apple, this technique was successfully used to transiently
express the
Malus COP1
gene (
Li et al., 2012
). It opened an
alternative for the functional characterization of apple genes in a
homologous system.
Cusin et al. (2017)
used the IL-60 plasmids
in apple for expression of the reporter gene,
GFP
. The
Gala
cultivar of apple treated with pIR-GFP plus p1470 showed a
stable, broad, and strong expression of GFP that spread
throughout all tissues over time and remained stable for up to
6 months after the plasmid treatment. This early success has
motivated the researchers to transfer scab resistance genes (such
as
Vf2
), as well as other genes of biotechnological interest, to elite
apple cultivars. The genes appeared to be stably expressed
throughout the plant and during the course of development.
This method eliminated the need for genetic crossing and plant
genetic transformation and presented an instant tool for
transferring the genetic traits of interest. This gene delivery
system has been recently used in grape to study the role of
the
VviAGL11
gene in seed morphogenesis (
Malabarba et al.,
2018
). Similarly, in a study conducted in 2018 in Brazil the IL-60
technology was used to introduce an herbicide resistance gene in
Eucalyptus
species.
This technology has opened exciting new possibilities for fast
trait delivery in woody fruit species (
Nagamangala Kanchiswamy
et al., 2015
). This episomal expression system, based on modi
fi
ed
viruses, may enable the expression of stable genetic traits that can
be introduced by treating scions prior to grafting to elite
genotypes, bypassing the need for backcrossing to recover the
original genetic background.
RNA virus-based delivery system
To improve the versatility of viral vectors, RNA virus-based
delivery methods were developed (
Zhang Y. et al., 2019
;
Ariga,
Toki and Ishibashi, 2020
;
Varanda et al., 2021
). Several RNA
viruses that were modi
fi
ed to deliver the components of the
genome editing machinery (Cas9 and gRNA) to plants are
summarized in
Table 1
. Similar to DNA viruses, RNA viruses
were also modi
fi
ed for VIGS. We dealt with it separately in a
paragraph; also, the topic was recently reviewed in
Rössner et al.
(2022)
.
Jiang et al. (2019)
developed a Beet Necrotic Yellow Vein
Virus (BNYVV)-based system to deliver gRNA targeting the
PDS
(
Phytoene Desaturase
) gene in Cas9-overexpressing
N
.
benthamiana
plants. It has resulted in 78% photobleaching of
the leaf area in the inoculated plants. Similarly, the gene editing
capabilities of barley stripe mosaic virus (BSMV) have been
demonstrated in
N
.
benthamiana,
wheat, and maize (
Hu et al.,
2019
). After successful delivery of
PDS
gRNA via BSMV to
N
.
benthamiana
plants were co-in
fi
ltrated with Cas9 constructs.
Hu
et al. (2019)
further assessed this system with transgenic Cas9-
expressing wheat and maize. In wheat, gRNAs targeting the
TaGASR7
gene, determining grain length and weight,
exhibited up to 78% mutation ef
fi
ciency as indicated by
restriction digestion of the target gene. Similarly, in maize
plants, gRNAs targeting the
ZmTMS5
gene
,
responsible for
the heat-induced male-sterile phenotype exhibited up to 48%
editing ef
fi
ciencies (
Hu et al., 2019
). In a subsequent study, it was
shown that multiple BSMV constructs could be co-inoculated to
simultaneously target multiple genes in wheat (
Li et al., 2021
).
Further, to address concerns of low gRNA expression by viral
vectors,
Cody, Scholthof and Mirkov (2017)
modi
fi
ed the tobacco
mosaic virus (TMV) vector by deleting a coat protein to prevent
its systemic movement through the plant and therefore enhanced
the local viral titer for transient expression assays.
GFP
gRNAs when co-in
fi
ltrated with the TMV-based
Cas9 delivery system showed nearly 70% editing ef
fi
ciency in
GFP-overexpressing
N
.
benthamiana
leaves. In a follow-up
study, RNA interference suppressors were used to further
optimize the system. In this study, delivering Cas9 and
gRNAs from a single TMV construct simultaneously
eliminated the need for producing transgenic plants
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expressing Cas9 or co-delivery of the components (i.e., Cas9 and
gRNA) from separate constructs (
Chiong, Cody and Scholthof,
2021
). Although editing ef
fi
ciency was lower when using a single
construct compared to co-delivery, it was still possible to obtain
almost 7% editing ef
fi
ciency in
N
.
benthamiana
even with a
4.2 kb insert (
Chiong, Cody and Scholthof, 2021
). In contrast,
Ma
et al. (2020)
reported the highest editing ef
fi
ciency to date using
Sonchus
Yellow Net Virus (SYNV). Since SYNV can carry a large
insert cargo, this characteristic makes it a good candidate for
expressing Cas9 as well as single or multiplexed gRNAs.
Ma et al.
(2020)
used this system in
N
.
benthamiana,
where an editing
ef
fi
ciency ranging from 40
91% in plants infected with gRNA
constructs targeting
GFP, NbPDS, NbRDR6,
or
NbSGS3
genes
was observed. Similar editing ef
fi
ciency was observed when
SYNV constructs designed for the multiplexed editing of the
NbRDR6
and
NbSGS3
genes were used.
Potexviruses (PVX) have also been used as gRNA delivery
vehicles.
Ariga, Toki and Ishibashi (2020)
showed the successful
delivery of Cas9 and gRNAs using PVX to
N
.
benthamiana
plants
via Agroin
fi
ltration. Furthermore, Cas9 was replaced by this
group with a larger base-editing version, which proved to be
stably integrated into the virus genome. The PVX vector did not
infect the germline or produce edited progeny. However, plants
regenerated from mechanically (rub)-inoculated tissues
contained
NbTOM1
edits, but with a lower ef
fi
ciency than
those regenerated from Agroin
fi
ltrated-plants (62%). A later
study revealed that PVX could be a useful vector to deliver
multiplexed gRNAs (
Uranga et al., 2021
).
In addition, there are two Tobraviruses, TRV and Pea Early-
Browning Virus (PEBV), that are currently being used as
CRISPR/Cas9 delivery vectors. TRV has a wide host range
and has an easily modi
fi
able bipartite positive-sense RNA
genome. It is also a proven VIGS vector for several crops.
Previous studies had shown that TRV could successfully edit
the
PDS3
and
PCNA
genes either singularly or simultaneously in
N
.
benthamiana
when gRNAs were co-delivered from separate
constructs (
Ali et al., 2015a
).
Ali et al. (2015a
,
b)
reported that
germline
PDS3
editing was observed in seeds collected from the
TABLE 1 RNA viruses modi
fi
ed to deliver Cas9 and/or gRNA constructs in plants.
Virus name
Inoculation
methods
Virus insert
cargo
Host gene
target and
editing ef
fi
ciency %
Plants used
Reference
Beet necrotic yellow
vein virus (BNYVV)
Agrobacteria
in
fi
ltration
Single gRNA
NbPDS3
: 85%
Cas9-overexpressing
N.
benthamiana
Jiang et al. (2019)
Sonchus
yellow net
rhabdovirus (SYNV)
Agrobacteria
in
fi
ltration, rub
inoculation
Cas9 and single or multiplexed
gRNAs
GFP
:77
91%
NbPDS
:40
79%
NbRDR6
:53
91%
NbSGS3
:79
91%
Multiplexed
NbRDR6
+
NbSGS3
:
60
96%
N. benthamiana
(WT
or GFP expressing)
Ma et al. (2020)
Barley stripe mosaic
virus (BSMV)
Agrobacteria
in
fi
ltration; rub
inoculation
Single gRNA (+/. FT)
TaPDS
: 3.8
96.1%
TaGW2
:
>
75%
TaGASR7
:
>
70%
N.benthamiana
; Cas9-
expressing wheat
Li et al. (2021)
Potato virus
X (PVX)
Agrobacteria
in
fi
ltration; rub
inoculation
Cas9 and gRNA
NbTOM1
N. benthamiana
Ariga, Toki and
Ishibashi, (2020)
Agrobacteria
in
fi
ltration
Single/multiplexed gRNAs+/
tRNA spacers, mobile FT
NbXT2B
:37
85%
NbPDS
:25
73%
NbFT
: 52%
Cas9-expressing
N.
benthamiana
Uranga et al.
(2021)
Tobacco mosaic
virus (TMV)
Agrobacteria
in
fi
ltration
Individual or simultaneous
delivery of Cas9 and single or
multiplex gRNAs
GFP
:61
63%
NbAGO1
:6
27%
Multiplexed: 11
64%
GFP-expressing
N.
benthamiana
Chiong et al.
(2021)
Cody et al. (2017)
Tobacco rattle
virus (TRV)
Agrobacteria
in
fi
ltration
Multiplexed gRNAs with
mobile FT or tRNA
modi
fi
cations
NbPDS3
: 58%
NbAG
:53
86%
Multiplexed: 10
95%
Cas9-expressing
N.
benthamiana
Ellison et al.
(2020)
Pea early browning
virus (PEBV)
Agrobacteria
in
fi
ltration, rub
inoculation
Single or multiple gRNAs
NbPDS
:36
72%
Cas9-expressing
N.
benthamiana
Ali et al. (2018)
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earliest
fl
oral buds, obviating plant regeneration from infected
tissue. In further testing of this system in Cas9-expressing
Arabidopsis
, TRV delivery of
AtGLI
or
AtTT4
gRNAs
produced indels at the target sites (
Ali et al., 2018
). A direct
comparison of TRV versus PEBV editing ef
fi
ciency of
PDS3
in
Cas9-expressing
N
.
benthamiana
revealed that PEBV had a
signi
fi
cantly higher editing ef
fi
ciency of 57
63% compared to
27
35% in the case of TRV (
Ali et al., 2018
). In recent studies,
TRV-delivered gRNAs produced heritable edits when fused with
mobile
FLOWERING LOCUS T
(FT) or tRNA sequences
targeting
NbPDS
and
NbAG
(
Ellison et al., 2020
). In another
study, TRV gRNA delivery was used to target viral pathogens
directly rather than focusing on plant defense-related genes to
increase resistance (
Ali et al., 2015c
). The majority of the research
using the TRV-based construct was performed in
N
.
benthamiana
; hence, more research is needed to validate these
fi
ndings in other crops.
DNA and RNA virus modi
fi
cations for
virus-induced gene silencing
VIGS is a reverse genetics tool for in vivo gene function studies
in plants (
Hayward et al., 2011
), which depends on post-
transcriptional gene silencing (PTGS) machinery in a sequence-
speci
fi
c manner. Brie
fl
y, in this process, fragments of a gene of
interest are cloned into a virual vector, and the endogenous RNA
silencing machinery of the host p
lant causes RNA degradation and
thus reduces the expression of the target gene (
Baulcombe, 2004
).
VIGS was demonstrated using numerous plant-virus types
(
Burch-Smith et al., 2004
;
Kant and Dasgupta, 2019
). In the past
decade, several viral genomes have been modi
fi
ed to create
powerful reverse genetic tools for the functional
characterization of genes in plants, such as Tobacco rattle
virus (RNA virus,
Liu et al., 2002
), Apple latent spherical
virus (RNA virus,
Gedling et al., 2018
;
Igarashi et al., 2009
),
African cassava mosaic virus (DNA virus,
Lentz et al., 2018
),
Cucumber mosaic virus (RNA virus,
Tzean et al., 2019
), barley
streak mosaic virus (RNA virus,
Bennypaul et al., 2012
), to name
a few. However, most of the reported VIGS vectors only silence a
single gene and VIGS vectors with visible indicators to evaluate
early penetrance of the plant tissue are lacking. Soon after its
discovery, the VIGS method gained immense popularity and was
readily adopted in many plants due to the ease of application and
ability to study gene knockout phenotypes (
Rössner et al., 2022
).
Later, some viral vectors were implemented for ectopic gene
expression and multigene silencing (
Xie et al., 2021
), and delivery
of guide RNA (
Gentzel et al., 2022
;
Rössner et al., 2022
).
However, plant studies are lacking in the area of virus-
mediated RNA activation (RNAa;
Voutila et al., 2012
).
Transgenerational inheritance of the silencing phenotype was
also demonstrated in a few studies (
Senthil-Kumar and Mysore,
2011
;
Bennypaul et al., 2012
). Given the large amount of
literature available on this topic, which has also been reviewed
extensively in several excellent reviews and books (
Courdavault
and Besseau, 2020
;
Rössner et al., 2022
), only a summary of some
commonly used viral VIGS vectors and delivery methods are
provided below.
The inoculation step is critical for the successful delivery of
the VIGS construct and subsequent steps involving viral
proliferation, spread, and gene silencing. The viral construct
can be delivered by three different methods:
Agrobacterium
-
mediated in
fi
ltration, in vivo/in vitro produced RNA in
fi
ltration,
and DNA in
fi
ltration. However,
Agrobacterium
-mediated
infection is the most common and convenient method. There
are several plant tissues that can be used for
Agrobacterium
infection including sprouts (
Yan et al., 2012
), roots (
Ryu et al.,
2004
), stems (
Wang et al., 2015
), leaves (
Liu et al., 2002
;
Liu et al.,
2012
), the carpopodium of young fruit (
Fu et al., 2005
), and fruits
(
Orzaez et al., 2006
).
VIGS offers several advantages, including the short period
after plant inoculation in which gene silencing effects can be
observed. Another advantage of VIGS is that it can be literally
applied to any plant species, monocots, and eudicots (
Becker
and Lange, 2010
;
Kant and Dasgupta, 2017
;
Zhang J. et al.,
2018
;
Zhang et al., 2018b
), due to the increasing availability of
viral vectors. Effective and un
iform gene silencing can be
achieved when performed at the initial stages of plant
development, but the method also works well when applied
to induce tissue-speci
fi
cgenesilencinglaterduring
development (
Stratmann et al., 2011
;
Bennypaul et al.,
2012
). VIGS allows the characterization of genes necessary
for plant survival that cannot be studied by generating
mutants or by stable genetic transformation (
Brigneti et al.,
2004
). With several listed advantages, VIGS also has some
limitations. In most cases, VIGS generally does not cause
complete silencing of the gene of interest (
Sahu et al., 2012
;
Singh et al., 2015
), and the procedure is genotype-dependent.
Lastly, the timing of the VIGS phenotype appearance and the
duration for which the gene sile
ncing effect lasts is species-
speci
fi
c. Also, an effect of the ambient temperature on VIGS
through secondary siRNA production was reported (
Fei, Pyott
and Molnar, 2021
).
Direct delivery
As mentioned earlier, many methods of gene editing
technologies focus on the introduction of raw polynucleotides
into a cell. These polynucleotides are then transcribed and/or
translated, allowing the native cellular machinery to assemble the
gene editing ribonucleoprotein (RNP) complex in vivo. Plasmid
expression, however, is not the only approach to introducing
RNPs into a cell. Gene editing RNP complexes can be pre-
assembled in vitro and then transfected into cells, a process
referred to as direct delivery.
Frontiers in
Genome Editing
frontiersin.org
11
Rustgi et al.
10.3389/fgeed.2022.1011934