International Journal of
Molecular Sciences
Review
Advances and Prospects of Virus-Resistant Breeding in Tomatoes
Zolfaghar Shahriari 1,2,† , Xiaoxia Su 1,† , Kuanyu Zheng 1, * and Zhongkai Zhang 1, *
1
2
*
†
Biotechnology and Germplasm Resources Research Institute, Yunnan Academy of Agricultural Sciences,
Yunnan Seed Laboratory, 2238# Beijing Rd, Panlong District, Kunming 650205, China;
shahriari225@gmail.com (Z.S.); sxx919@163.com (X.S.)
Crop and Horticultural Science Research Department, Fars Agricultural and Natural Resources Research and
Education Center, Agricultural Research, Education and Extension Organization (AREEO),
Shiraz 617-71555, Iran
Correspondence: zky@yaas.org.cn (K.Z.); zzk@yaas.org.cn (Z.Z.); Tel.: +86-871-6512-3133 (K.Z. & Z.Z.)
These authors contributed equally to this work.
Abstract: Plant viruses are the main pathogens which cause significant quality and yield losses in
tomato crops. The important viruses that infect tomatoes worldwide belong to five genera: Begomovirus, Orthotospovirus, Tobamovirus, Potyvirus, and Crinivirus. Tomato resistance genes against
viruses, including Ty gene resistance against begomoviruses, Sw gene resistance against orthotospoviruses, Tm gene resistance against tobamoviruses, and Pot 1 gene resistance against potyviruses,
have been identified from wild germplasm and introduced into cultivated cultivars via hybrid breeding. However, these resistance genes mainly exhibit qualitative resistance mediated by single genes,
which cannot protect against virus mutations, recombination, mixed-infection, or emerging viruses,
thus posing a great challenge to tomato antiviral breeding. Based on the epidemic characteristics
of tomato viruses, we propose that future studies on tomato virus resistance breeding should focus
on rapidly, safely, and efficiently creating broad-spectrum germplasm materials resistant to multiple viruses. Accordingly, we summarized and analyzed the advantages and characteristics of the
three tomato antiviral breeding strategies, including marker-assisted selection (MAS)-based hybrid
breeding, RNA interference (RNAi)-based transgenic breeding, and CRISPR/Cas-based gene editing.
Finally, we highlighted the challenges and provided suggestions for improving tomato antiviral
breeding in the future using the three breeding strategies.
Citation: Shahriari, Z.; Su, X.; Zheng,
K.; Zhang, Z. Advances and
Keywords: virus resistance gene; RNAi; tomato breeding; CRISPR-Cas; genome editing
Prospects of Virus-Resistant Breeding
in Tomatoes. Int. J. Mol. Sci. 2023, 24,
15448. https://doi.org/10.3390/
ijms242015448
Academic Editors: Yong-Gu Cho
and Yangyong Zhang
Received: 1 August 2023
Revised: 15 October 2023
Accepted: 16 October 2023
Published: 22 October 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1. Introduction
Tomato (Solanum lycopersicum L.) is one of the most economically valuable fruit or
vegetable crops worldwide. According to the Food and Agriculture Organization of
the United Nations (FAO) Statistical report, the total worldwide production of tomatoes
was 189.23 million tons in 2021, with a value of over USD 30 billion. Viral diseases
can significantly decrease the yield and quality of tomatoes [1]. According to the new
classification system (2022) approved by the International Committee for the Classification
of Viruses (ICTV), there are 181 viral species infecting tomato crops. The major tomato viral
pathogens that have been emerging worldwide over the past 20 years include the following
genera: Begomovirus, Orthotospovirus, Tobamovirus, Potyvirus, and Crinivirus [1–3].
Considering that most of these viruses are transmitted by insect vectors, the main
agronomic and classic management of viral diseases involves controlling the vector with
insecticides and uprooting symptomatic plants or those with sanitary voids to reduce the
incidence of the virus [4,5]. However, these controlling methods only reduce the viral
effects to some extent and cannot efficiently eliminate tomato virus disease [6]. Virus
resistance breeding is the most promising method for controlling viral diseases [7,8].
Combining conventional hybrid breeding with marker-assisted selection (MAS) to introduce resistance genes from wild germplasm into cultivated cultivars has proven to
Int. J. Mol. Sci. 2023, 24, 15448. https://doi.org/10.3390/ijms242015448
https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2023, 24, 15448
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be effective for virus resistance breeding in tomatoes [8–10]. However, most of the virus
resistance genes currently found in tomatoes are single-gene-mediated with qualitative
resistance, and thus, virus mutation and mixed infection can easily lead to resistance
breakdown [11–13]. In addition, some emerging tomato viruses, such as tomato chlorosis
virus (ToCV), tomato brown rugose fruit virus (ToBRFV), and tomato mottle mosaic virus
(ToMMV), still lack relevant natural resistance genes, making conventional virus resistance
breeding in tomatoes challenging [14–16].
This review summarizes tomato viruses and their characteristics. According to the
epidemic characteristics of tomato viruses, we propose that tomato virus resistance breeding should focus on rapidly, safely, and efficiently creating broad-spectrum germplasm
materials resistant to multiple viruses. Based on this proposition, we summarize and
analyze the advantages and characteristics of three tomato antiviral breeding strategies:
MAS-based hybrid breeding, RNA interference (RNAi)-based transgenic breeding, and
CRISPR/Cas-based gene editing. Finally, we discuss the challenges and provide suggestions for improving these three breeding strategies in the future.
2. Tomato Viruses and Their Epidemic Characteristics
Currently, the main tomato epidemic viruses worldwide include begomoviruses,
orthotospoviruses, tobamoviruses, potyviruses, and criniviruses. The classification and
details of these viruses are listed in Table 1. These major epidemic viruses generally cause
tomato plant leaf shrinkage, chlorosis, leaf and fruit necrosis, spotting, and other symptoms,
which significantly impact tomato quality and yield (Figure 1).
Figure 1. Symptoms of viral diseases in tomatoes. (A) Symptoms of TSWV on tomato fruits.
(B) Symptoms of ToBRFV on tomato fruits. (C) Symptoms of TYLCV on tomato leaves. (D) Symptoms
of ChiVMV on tomato leaves. (E) Symptoms of ToCV on tomato leaves.
A typical characteristic of plant viruses is the rapid nucleotide mutation and genome
recombination rates, which contribute to the emergence of novel viral strains or species,
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increase the virulence of the virus, and cause the breakdown of host resistance, resulting in
severe symptoms in the host plant [17–19].
Another characteristic of plant viruses, as revealed by high-throughput sequencing
or other means of detection, is the presence of high mixed-infection incidence in the field
between viruses or between viruses and other pathogens [2,20,21]. Mixed infections usually
increase synergies between pathogens and the breakdown of host resistance, which has
been reported in many cases [13,22]. Therefore, developing a quick response against the
resistance breakdown caused by virus mutation, recombination, and mixed infection to
efficiently create broad-spectrum and persistent antiviral germplasm materials has become
a major challenge and the primary objective in tomato antiviral breeding.
Table 1. Classification and details of the major epidemic viruses of tomato.
Genus
Epidemic Species
Worldwide
Genome
Transmission
Symptoms
References
Whitefly, seed
Yellowing, curling, and a
significant loss in apical
leaf. Early-infected plants
are frequently infertile.
Since most blooms (>90%)
droop after infection, there
is almost no or fewer
small fruit.
[23–26]
[27–29]
Tomato yellow leaf
curl virus
(TYLCV)) and
tomato leaf curl
virus (ToLCV)
Single-stranded
DNA (ssDNA)
Orthotospovirus
Tomato spotted
wilt virus (TSWV)
Negative-sense
single-stranded
ambisense
(-ssRNA) RNA
Thrips, seed
Stunting, necrosis,
bronzing, chlorosis, ring
spots, and ring patterns on
the leaves, stems,
and fruits.
Tobamovirus
Tobacco mosaic
virus (TMV),
tomato mosaic
virus (ToMV), and
tomato brown
rugose fruit virus
(ToBRFV)
Single-stranded
positive-sense
RNA (+ssRNA)
Seed, mechanical
transmission such
as by hand,
pruning tools,
soil, etc.
Yellow–green mottling on
the leaves; stunted growth;
flowers and leaflets may
be curled, distorted, and
smaller than normal
in size.
[15,30–34]
Potyvirus
Potato virus Y
(PVY), and, chilli
veinal mottle virus
(ChiVMV)
Single-stranded
positive-sense
RNA (+ssRNA)
viruses
Aphid, seed
Leaf mosaic, mottle and
crinkling, vein necrosis
and necrotic spots, stem
and petiole necrosis, leaf
drop, and yield reduction.
[2,21,35,36]
Crinivirus
Tomato chlorosis
virus (ToCV)
Single-stranded
positive-sense
RNA (+ssRNA)
viruses
Whitefly
Leaf chlorosis, chlorotic
flecking, and bronzing.
Fruits are symptomless
but with reduced yield.
[37,38]
Begomovirus
3. Strategies of Tomato Resistance Breeding against Viruses
At present, there are three main strategies for tomato antiviral breeding: MAS-based
hybrid breeding, RNAi-based transgenic breeding, and CRISPR/Cas-based gene editing.
(1) MAS-based hybrid breeding. In this strategy, wild or domestic germplasm with resistance genes is hybridized with non-resistance germplasm materials, and F1 hybrid
generation is further selfed for multiple generations. By serving as a resistance selection
method for the selfed generations, MAS allows for the rapid creation of new resistance
germplasm (Figure 2A) [39]. (2) RNAi-based transgenic breeding. In this strategy, a
targeted virus gene/dsRNA/microRNA sequence is transferred into the non-resistant
germplasm material by the Agrobacterium-mediated transformation method so as to induce the RNA silencing effect in the host plant to resist virus infection (Figure 2B) [40].
Int. J. Mol. Sci. 2023, 24, 15448
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(3) CRISPR/Cas-based gene editing. In this strategy, a CRISPR/Cas vector is designed to
target the DNA/RNA sequence of the virus or the host’s susceptible genes, thus interrupting the replication and assembly process of the virus in the host, ensuring resistance against
the virus (Figure 2C) [41]. These three strategies are discussed in the following sections.
Figure 2. Strategies of tomato resistance breeding against viruses. (A) Marker-assisted selection (MAS)-based hybrid breeding. (B) RNA interference (RNAi)-based transgenic breeding.
(C) CRISPR/Cas-based gene editing.
4. Virus Resistance Gene and MAS-Based Hybrid Breeding in Tomatoes
4.1. The Tomato Ty Gene Family Encoding for Resistance against Begomoviruses
At present, six begomoviruses resistance genes, all belonging to the Ty gene family,
have been identified, namely, Ty-1, Ty-2, Ty-3, Ty-4, Ty-5, and Ty-6 (Table 2). Ty-1, Ty-3,
Ty-4, and Ty-6 are derived from the wild tomato species S. chilense, while Ty-2 and Ty-5 are
obtained from S. habrochaites and the commercial tomato cultivar Tyking, respectively [42,43].
Ty-1 is allelic with Ty-3, and the two genes are located on chromosome 6 of S. chilense [44].
Ty-1/3 increases the cytosine methylation of viral genomes and induces a hypersensitive
response to viral infections, conferring plants with TYLCV resistance [45]. Although
Ty-1 exhibits broad-spectrum begomoviruses resistance, recent studies have shown that
resistance is compromised by the co-infection with a beta satellite [46]. Gene Ty-2 located
on the long arm of S. habrochaites chromosome 11 has been identified as the nucleotidebinding domain and a leucine-rich repeat-containing (NB-LRR) gene [47]. Moreover, the
Ty-4 gene maps to chromosome 3 of S. chilense and has been reported to increase virus
resistance in combination with Ty-3 [42]. The recessive TYLCV resistance gene Ty-5 located
on chromosome 4 of the commercial tomato cultivar Tyking encodes the mRNA surveillance
factor Pelota [8,42]. A recent study showed that Ty-5 confers broad-spectrum resistance to
two representative begomoviruses occurring in China [8]. Ty-6, located on chromosome
10 of S. chilense, effectively complements the resistance conferred by Ty-3 and Ty-5 [48]. It
was reported that Ty-6 also confers resistance to tomato mottle virus (ToMoV), suggesting
that the gene inhibits both mono- and bi-partite begomoviruses in tomatoes [48]. Although
PCR-based markers have been identified and developed for Ty-1, Ty-2, Ty-3, and Ty-4
TYLCV-resistant loci, these markers are inconsistent, thus limiting their application in
MAS [49,50].
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Table 2. A brief review of virus resistance gene families in tomatoes.
Resistance Gene
Family
Ty gene family
(against
begomoviruses)
Sw gene family
(against orthotospoviruses)
Tm gene family
(against
tobamoviruses)
Pot-1 gene
(against
potyviruses)
Resistance
Genes
Source of
Resistance Genes
Location on
Chromosome
Gene Action
Efficiency
Resistance Mechanism
Ty-1
Solanum chilense
6
Dominant
Broad-spectrum
begomoviruses resistance
Ty-1 encodes an RNA-dependent RNA polymerase (RDR) involved in the RNA silencing
pathway, increasing antiviral RNAi responses and the viral genome’s
cytosine methylation.
Ty-2
S. habrochaites
11
(Long arm)
Dominant
TYLCV resistance
Ty-2 encodes a nucleotide-binding leucine-rich repeat (NLR) protein. The Ty-2 could
recognize TYLCV Rep/C1 protein and induce hypersensitive responses (HR) in host plant.
Ty-3
S. chilense
6
Dominant
Complementary resistance
Ty-3 encodes an RNA-dependent RNA polymerase (RDR) involved in the RNA
silencing pathway.
Ty-4
S. chilense
3
Dominant
Increase virus resistance in
combination with Ty-3
Not reported.
Ty-5
Tyking
4
Recessive
Broad-spectrum resistance
Encodes messenger RNA (mRNA) surveillance factor Pelota. Silencing of Pelota in a
susceptible line rendered the transgenic plants highly resistant.
Ty-6
S. chilense
10
Dominant
Complements the
resistance conferred by
Ty-3 and Ty-5
Not reported.
Sw-1a
Lycopersicum
pimpinellifolium
Not reported
Dominant
Some degree of resistance
to specific TSWV
Not reported.
Sw-1b
L. pimpinellifolium
Not reported
Dominant
Some degree of resistance
to specific TSWV
Not reported.
Sw-2
L. pimpinellifolium
Not reported
Recessive
Some degree of resistance
to specific TSWV
Not reported.
Sw-3
L. pimpinellifolium
Not reported
Recessive
Some degree of resistance
to specific TSWV
Not reported.
Sw-4
L. pimpinellifolium
Not reported
Recessive
Some degree of resistance
to specific TSWV
Not reported.
Sw-5
S. peruvianum
9
Dominant
High level of resistance to
a wide range of TSWV
Sw-5 belongs to nucleotide-binding leucine-rich repeat (NB-LRR) type R gene. Sw-5
confers resistance by recognizing a 21-amino-acid peptide region of the viral movement
protein NSm, triggering immunity response.
Sw-6
L. pimpinellifolium
Not reported
Incompletely
Dominant
Some degree of resistance
to specific TSWV
Not reported
Sw-7
L. chilense
12
Dominant
Resistance to a wide range
of TSWV
Involved in pathogenesis-related (PR) proteins PR1 and PR5-related resistance process.
Tm-1
S. habrochaites
2
Incompletely
Dominant
TMV partial resistance
Tm-1 encodes a protein that binds ToMV replication proteins and inhibits the
RNA-dependent RNA replication of ToMV.
Tm-2
S. peruvianum
9
Dominant
TMV partial resistance
Tm-2 belongs to nucleotide-binding leucine-rich repeat (NB-LRR) type R gene, which can
recognize movement proteins (MPs) of TMV and ToMV and activate a resistance response.
References
[8,42,44–47,51–54]
[10,50,52–54]
Tm-22
S. peruvianum
9
Dominant
Confers a more effective
TMV resistance
Tm-2 belongs to nucleotide-binding leucine-rich repeat (NB-LRR) type R gene, which can
recognize movement proteins (MPs) of TMV and ToMV and activate a resistance response.
Pot-1
L. hirsutum
3
Recessive
PVY resistance
Tomato Pot-1 is the orthologue of the pepper pvr2-eIF4E gene, encoding the
plant-susceptible eIF4E1 translation initiation factor protein. Duplicate recessive Pot-1
genes interrupt the interaction of the potyviruses VPg protein with the eIF4E1,
suppressing virus replication.
[14,55–60]
2
[61–63]
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4.2. The Tomato Sw Gene Family Conferring Resistance against Orthotospoviruses
S. Peruvianum, reported as the first wild tomato with a broad-spectrum resistance to
tomato spotted wilt virus (TSWV), has been widely crossed with commercial cultivars since
the 1930s [7]. Studies on molecular genetics indicated that this resistance was conferred
by a single dominant gene/locus named Sw-5, which was initially found to be effective
against several TSWV isolates from the United States [64] and Brazil [65]. Sw-5 also exhibits
a broad spectrum and high level of resistance to other orthotospoviruses, including tomato
chlorotic spot virus (TCSV), chrysanthemum stem necrosis virus (CSNV), and groundnut
ringspot virus (GRSV), and is widely used in tomato breeding [7,66]. Sw-5 was discovered
near the telomeric area of chromosome 9 between the CT71 and CT220 restriction fragment
length polymorphism (RFLP) markers [67]. The Sw-5 locus is part of a loosely clustered
gene family containing six paralogous genes: Sw-5a, Sw-5b, Sw-5c, Sw-5d, Sw-5e, and Sw5f [7,10]. Among these genes, only Sw-5b has universal resistance to various TSWV isolates,
although Sw-5a and Sw-5b are highly homologous (95%) [10,68]. In addition to the Sw-5
gene, seven other genes resistant to TSWV belonging to the Sw gene family have been
currently identified in tomatoes, and they include Sw-1a, Sw-1b, Sw-2, Sw-3, Sw-4, Sw-6, and
Sw-7 (Table 2). The introgression/incorporation of these resistance alleles in the commercial
varieties could create materials with broad-spectrum resistance [69]. Molecular markers
associated with TSWV resistance in tomatoes were summarized in a previous study that
developed more than 20 molecular linkage markers for Sw-5 and Sw-7 TSWV resistance
genes [10]. Some of these linkage markers included randomly amplified polymorphic
DNA (RAPD), a sequence-characterized amplified region (SCAR), amplified fragment
length polymorphisms (AFLP), cleaved amplified polymorphic sequence (CAPS), insertion–
deletion (In-DEL), SNP, competitive allele-specific PCR (KASP), RFLP, and simple sequence
repeats (SSR). However, the linkage molecular markers of Sw-1a, Sw-1b, Sw-2, Sw-3, Sw-4,
and Sw-6 have not been reported yet [10].
4.3. The Tomato Tm Gene Family Conferring Resistance against Tobamoviruses
For more than five decades, three resistance genes, Tm-1, Tm-2, and Tm-22 , have been
considered as the resistance factors against tobamoviruses in tomatoes (Table 2) [55,56,70].
Tm-1 is an incompletely dominant gene derived from the wild tomato S. habrochaites [14,55].
Conversely, Tm-2 and Tm22 are completely dominant genes introgressed from S. peruvianum
and are considered allelic [14]. Tm-22 confers more effective resistance than Tm-1 or Tm-2
and has shown durable ToMV resistance for the last 60 years, explaining why it is the
most currently and widely utilized in breeding tomato cultivars [71]. Reports showed
that ToBRFV overcomes all the tobamoviruses resistance genes in tomatoes, including the
durable Tm-22 resistance gene [57]. The PCR-based markers for Tm-1, Tm-2, and Tm-22
resistant genes have also been reportedly used for MAS [50,72,73]. Despite the many
advantages and several reports on its application in studying virus resistance genes, MAS
is still in the research stage of its utilization in breeding tomatoes for virus resistance [50].
4.4. The Tomato Pot 1 Gene Conferring Resistance against Potyviruses
The wild tomato relative Lycopersicon hirsutum PI247087 was identified as the source of
resistance to potyviruses [61,62]. Analysis indicated that resistance is conferred by a single
recessive gene, pot-1, mapped to the short arm of tomato chromosome 3 in the vicinity
of the recessive py-1 locus for resistance to corky root rot [61]. Studies revealed that the
recessive pot-1 gene is the orthologue of the pepper (Capsicum annuum) pvr2 gene [63].
4.5. The Challenges in Breeding Tomato Hybrid Cultivars against Viruses
Although using natural virus-resistant germplasm resources for hybrid breeding is
the preferred and most effective way for obtaining resistant commercial varieties, only a
few virus-resistant tomato germplasm resources are available, and the resistance is limited
to a few viruses [74]. For example, virus resistance hybrid breeding cannot be applied
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against emerging viruses, such as ToCV, ToBRFV, ToMMV, etc., due to the lack of their
corresponding natural resistance genes [14–16].
The other challenge is that most of the virus-resistance genes discovered so far exhibit
single-gene-mediated qualitative resistance, which can be easily broken by virus mutations and mixed-infection, a phenomenon that has been commonly reported [12,13,75].
Improving multiple virus-resistant lines and cultivars by introducing several resistance
genes targeting different viruses in a cultivar may play an important role in future tomato
improvement projects [76–79]. Though promising, this process is complex and requires
very long cycles of hybrid breeding. One example is a homozygous breeding line UMH
1203 carrying the Tm-2a , Ty-1, and Sw-5 genes, which took 10 years to successfully develop
multiple resistance against ToMV, TSWV, and TYLCV from several tomato landraces [78,80].
Nevertheless, a yield reduction of 40–50% has been reported for this breeding line at lowvirus-incidence conditions [80]. Several reports indicated that the introgression of TYLCV
resistance caused most of the yield reduction observed in fresh tomatoes due to the introgressed genes and/or linkage drag from the wild tomato species [80,81]. This reduction in
agricultural yield would only be acceptable when cultivating under high levels of virus
infection, and these new multi-resistance lines should only be used to develop cultivars for
highly virus-infected areas [81].
5. RNAi-Based Transgenic Breeding
RNA silencing, also known as RNAi, is a conserved defense mechanism that suppresses the expression of viral nucleic acids, transposable elements, or host genes that need
to be regulated [40,82]. The principle is based on the recognition and splicing of the doublestranded (ds) or hairpin (hp) RNA by Dicer-like (DCL) proteins into 21- to 24-nucleotide (nt)
small RNAs (sRNAs). The subsequent steps involve a series of sRNA signal amplification
and cleavage target mRNA processes with the participation of RNA-induced silencing
complex (RISC) [83]. RNAi is an important pathway for plants resisting viral infections
through the sequence-specific degradation of target viral RNA [40,74,84–86].
In the first-generation antiviral transgenic strategy, a single-stranded sequence of a
viral gene, such as viral coat protein (CP) gene, RNA dependent RNA polymerase (RdRp)
gene, etc., is transferred into the host plant genome, inducing the RNA silencing effect
against the target virus by the host (Table 3). Subsequent studies found that transferring
double-stranded RNA (dsRNA) or hairpin RNA (hpRNA) constructed based on viral
sequences was more effective in inducing the RNA silencing effect than single-stranded
viral sequences, making it a second-generation antiviral transgenic strategy (Table 3). With
the recent depth of research on sRNAs, a new antiviral gene transfer strategy based on
artificial sRNA engineering technology has been developed [87]. This third-generation
antiviral transgenic strategy is based on artificial microRNAs (amiRNAs) or synthetic transacting small interfering RNAs (syn-tasiRNAs), which are 21 nt and artificially engineered
to be highly specific to ensure a high sequence complementarity with target virus RNA
and overcome the limited specificity of RNAi [88,89]. AmiRNAs and syn-tasiRNAs are
functionally similar but are generated differently. AmiRNAs are derived from the DCL1
cleavage of miRNA precursors with foldback structures, while syn-tasiRNAs are produced
in a multi-step RNAi process [90].
The biggest advantage of the third-generation antiviral transgenic strategy based on
amiRNAs and syn-tasiRNAs is that it aggregates multiple-virus-targeting, which rapidly
creates broad-spectrum resistance [91–93]. Many successful cases have been reported in
this regard. For example, Arabidopsis miR159 was used as a backbone to express genes
targeting P25, HC-Pro, and Brp1 of potato virus X (PVX), potato virus Y (PVY), and
potato spindle tuber viroid (PSTVd) via the third-generation antiviral transgenic strategy,
demonstrating resistance against PVX, PVY, and PSTVd co-infection simultaneously [92].
Another study showed that the Arabidopsis TAS3a gene was engineered to express syntasiRNAs targeting the genome of turnip mosaic virus (TuMV) and cucumber mosaic virus
Int. J. Mol. Sci. 2023, 24, 15448
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(CMV). The transgenic Arabidopsis thaliana plants expressing these syn-tasiRNAs showed
high levels of resistance to both viruses [91].
In general, the third-generation antiviral transgenic strategy has shown high potential
for virus resistance breeding. However, public concern and controversy over genetically
modified (GM) crops and the strict regulation of policies have greatly inhibited the commercial development potential of GM crops. Therefore, some researchers opt to apply amiRNAs
or dsRNA exogenously as crude extracts to prevent virus infection, which can also effectively induce the gene-silencing pathway in host plants against virus infection [94,95].
Table 3. RNAi-based transgenic virus breeding methods in plants.
Strategy
First-generation
antiviral
transgenic
strategy
Secondgeneration
antiviral
transgenic
strategy
Target Virus
Genus
RNAi Induction
Method
Targeted
Region
Precursor(s)
Efficiency
Reference
TMV
Tobamovirus
ssRNA
CP
cDNA
Delayed symptom development; 10 to
60 percent of the transgenic plants failed
to develop symptoms.
[96]
TMV
Tobamovirus
ssRNA
CP
cDNA
The resistance level of expression TMV
CP from the pal2 promoter is less than
that of the 35S promoter.
[97]
TSWV
Orthotospovirus
ssRNA
N
cDNA
Lack of systemic symptoms and little or
no systemic accumulation of virus.
[98]
ToLCV
Begomovirus
ssRNA
Rep
cDNA
A high level of resistance and
inheritability of the transgene was
observed up to T2.
[99]
TLCV
Begomovirus
ssRNA
CP
cDNA
T1-generation
transgenic plants were showed variable
degrees of disease resistance/tolerance
compared to the untransformed control.
[100]
ToLCNDV
Begomovirus
ssRNA
AV2
cDNA
Transgenic plants showed symptomless,
although viral DNA could be detected
in some plants by PCR.
[101]
PRSV
Potyvirus
ssRNA
CP
cDNA
PRSV infection was not observed on any
of the transgenic resistance (TR) plants.
TR plant yields were at least three times
higher than the industry average.
[102]
TMV
Tobamovirus
dsRNA
CP, p126
dsRNA
The application of TMV p126 dsRNA
onto tobacco plants induced greater
resistance against TMV infection as
compared to CP dsRNA (65 vs. 50%).
[103]
ToLCV
Begomovirus
hpRNA
AC1,
AC4
hpRNA
Provides a promising approach to
suppress a wide spectrum of ToLCV
infection in the tomato.
[104]
ToLCV
Begomovirus
dsRNA
AC4
dsRNA
Absolute absence of leaf curl virus
disease symptoms and reduction in
nematode symptoms.
[105]
ToCMoV
Begomovirus
hpRNA
AC1,
AC4,
AV1,
AC5
hpRNA
Most transgenic lines showed significant
delays in symptom development, and
two lines had immune plants.
[106]
PVY
Potyvirus
dsRNA
CP
dsRNA
Highly resistant to three strains of PVY.
[107]
[108]
[109]
PVY
Potyvirus
CaCV
GBNV
CMV
ChiVMV
Orthotospovirus
Cucumovirus
Potyvirus
hpRNA
CP
hpRNA
Nine of the ten transgenic lines showed
no infection by PVYO , and six of the
nine showed no infection by PVYNTN .
hpRNA
viral
silencing
suppressors
gene
hpRNA
Efficiently controls multiple viruses
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Table 3. Cont.
Strategy
Third-generation
antiviral
transgenic
strategy
Target Virus
Genus
RNAi Induction
Method
Targeted
Region
Precursor(s)
Efficiency
Reference
ToLCNDV
Begomovirus
amiRNA
AV1,
AV1 +
AV2
AthmiR319a
High tolerance when targeting AV1 +
AV2. Moderate tolerance when
targeting AV1.
[87]
TYLCV
Begomovirus
amiRNA
AC1+Rep
AthmiR159a
Confer resistance to TYLCV.
[110]
TSWV
Orthotospovirus
syn-tasiRNA
TAS1c
100% of the plants were resistant.
[111]
TSWV
Orthotospovirus
syn-tasiRNA
RdRP
TAS1c
Delay of viroid accumulation
[112]
amiRNA
L, M, G
TomiR6026
Bioinformatic assay showed successful
results in controlling both viruses.
[93]
PhCMoV,
ToBRFV
Alphanucleorhabdovirus
Tobamovirus
NSm+RdRP
PVY
Potyvirus
amiRNA
CI, NIa,
NIb, CP
AthmiR319a
Higher protection when targeting NIb
or CP.
[113]
PVY,
PVX,
PSTVd
Potyvirus
Potexvirus
Pospiviroid
amiRNA
P25,
HC-Pro,
Brp1
AthmiR159a
Resistance against PVX, PVY, and
PSTVd coinfection simultaneously,
whereas the untransformed controls
developed severe symptoms.
[92]
PRSV: papaya ringspot virus; ToLCV: tomato leaf curl virus; ToLCNDV: tomato leaf curl New Delhi virus;
ToCMoV: tomato chlorotic mottle virus; CaCV: capsicum chlorosis virus; GBNV: groundnut bud necrosis virus;
PhCMoV: physostegia chlorotic mottle virus; ToBRFV: tomato brown rugose fruit virus.
6. Virus Resistance Breeding Based on the CRISPR/Cas Genome Editing
CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPRassociated) systems have recently emerged as efficient genome editing tools that provide a
new breeding strategy for crop breeding against pathogens [114]. CRISPR/Cas has been
successful in breeding some crops for pathogen resistance, such as wheat resistant to rust
fungi [115], rice resistant to bacterial blight [116], and tomatoes resistant to viruses [114,117].
The CRISPR/Cas system comprises two key components: the guide RNA (gRNA)
that complements the target editing sequence and the Cas endonuclease that cleaves the
sequences targeted by the gRNA [118]. Cas endonuclease can be divided into two distinct
classes (I and II) and six types (I to VI) based on their functional mechanisms [119]. Class
I includes types I, III, and IV, which utilize a multi-protein effector complex, while Class
II includes types II, V, and VI, which utilize a single effector protein, conferring it a wider
adaptability than Class I [120,121]. The CRISPR/Cas system is utilized in two ways in
virus resistance breeding: (1) targeting the viral genomic sequence for gene editing by
cleaving or mutating the viral genome to inhibit viral replication in the host [41,122,123] and
(2) knocking out or mutating the host susceptibility genes involved in virus infection and
replication process to reduce the compatible interaction between host and the virus [124].
6.1. CRISPR/Cas Genome Editing Targeting DNA Viruses
CRISPR/Cas9, which is currently widely used in gene editing, belongs to Class II and
Type II [121]. Since DNA viruses can form dsDNA intermediates during replication, the
CRISPR/Cas system can be used to target viral DNA sequences for cleavage or mutation to
inhibit viral replication. TYLCV was the first geminivirus to be edited by CRISPR/Cas9
for TYLCV-resistant tobacco breeding [125]. This method has been successfully used on
tobacco, Arabidopsis, and tomato to generate multi-generational stable resistance against
TYLCV, demonstrating the great potential of CRISPR/Cas in anti-geminivirus breeding
(Table 4).
For the CRISPR/Cas-mediated engineering of tomato against geminiviruses, the
intergenic (IR), CP, and replication (Rep) regions of the geminiviruses were selected as
target sites of gRNA (Table 4). The genus Geminivirus has a conserved sequence (5′ TAATATAC-3′ ) in the IR region. Therefore, an IR-gRNA targeting this conserved sequence
could be used to develop a broad-spectrum resistant tomato that is resistant to various
geminiviruses, including TYLCV, cotton leaf curl kokhran virus (CLCuKoV), and merremia
mosaic virus (MeMV). This could significantly reduce the virus accumulation and alleviate
disease symptoms in tomatoes [125,126].
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6.2. CRISPR/Cas Genome Editing Targeting RNA Viruses
The Cas endonucleases of the CRISPR/Cas system targeting plant RNA viruses mainly
include FnCas9 (discovered from Francisella novicida) belonging to Type II of Class II of the
Cas nuclease family [127] and CRISPR/Cas13 (formerly known as C2c2) belonging to Type
VI of Class II [128]. Cas13 can be divided into several groups, including Cas13a, Cas13b,
Cas13c, etc. [129–131]. Cas13a is the first direct homolog of the Cas13 family used to cleave
single-stranded RNA (ssRNA) fragments in CRISPR/Cas-mediated gene editing [129].
So far, the CRISPR/FnCas9 and CRISPR/Cas13a systems have been successfully used to
edit potatoes, tobacco, rice, sweet potato, and other crops for resistance against various
RNA viruses. These viruses include PVY, TMV, CMV, southern rice black-streaked dwarf
virus (SRBSDV), rice stripe mosaic virus (RSMV), etc. (Table 4). The gRNA target site
in the RNA viruses is similar to that in DNA viruses, which is mainly located in the IR
region and some key regions of the RNA virus coding protein (Table 4). Unfortunately,
CRISPR/Cas-mediated editing against RNA viruses has not been reported in tomatoes.
Int. J. Mol. Sci. 2023, 24, 15448
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Table 4. CRISPR/Cas gene editing system used to create virus resistance in tomatoes and other crops.
Target Virus
Genus
Plant
Targeted
Genome
CRISPR/Cas
TYLCV
Begomovirus
Tomato
Viral DNA
TYLCV
Begomovirus
Tomato
Viral DNA
TYLCV
Begomovirus
Tomato
CLCuKoV
Begomovirus
MeMV
Begomovirus
CMV
Targeted Region
Efficiency
Reference
CRISPR/Cas9
CP, IR
Significant reduction or delayed accumulation of viral DNA compared to the control plants.
[132]
CRISPR/Cas9
CP, Rep
Low accumulation of the viral DNA genome compared to the control plants.
[114]
Viral DNA
CRISPR/Cas9
IR, CP, RCRII
Reduction or delayed accumulation of viral DNA, abolishing or significantly attenuating symptoms
of infection.
[125]
Tomato
Viral DNA
CRISPR/Cas9
IR, CP, RCRII
Tomato
Viral DNA
CRISPR/Cas9
IR, CP, RCRII
Significantly limits CLCuKoV and MeMV replication and systemic infection.
[126]
Cucumovirus
Arabidopsis
Viral RNA
CRISPR/FnCas9
ORF1a, ORF CP,
3′ UTR
Tobamovirus
Tobacco
Viral RNA
CRISPR/FnCas9
Significantly attenuated infection symptoms and reduced viral RNA accumulation. The resistance was
inheritable, and the progenies showed significantly low virus accumulation.
[133]
TMV
3′ ORFs
TuMV
Potyvirus
Tobacco
Viral RNA
CRISPR/LshCas13a
HC-Pro, CP
Targeting the HC-Pro rather than those targeting the coat protein (CP) sequence significantly inhibits
TuMV-GFP accumulation and systematic movement.
[134]
TuMV
Viral RNA
CRISPR/LshCas13a
HC-Pro, CP
Significant inhibition of TuMV-GFP accumulation level and systematic movement in T1 and T2 plants.
[135]
[136]
Potyvirus
Arabidopsis
PVY
Potyvirus
Potato
Viral RNA
CRISPR/LshCas13a
P3, CI, NIb, CP
Specifically resistant to multiple PVY strains while having no effect on unrelated viruses such as PVA or
Potato virus S.
TMV
Tobamovirus
Tobacco
Viral RNA
CRISPR/LshCas13a
RdRp, MP, CP
Significant reduction or delayed accumulation of viral RNA compared to the control plants.
[137]
SRBSDV
Fijivirus
Rice
Viral RNA
CRISPR/LshCas13a
ORF
Abolishing or significantly attenuating symptoms of infection. T3 transgenic plants we tested showed
stable resistance to SRBSDV.
[137]
RSMV
Cytorhabdovirus
Rice
Viral RNA
CRISPR/LshCas13a
ORF
Abolishing or significantly attenuating symptoms of infection. T3 transgenic plants we tested showed
stable resistance to RSMV.
[137]
SPCSV
Crinivirus
Sweet
potato
Viral RNA
CRISPR/LwaCas13a
CRISPR/13d
RNase3
Transgenic plants and their grafted plants showed a significant reduction in virus accumulation and
were asymptomatic.
[138]
TYLCV
Begomovirus
Tomato
Tomato genome
CRISPR/Cas9
SlPelo
Knocking out the bialleles of SlPelo proved to suppress systematic infection of TYLCV.
[117]
PVY
Potyvirus
Tomato
Tomato genome
CRISPR/Cas9
eIF4E1
Significant reduction in susceptibility to the N strain (PVY-N) but not to the ordinary strain (PVY-O).
[139]
CMV
Cucumovirus
Tomato
Tomato genome
CRISPR/Cas9
eIF4E1
Viral aphid transmission from an infected susceptible plant to gene-edited plants was reduced compared
with the parental control.
[139]
TEV
Potyvirus
Tomato
Tomato genome
CRISPR/Cas9
eIF4E1
A combination of mutations in regions I and II of eIF4E1 associates with resistance to several isolates
of potyviruses.
[140]
[140]
PVY
Potyvirus
Tomato
Tomato genome
CRISPR/Cas9
eIF4E1
Differences in silent targets showed differences in resistance levels.
PepMoV
Potyvirus
Tomato
Tomato genome
CRISPR/Cas9
eIF4E1
Knocking out eIF4E1 exhibited a significant reduction accumulation of PepMoV but not TEV.
[141]
PVMV
Potyvirus
Tomato
Tomato genome
CRISPR/Cas9
4E2 (eIF4E2)
Knocking out eIF4E2 exhibited resistance to six of the eight PVMV isolates but not to other potyviruses.
[142]
TuMV
Potyvirus
Tomato
Tomato genome
CRISPR/Cas9
eIF(iso)4E
Homozygous mutations and transgene-free T2 and T3 generation in self-pollinating species showed no
differences in dry weights and flowering times with wild-type plants under standard growth conditions.
[143]
ToBRFV
Tobamovirus
Tomato
Tomato genome
CRISPR/Cas9
SlTOM1a-e
Quadruple-mutant plants did not show detectable ToBRFV CP accumulation or obvious defects in growth
or fruit production. The quadruple-mutant plants also showed resistance to three other tobamoviruses.
[31]
ToBRFV
Tobamovirus
Tomato
Tomato genome
CRISPR/Cas9
SlTOM1
SlTOM3
SlTOM1a and SlTOM3 are essential for the replication of ToBRFV but not for ToMV and TMV.
[144]
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6.3. CRISPR/Cas Genome Editing Targeting Host Susceptible Genes
Since plant viruses are highly dependent on the host to complete their replication cycle,
the replication, assembly, and movement of viruses in plant cells require interaction with
host–plant-specific factors, often referred to as susceptibility genes (S genes), for successful
infection [145]. Editing these susceptible genes via CRISPR/Cas to break compatible
interactions between the virus and host can help develop resistance in the host plants [146].
A well-known S gene that confers resistance to potyviruses is the eukaryotic translation initiation factor 4E (eIF4E). eIF4E is a mRNA cap-binding protein that plays a critical
role in initiating mRNA translation and regulating protein synthesis [124]. Studies have
shown that eIF4E can interact with the viral protein genome-link (VPg) of PVY to promote
the translation, replication, and intercellular movement of the PVY [124,147]. In recent
years, there have been many reports of using CRISPR/Cas to knock out or mutate the
eIF4E homologous gene in tomatoes to obtain tomato lines with complete resistance to
potyviruses (Table 4). Tobamoviruses, especially TBoRFV, which emerged recently, are
another important group of viruses that threaten tomato production. The TOBAMOVIRUS
MULTIPLICATION1 (TOM1) gene encoded by Arabidopsis is required for the replication
of TMV [148]. When TOM1 was mutated in Arabidopsis, the accumulation of TMV was
significantly inhibited [148]. The CRISPR/Cas9-mediated knockout of TOM1 homologs,
including SlTOM1a-e and SlTOM3, in tomatoes resulted in ToBRFV resistance in tomato
plants [31,144].
In addition, SlPelo, a TYLCV susceptibility gene in tomatoes [149], has been successfully used for CRISPR/Cas-mediated antiviral breeding [117]. Tomatoes with SlPelo
knockout showed significant inhibition and limited spread of TYLCV [117]. These results
show great potential for CRISPR/Cas antiviral breeding targeting host-susceptible genes,
but only if the molecular interactions between viruses and hosts are well-understood.
7. Future Prospects
7.1. CRISPR/Cas-Mediated Tomato Breeding for Resistance against Orthotospoviruses
Orthotospoviruses cause significant yield and quality reduction in tomatoes [150];
however, there are limited technologies to control these viruses at present. Some progress
has been made in the study of TSWV resistance genes, particularly the Sw-5 gene and its
homologs, which have been identified in tomatoes and are widely used in tomato hybrid
breeding. However, many TSWV strains have been reported to break the Sw-5-mediated
resistance in tomatoes worldwide [12,151]. In addition, some orthotospoviruses, such as
capsicum ring spot virus (CYRSV), have been reported to break down the Sw-5-mediated
resistance in tomatoes [152]. These results indicate that it is urgent to develop new methods
for tomato resistance breeding against orthotospoviruses.
So far, gene editing-mediated tomato breeding has achieved great success in developing resistance against geminiviruses, tobamoviruses, and potyviruses. However, there
are no reports on gene editing-mediated breeding for resistance against orthotospoviruses.
The gRNA targeting some critical genes of orthotospoviruses, such as nuclear protein N
and RNA silencing suppressor NSs, may be a good strategy to create tomato lines resistant
against orthotospoviruses.
Targeting host-susceptible genes via the gene-editing method is also a viable strategy
for tomato breeding against orthotospoviruses. Several host factors interacting with TSWV
have been identified, one such factor being the eukaryotic translation elongation factor 1A
(eEF1A). eEF1A interacts with the RNP of TSWV, and silencing eEF1A via virus-induced
gene silencing (VIGS) significantly inhibits TSWV replication in tobacco [153,154]. Ribosomal protein S6 (RPS6) is a host factor that is part of the 40S ribosomal subunit. Silencing
RPS6 showed high resistance to TSWV in tobacco [155]. The suppressor of the G2 allele
of skp1 (SGT1) is a co-chaperone that interacts with Hsp70 [156]. Tobacco SGT (NbSGT1)
interacts with TSWV NSm to promote intercellular and systemic motility of the virus [157].
Int. J. Mol. Sci. 2023, 24, 15448
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Thus, the homologs of these susceptibility factors in tomatoes could be a potential target
for gene editing against orthotospoviruses.
7.2. Challenges of the CRISPR/Cas-Mediated Antiviral Breeding in Tomato
CRISPR/Cas genome editing technology has great advantages compared with conventional breeding techniques. These advantages include shortening breeding cycles and
saving breeding costs. However, at present, CRISPR/Cas mainly relies on the Agrobacterium-mediated transgene method to integrate exogenetic DNA segments into the plant
genome, making its application controversial and as strictly regulated as that of GM crops.
This also restricts the future commercial application of CRISPR/Cas in resistance breeding.
Therefore, the development of DNA-free genome editing methods can help to avoid the
abovementioned problems. The current DNA-free genome editing techniques include
four main methods. (1) Selecting mutant plants without CRISPR/Cas elements from the
gene-edited selfed or hybrid progenies. The selection process usually takes a lot of time and
effort; therefore, new rapid selection techniques have been developed, such as inserting
visible fluorescent markers into CRISPR/Cas vectors to improve selection efficiency [158].
(2) Polyethylene glycol (PEG)-mediated transient expression of CRISPR/Cas system in
plant protoplast. The biggest challenge of this method is the difficulty of plant regeneration
from protoplasts. Fortunately, gene editing in wild tomatoes has been successful with
the PEG-mediated protoplast method [159]. (3) Modifying the plant virus as the vector to
deliver the CRISPR/Cas system. So far, various plant viruses have been modified to deliver
gRNA, including DNA viruses such as TYLCV, Bean yellow dwarf virus (BeYDV) [160,161],
and wheat dwarf virus (WDV) [162]; RNA viruses such as TSWV [163], barley stripe
mosaic virus (BSMV) [164], tobacco rattle virus (TRV) [165], and sonchus yellow net rhabdovirus (SYNV) [166] have also been used for gRNA delivery. Plant viruses have many
advantages as delivery vectors, including their ease of manipulation, high accumulation
levels (including gRNA and repair templates), and systemic movement across host plants,
leading to high expression levels of the gRNA in specific tissues, such as flowers, fruits,
buds, etc. However, as gRNA carriers, the carrying capacity of plant viruses is greatly
limited (typically < 1 kb) [167]. For these reasons, the future development direction of
a plant-virus-based CRISPR/Cas delivery system for tomatoes would be to expand the
delivery capacity by modifying more potential viruses that can carry large DNA/RNA
sequences. (4) Utilizing endogenous mobile mRNA from plants as carriers for gRNA
delivery. Some plant endogenous mRNAs can be used for long-distance transport through
the phloem. Studies found that a key motif of mRNA, tRNA-like sequence (TLS), is critical
for the long-distance transport of mRNA in the phloem [168]. The studies constructed TLS
into the Cas9 vector and expressed Cas9/gRNA-TLS construct in the rootstock through
transgenic methods. Thus, the gRNA could also be delivered from rootstock to the scion
with the assistance of TLS sequences, and the DNA-free genome editing seeds can be
selected from the scion plants [169]. This method utilizes the plant endogenous delivery
system, making it much more effective than the exogenic delivery system. In addition, this
method does not require additional removal of transgenes or regeneration of plants from
the transfected protoplasts, meaning that the breeding cycle is greatly shortened and the
efficiency of gene editing is improved. Tomato is also a very suitable crop for grafting, and
grafting is conducive to the polymerization of multiple resistances in commercial tomato
production [169]. However, the current research on mobile mRNA has mainly focused
on model plants such as Arabidopsis and tobacco, and little is known about the tomato’s
mobile mRNA. Therefore, more research needs to be carried out in the future to explore the
potential of the mobile mRNA of tomatoes as a gRNA delivery vector.
A potential risk of CRISPR/Cas-mediated antiviral breeding is that targeting the viral
genetic sequence could potentially accelerate virus evolution and the breakdown of the
host’s resistance. For example, the gene-edited cassava materials targeting the AC2 and AC3
sequences of the African cassava mosaic virus (ACMV) did not show significant resistance
compared with the control group. Further sequencing studies found that 33% to 48% of the
Int. J. Mol. Sci. 2023, 24, 15448
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edited viruses evolved a conserved single-nucleotide mutation at the target recognition
site to evade the gRNA recognition and avoid CRISPR/Cas9 cleavage [170]. Therefore,
simultaneously targeting multiple sites of the viral genome may be a better way to reduce
the risk of viruses evading recognition by gRNA due to single-site mutations [171].
In addition, there is another practical issue that needs to be considered; that is, the
possible negative effects of antiviral gene editing on plants need to be evaluated. Balancing
the yield, quality, and resistance to disease is a daunting challenge in crop breeding due
to the negative relationship among these traits [172,173]. Genome editing in dealing with
viruses for virus resistance may also affect other important agricultural traits and agronomic
traits of crops, such as yield, biotic and abiotic resistance, etc. [174,175]. Unfortunately, no
detailed data on antiviral gene-edited field crops in the field have been reported so far.
7.3. Suggestions for Improving Tomato Antiviral Breeding in the Future
We summarized the advantages, challenges, and prospects of three different antiviral breeding strategies, including MAS-based hybrid breeding, RNAi-based transgenic
breeding, and CRISPR/Cas-based gene editing, as presented in Table 5.
Table 5. Suggestions for improving tomato antiviral breeding in the future.
Methods
Characteristics
Challenges
Suggestions and Future Prospects
i.
i.
ii.
i.
MAS-based hybrid
breeding
ii.
Relay on conventional
breeding method.
Widely used in commercial
breeding.
iii.
iv.
v.
i.
RNAi-based transgenic
breeding
ii.
Genetic
engineering
method.
Rapidly and efficiently
creates
broad-spectrum
resistance against multiple
viruses.
i.
ii.
Long breeding cycles compared with genetic engineering breeding.
Lack of natural resistance
genes.
Single-gene-mediated resistance is easily broken down
by viruses.
Difficulty in developing
broad-spectrum resistance
against multiple viruses.
Needs appropriate and reliable DNA markers.
Public controversy and
strict regulation of policies
on GM crops.
Homology-dependent gene
silencing and unwanted recombination, non-target effects and off-target effects.
ii.
iii.
iv.
i.
ii.
i.
i.
i.
ii.
CRISPR/Cas-based
gene editing
iii.
Genetic engineering method.
Relies on Agrobacteriummediated transgene technology.
Shows great potential in antiviral breeding.
ii.
iii.
iv.
Controversial and strictly
regulated like GM crops.
The technology needs to be
improved to meet the needs
of commercial breeding.
Has the potential risk of
accelerating virus mutation
and evolution.
Inefficient targeting of the S
gene for editing.
ii.
iii.
iv.
Reinforce integration of ecological, biogeographical, and genetic discipline of
tomato and tomato viruses, which will
help to identify tomato local adaption
and source of resistance against emerging virus species or strains.
Utilizing selection and breeding methods, such as genomic prediction
(GP), genome-wide association studies (GWAS), and major QTL mapping
to identify potential new resistance
genes.
Finding closed, reliable, and effective
new molecular markers for virus resistance genes.
Using the gene pyramiding method to
polymerize multiple resistances in the
shortest possible time.
The third-generation antiviral transgenic technology (amiRNAs and syntasiRNAs) has improvements, including more specialized in-targets, to overcome the unwanted recombination
and off-target effect.
Exogenous application of RNAi. research and development.
Developing DNA-free gene editing technology, including DNAfree selection techniques, and a
PEG/virus/endogenous
mRNA
mediated delivery system.
Improving the efficiency of gene editing and reducing the off-target rate
by utilizing endogenous promoter and
optimized delivery system.
Developing gene editing techniques
targeting multiple viruses simultaneously or targeting multiple sites of a
virus simultaneously.
Developing the S gene editing targets
based on the molecular interaction
mechanism between viruses and hosts.
Int. J. Mol. Sci. 2023, 24, 15448
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For future MAS-based hybrid breeding against tomato viruses, the biggest challenge
is how to identify novel antiviral genes in tomatoes to address emerging viruses or strains.
Based on the arms race pathogen–host model of concerted evolution, we suggest a reinforced integration of ecological, biogeographical, and genetic discipline of tomato and
tomato viruses, which will help us identify tomato’s local adaption and source of resistance [176]. Gene regulatory networks via monogenetic methods will help to explain the
heritability of antiviral traits [176]. Selection of breeding methods, such as genomic prediction (GP), genome-wide association studies (GWAS), and major QTL mapping, should also
be utilized to identify potential new resistance genes. These techniques have already been
successful in identifying new virus resistance genes [176–179]. To overcome the challenges
of developing broad-spectrum resistance against multiple viruses, we suggest utilizing the
gene pyramiding method through MAS to integrate multiple resistance genes into a single
plant in the shortest possible time [79].
For RNAi-based transgenic breeding against tomato viruses, public controversy and
strict regulation of policies on GM crops is the biggest challenge. How to improve transgenic
technology from the technical level to eliminate public concerns becomes the key focus of
future research.
Although CRISPR/Cas-based gene editing has great potential for breeding tomatoes
against viruses, several challenges need to be addressed to ensure its application in future
antiviral breeding. These include developing DNA-free gene editing technology, improving
the efficiency of gene editing and reducing the off-target rate, and developing multiple
targets gene editing technology and the S gene editing targets.
8. Conclusions
Despite many years of agricultural research, there is still a lack of effective chemical
control or agronomic management measures to curb viral diseases. The most effective and
suitable method is utilizing natural genetic resistance resources or genetic tools to obtain
cultivars resistant to viruses. However, identifying the resistance sources/genes for all plant
viruses is difficult, and natural sources/genes could be broken by viral mutation. RNAi
transgenic research presents a new, useful way to control viral diseases, but the technique
is faced with concerns about GM crops, off-target results, and unwanted recombination. A
technology that could provide a viable alternative for virus resistance breeding in the future
is CRISPR/Cas-mediated gene editing. The CRISPR/Cas gene-editing technology allows
for the removal of the exogenous DNA via technical means, thus ensuring DNA-free editing
and avoiding the controversy and policy restriction associated with GM crops. However,
there is still a need to further explore the virus–plant molecular interactions, develop the
endogenous delivery system based on DNA-free genome editing technology, and evaluate
how the technology affects other important crop traits such as yield and tolerance to other
biotic and abiotic stresses to optimize the efficiency of gene editing for tomatoes.
Author Contributions: Z.Z. conceived and designed the manuscript; K.Z. wrote Sections 5–8 of
the manuscript; Z.S. wrote Sections 3 and 4 of the manuscript, Figure 2, Tables 2 and 3; X.S. wrote
Sections 1 and 2 of the manuscript and generated the data shown in Tables 1, 4 and 5. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Yunnan Seed Laboratory (grant number 202205AR070001),
the Major Science and Technology Program of Yunnan Province (grant number 202202AE090022), the
National Natural Science Foundation of China (grant number 32160620 and 31660508), and the Fund
for Reserve Talents of Young and Middle-aged Academic and Technical Leaders of Yunnan Province
(grant number 202305AC160026).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Int. J. Mol. Sci. 2023, 24, 15448
16 of 22
Conflicts of Interest: We declare that we have no conflict of interest nor any commercial or associative
interest that represents a conflict of interest in connection with the work submitted.
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