ArticlePDF Available

Knockout of elF4E using CRISPR/Cas9 for large-scale production of resistant cucumber cultivar against WMV, ZYMV, and PRSV



CRISPR/Cas9 is one of the most robust technologies for plant breeding enabling precise and efficient modifications in a genome. This technology is being used for the manipulation of target genes in a host to develop resistance against the plant pathogens. Cucumis sativus elF4E is one of the target genes playing a key role in viral infection during interaction with potyvirus viral proteins genome linked (VPg). Nevertheless, the allelic and positional effect of elF4E mutations in C. sativus is to be clarified in elF4E-VPg interaction. In addition, there are entanglements in the massive production of pathogen-resistant cultivars suitable for commercial production using CRISPR/Cas9 technology. Therefore, we targeted different positions of the elF4E in G27 and G247 inbred lines, using specific gRNA1 and gRNA2 for the first and third exons, respectively, and 1,221 transgene-free plants were selected in segregated T1 generation, where 192 G27 and 79 G247 plants had the least mutation at Cas9 cleavage site of gRNA1 or gRNA2. Crossing was performed to see allelic effects of elfF4E mutations in F1 populations, which were homozygous and heterozygous single (elF4E_1DEL or elF4E_3DEL) and double (elF4E_1-3DEL) mutants. Disease symptoms of watermelon mosaic virus (WMV), papaya ringspot virus (PRSV), and zucchini yellow mosaic virus (ZYMV) were evaluated in both non-edited and edited F1 plants, and we did not observe any symptom in homozygous elF4E_1-3DEL and elF4E_1DEL mutants. However, homozygous elF4E_3DEL was positive in reverse transcription polymerase chain reaction (RT-PCR), even if there were no significant symptoms on the inoculated leaves. ELISA and qRT-PCR indicated lower viral accumulation in homozygous elF4E_3DEL than heterozygous and non-edited plants. Regeneration and transformation protocols were also optimized comprehensively for both the genotypes. The average number of shoots/100 explants was determined for both G27 and G247 as 13.6 and 18.0, respectively. We could not detect any distinguishing difference between the non-edited and edited F1 plants for yield and morphology. Our results demonstrate an effective route for mass production of viral resistant cultivars of cucumber to WMV, ZYMV, and PRSV. In this way, the pathogen-resistant cultivars could be generated to reduce the losses caused by these pathogens in cucumber production.
Knockout of elF4E using CRISPR/
Cas9 for large-scale production
of resistant cucumber cultivar
against WMV, ZYMV, and PRSV
Hakan Fidan
, Ozer Calis
*, Esin Ari
, Aydin Atasayar
Pelin Sarikaya
, Mumin Ibrahim Tek
, Ahmet Izmirli
Yasemin Oz
and Gulsah Firat
Plant Protection Department Faculty of Agriculture Akdeniz University, Antalya, Türkiye,
Biotechnology Department, Faculty of Agriculture, Akdeniz University, Antalya, Türkiye,
Research and
Development Department AD ROSSEN Seeds, Antalya, Türkiye
CRISPR/Cas9 is one of the most robust technologies for plant breeding enabling
precise and efcient modications in a genome. This technology is being used
for the manipulation of target genes in a host to develop resistance against the
plant pathogens. Cucumis sativus elF4E is one of the target genes playing a key
role in viral infection during interaction with potyvirus viral proteins genome
linked (VPg). Nevertheless, the allelic and positional effect of elF4E mutations in
C. sativus is to be claried in elF4E-VPg interaction. In addition, there are
entanglements in the massive production of pathogen-resistant cultivars
suitable for commercial production using CRISPR/Cas9 technology. Therefore,
we targeted different positions of the elF4E in G27 and G247 inbred lines, using
specic gRNA1 and gRNA2 for the rst and third exons, respectively, and 1,221
transgene-free plants were selected in segregated T1 generation, where 192 G27
and 79 G247 plants had the least mutation at Cas9 cleavage site of gRNA1 or
gRNA2. Crossing was performed to see allelic effects of elfF4E mutations in F1
populations, which were homozygous and heterozygous single (elF4E_1
) and double (elF4E_1-3
) mutants. Disease symptoms of
watermelon mosaic virus (WMV), papaya ringspot virus (PRSV), and zucchini
yellow mosaic virus (ZYMV) were evaluated in both non-edited and edited F1
plants, and we did not observe any symptom in homozygous elF4E_1-3
mutants. However, homozygous elF4E_3
was positive in reverse
transcription polymerase chain reaction (RT-PCR), even if there were no
signicant symptoms on the inoculated leaves. ELISA and qRT-PCR indicated
lower viral accumulation in homozygous elF4E_3
than heterozygous and
non-edited plants. Regeneration and transformation protocols were also
optimized comprehensively for both the genotypes. The average number of
shoots/100 explants was determined for both G27 and G247 as 13.6 and 18.0,
Frontiers in Plant Science frontiersin.org01
Rajib Roychowdhury,
Volcani Center, Israel
Ashish Prasad,
Kurukshetra University, India
Umesh K. Reddy,
West Virginia State University, United States
Ozer Calis
This article was submitted to
Plant Biotechnology,
a section of the journal
Frontiers in Plant Science
RECEIVED 13 January 2023
ACCEPTED 21 February 2023
PUBLISHED 17 March 2023
Fidan H, Calis O, Ari E, Atasayar A,
Sarikaya P, Tek MI, Izmirli A, Oz Y and
Firat G (2023) Knockout of elF4E
using CRISPR/Cas9 for large-scale
production of resistant cucumber
cultivar against WMV, ZYMV, and PRSV.
Front. Plant Sci. 14:1143813.
doi: 10.3389/fpls.2023.1143813
Tek, Izmirli, Oz and Firat. This is an open-
access article distributed under the terms o f
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.
TYPE Original Research
PUBLISHED 17 March 2023
DOI 10.3389/fpls.2023.1143813
respectively. We could not detect any distinguishing difference between the
non-edited and edited F1 plants for yield and morphology. Our results
demonstrate an effective route for mass production of viral resistant cultivars
of cucumber to WMV, ZYMV, and PRSV. In this way, the pathogen-resistant
cultivars could be generated to reduce the losses caused by these pathogens in
cucumber production.
tissue culture
Plant viruses are responsible for economic losses to agriculture
production worldwide, with over 1,500 viruses belonging to 26
families (Cao et al., 2020). Members of the Potyviridae family,
which includes zucchini yellow mosaic virus (ZYMV), papaya
ringspot virus (PRSV), and watermelon mosaic virus (WMV), are
detrimental pathogens particularly to cucurbit crops including
cucumber. Potyviruses are single-positive stranded RNA viruses
with relatively larger genome size, typically around 10 kb, than
other plant pathogenic viruses (Revers and Garcia, 2015). Although
various precautions such as sanitation are being implemented to
control viral diseases, using virus-resistant cultivars is the most
effective method to control them. However, traditional breeding
approaches are inadequate and time consuming for developing
resistant cultivars. Most of the dominant resistance (R) genes confer
resistance against fungal and bacterial plant pathogens, rather than
viruses (Truniger and Aranda, 2009;Wang and Krishnaswamy,
2012). On the other hand, several factors in the plant could facilitate
the infection and increase susceptibility within hostvirus
interactions (Diaz-Pendon et al., 2004).
Eukaryotic translation initiation factors (eIFs) such as elF4E and
elF4G have been extensively studied for their role in hostvirus
interactions in various plant species over the past two decades. It
has been established that elF4E plays a key role in determining a
hosts susceptibility or resistance to pathogenic viruses, despite its
primary function as a regulator of cellular translation (Wang and
Krishnaswamy, 2012). elF4E is known as the cap-binding protein
and interacts with mRNAs5-terminal cap and nuclear protein
(Sonenberg and Gingras, 1998;Sonenberg and Dever, 2003).
However viral proteins (VPg) encoded by viruses interact with
elF4E by binding covalently to the hosts mRNA 5-terminal cap
(Murphy et al., 1996). Multiple research groups have demonstrated
that VPg-elF4E interaction is essential for potyvirus infection, and
loss of elF4E function confers recessive resistance against
potyviruses in the host (Wittmann et al., 1997;Leonard et al.,
2000;Ruffel et al., 2002). Naturally occurring mutants for elF4E
variants have also been identied in plants, such as the pvr2 allele in
pepper, as well as controlled mutations that suppress elF4E function
in plants (Ruffel et al., 2006).
Furthermore, identied elFs are not limited to pvr2 allele in
pepper; many elFs and their interactions were characterized in
various studies. Most of the characterized recessive genes associated
with the hostvirus interactions are responsible for encoding eIF4E,
eIF4G, and their isoforms. For instance, pot-1 in tomato (Solanum
lycopersicum), rym4,rym5, and rym6 in barley (Hordeum vulgare),
and mo1 in lettuce (Lactuca sativa) were characterized as recessive
genes encoding elF4E variants in plantvirus interactions (Nicaise
et al., 2003;Kanyuka et al., 2005;Ruffel et al., 2006). The most
critical characteristic of elF4E is responsible for susceptibility or
resistance against viral pathogens, even if it contributes to cellular
translation with its cellular translation function. Therefore, elF4E-
mediated resistance is a valuable alternative to control plant viruses
in agricultural production (Diaz-Pendon et al., 2004).
Developing new resistant cultivars through traditional
breeding and introgression of resistance (R)genesfromwild
ancestors of commercial cultivars can be challenging, and
pathogens can also overcome the R-genemediated resistance.
Alternative approaches, such as using the loss of susceptibility (S)
function mutants have been proposed to reduce host
susceptibility. Some host proteins, known as S proteins, can
increase infection rate and facilitate pathogen growth. Loss of S
function can provide durable, broad-spectrum resistance in
plants, because the viability of the obligate pathogens such as
viruses depends on the host factors (de Almeida Engler et al., 2005;
van Schie and Takken, 2014).
It has been demonstrated that homozygous elF4E mutations can
confer resistance against potyviruses in various plant species. For
example, deletion mutations in Arabidopsis thaliana elF4E and elF
(iso)4E provide complete resistance to turnip mosaic virus (TuMV)
without affecting plant vigor (Pyott et al., 2016). Induced deletion
mutations in tobacco elF4E genes (elF4E1-S,elF4E1-T,elF4E2-S,
and elF4E2-T) also conferred higher level of resistance to potato
virus Y (PVY), another member of the potyvirus family (Le et al.,
2022). Silencing of elF4E has shown broad-spectrum resistance
against RNA viruses in tomato (Mazier et al., 2011), besides
determining resistance to potyvirus in naturally occurring mutant
plants for elF4E and eIF(iso)4E (Gomez et al., 2009). Additionally,
cucumber vein yellowing virus (CVYV), ZYMV, and PRSV-
resistant cucumber plants with homozygous substitutions and
Fidan et al. 10.3389/fpls.2023.1143813
Frontiers in Plant Science frontiersin.org02
deletions in elF4E have been generated using CRISPR/Cas9
(Chandrasekaran et al., 2016).
The allelic and positional effects of elF4E mutations on
potyvirus resistance remain unclear in C. sativus,despite
previous reports of loss of elF4E function in various plants
including cucumber. Additionally, the mass production of
CRISPR/Cas9-edited plants resistant to plant pathogens has not
been extensively studied. Therefore, we have conducted this study
to investigate the positional and allelic effects of elF4E mutations
in C. sativus and to demonstrate an effective method for
generating pathogen-resistant mutant cultivars with CRISPR/
Cas9, suitable for commercial use in agricultural production.
We selected two inbred lines, G27 and G247, which were
regenerated after transformation and determined their
regeneration and transformation efciencies based on the
comprehensive optimization trials. Homozygous and
heterozygous single and double non-transgenic mutants in T2
were used to determine the allelic and positional effects of elF4E
mutations. We compared the F1 plants for agronomic value,
morphology, and virus resistance. This allowed us to examine
the loss of elF4E function in cucumber not only for potyvirus
resistance but also for its effects on plant morphology and
agronomic traits such as yield, fruit, and plant size.
Transformation and regeneration protocol
for G27 and G247 inbred lines
Comprehensive protocol optimization of regeneration and
transformation was performed for both G27 and G247 genotypes,
with 30 transformation experiments. The most optimal conditions
for the transformation of G27 and G247 were determined using
EHA105 strain of Agrobacterium tumefaciens, 1-day-old seedlings
(plant age), cotyledons with proximal ends as the explant type, and
300 mg l
timentin antibiotic in medium to suppress bacterial
growth. Additionally, 1.5 mg l
BAP (6- benzylaminopurine) and
1.0 mg l
ABA (abscisic acid) were found the most effective in
inducing shoot growth for both genotypes without preculture.
Under these optimized conditions, the average number of shoots
per 100 explants was 13.6 for G27 and 18 for G247 genotypes
(Supplementary Tables S1,S2). The transformation efciency of
G247 was higher than G27. Following the acclimatization stage, a
total of 174 ex-vitro plants were transferred to a greenhouse
(Figure 1). Meanwhile, PCR using Ag-CT0 primers was carried
out for screening of 64 T0 plants to conrm T-DNA insertion into
G27 and G247 inbred lines. Among these, 34 G27 and 22 G247
Plant regeneration from transformed cucumber cotyledon leaves. Cotyledon leaves are dissected into a base and two distal pieces (A). The
cotyledons, their proximal ends, and hypocotyl pieces are incubated in co-culture medium with Agrobacterium tumefaciens EHA105 cells with
pFGC-pcoCas9 plasmid T-DNA vector (B). Shoot formations from cotyledon leaves (C, D).In-vitro plants ready for acclimatization (E). Acclimatized
cucumber regenerants (F). Growth of the cucumber plants 1 month after potting in the greenhouse (G).
Fidan et al. 10.3389/fpls.2023.1143813
Frontiers in Plant Science frontiersin.org03
plants were found to be T-DNA positive and then we harvested T1
seeds from these plants.
CRISPR-induced mutations of G27 and
G247 in T1 generation
The screening was performed in T1 population; 2,315 plants of
G27 and 1,639 plants of G247 were T-DNA positive, while 751 of
G27 and 470 of G247 were transgene free. The number of
transgene-free plants was evaluated with the chi-square test, and
results were t with Mendelian segregation (Supplementary Table
S3). A total of 357 T1 plants composed of 251 of G27 and 106 of
G247 were also screened to detect homozygous or heterozygous
mutant plants with PCR using MC1F/R and MC2F/R primers and
digested with MvaI and PsuI restriction enzymes. The results
indicated that 192 of G27 and 79 of G247 had the least mutation
at Cas9 cleavage site of gRNA1 or gRNA2 position on elF4E. The
total number of homozygous mutations to gRNA1 was 42 for G27
and 16 for G247, which were higher than the gRNA2 position. Most
of the mutations were heterozygous at Cas9 cleavage site of gRNA2
in G27 and G247, and their number was 39 for G27 and 16 for
G247. Furthermore, gRNA1 and gRNA2 positions of selected 40
transgene-free plants in G27 and G247 were amplied with MC1F/
R and MC2F/R primers in PCR followed by the Sanger sequencing
to determine insertion, deletion, and substitution caused by non-
homologous end joining (NHEJ). The mutation types were
determined at the gRNA1 position in 21 plants and the gRNA2
position in ve plants, respectively. The largest deletions were
detected in gRNA1 of G27-M98 with 5 bp deletion, and 1 bp
deletion was the smallest in G27-M70. The most common deletions
were 2 bp and were detected on G27-M36, G27-254, G27-M485,
G247-M4464, and G247-M4591 for gRNA1. The deletions have
appeared at gRNA2, and were maximum of 4 bp for G27-M36 and
G247-M4464, and minimum of 2 bp for G27-MB7 and G247-
M4591 (Supplementary Figure S2). Although there was no insertion
mutation at the gRNA1 or gRNA2 cleavage site of Cas9 in
sequenced T1 plants, 1 bp (C/T) transition mutation was detected
on the gRNA1 position of G27-M98 (Supplementary Table S4).
F1 populations were generated to observe
positional and allelic effects of mutations
T2 seeds were harvested from T1 G27 and G247 mutant plants,
and selected T2 plants were used for crossing combinations to
generate F1 populations (Table 1). The G27-M36 and G247-M4464
had 2 and 4 bp deletion at the Cas9 cleavage site of gRNA1 and
gRNA2, respectively, and they were used to generate homozygous
F1 populations, while as G27-M36 was crossed with
non-edited G247 to obtain heterozygous elF4E_1-3
F1 mutants.
The G27-254 and G247-M4591 had 2 bp deletions at the gRNA1
site, so they were crossed to obtain homozygous elF4E_1
plants, and G27-254 was used in crossing combinations with non-
edited G247 for heterozygous elF4E_1
genotype. Similarly, G27-
MB7 and G247-M398 that had two deletions at the gRNA2 position
were used to generate homozygous elF4E_3
F1 populations, and
G27-MB7 was crossed with non-edited G247 for heterozygous
. Their F1 genotypes were conrmed with MvaI and
PsuI restriction enzymes after amplication of gRNA targets using
PCR with MC1F/R and MC2F/R primers.
Deletions causing amino acid alteration
and stop codon formation in edited F1
The same genotypes of induced mutation type by Cas9 cleavage
of elF4E have been selected for crossing plots to prevent chimerism
in F1 populations (Table 1). Edited F1s amino acid and nucleotide
sequences of elF4E were aligned with non-edited F1 plants
(Figure 2). However, there was no difference between the amino
acid sequence of G27-M36 × G247-M4464 and G27-254 × G247-
M4591, even if they are different type of mutants as well as double
TABLE 1 Crossing plots to generate different F1 genotypes.
G27-M36 × G247-M4464 G27-M36 × G247-NE
gRNA1 2 del 2 del 2 del WT
gRNA2 4 del 4 del 4 del WT
F1 Homozygous elF4E_1-3
Heterozygous elF4E_1-3
G27-254 × G247-M4591 G27-254 × G247-NE
gRNA1 2 del 2 del 2 del WT
F1 Homozygous elF4E_1
Heterozygous elF4E_1
G27-MB7 × G247-M398 G27-MB7 × G247-NE
gRNA2 2 del 2 del 2 del WT
F1 Homozygous elF4E_3
Heterozygous elF4E_3
Fidan et al. 10.3389/fpls.2023.1143813
Frontiers in Plant Science frontiersin.org04
and single mutants according to nucleotide sequence, respectively.
The alteration was started at the 80
amino acid of elF4E, which
was the Cas9 cleavage site of gRNA1 for G27-M36 × G247-M4464
and G27-254 × G247-M4591. The stop-codon formation was
detected at the 116
amino acid for G27-M36 × G247-M4464
and G27-254 × G247-M4591. Another single mutant F1 for the
gRNA2 position of elF4E was generated with the crossing of G27-
MB7 and G247-M398, which had 2 bp deletions. The deletion
caused stop-codon formation at the 181
amino acid position, after
the altered amino acids IWAGrather than RSGQ(Figure 2).
Homozygous elF4E_1-3
, elF4E_1
and elF4E_3
did not show symptoms
associated with inoculated viruses
The leaves of generated F1 plants including control non-edited F1
plants and resistant plants were inoculated with WMV, ZYMV, and
PRSV; each genotype was evaluated according to 05, 19, and 04
scales, respectively (Wai and Grumet, 1995;Guner et al., 2002;
Palomares-Rius et al., 2011). The WMV and ZYMV symptoms
appeared on non-edited F1 plants at 7 dpi, while PRSV symptoms
were detected on leaves of non-edited F1 at 20 dpi. Evaluation of the
virus symptoms of non-edited plants using different scales revealed:
4.60 for ZYMV (Figure 3), 7.68 for PRSV (Figure 4), and 3.84 for
WMV (Figure 5). The results were similar with the heterozygous
, and elF4E_3
F1 plants. Their scores
were WMV = 4.24, ZYMV = 6.64, and PRSV = 3.4 for heterozygous
; WMV = 4.24, ZYMV = 7.12, and PRSV = 3.24 for
heterozygous elF4E_1
; WMV = 4.32, ZYMV = 7.28, and PRSV =
3.32 for heterozygous elF4E_3
. We did not nd any resistance
reaction in heterozygous mutations in these genotypes. However, the
lowest scores were measured in homozygous mutants for elF4E_1-
, elF4E_1
, and elF4E_3
. There were no symptoms in
homozygous mutants elF4E_1-3
and elF4E_1
F1 plants for
each inoculated virus, while as restricted small lesions associated with
ZYMV were detected on 1-2 leaves of homozygous elF4E_3
Homozygous elF4E_1-3
and elF4E_1
plants that were negative in reverse
transcription polymerase chain reaction
and enzyme-linked immunosorbent assay
The leaves were harvested from inoculated F1 plants including
edited as well as control non-edited plants, and the samples were
puried and prepared for reverse transcription polymerase chain
reaction (RT-PCR), quantitative RT-PCR (qRT-PCR), and enzyme-
linked immunosorbent assay (ELISA). The results have shown that
homozygous elF4E_1-3
and elF4E_1
plants were negative in
RT-PCR, while other heterozygous and non-edited plants were
positive for ZYMV (Figure 3), PRSV (Figure 4), and WMV
(Figure 5). However, homozygous elF4E_3
was positive in RT-
PCR, while they did not show any symptoms after the WMV, ZYMV,
and PRSV inoculation. Viral loads were also evaluated with qRT-PCR
and ELISA for inoculated non-edited and edited F1s. ELISA results
were determined as positive for homozygous elF4E_3
, and only
homozygous elF4E_1-3
and elF4E_1
plants were negative in
ELISA. The absorbance values at 405 nm of WMV, ZYMV, and
PRSV inoculated plants indicated that the viral load of homozygous
was less than heterozygous and non-edited F1s. We did
not detect any viral loads in homozygous elF4E_1-3
plants according to qRT-PCR. However, relative
ZYMV, WMV, and PRSV loads were higher in homozygous
than homozygous elF4E_1-3
and elF4E_1
even if viral load of homozygous elF4E_3
was lower than
heterozygous mutants and non-edited plants (Figure 6).
Yield and morphological difference in
edited and non-edited F1 plants
The edited F1s and non-edited F1 populations were compared
for various morphological criteria including internode, plant, leaf,
fruit lengths, yield, single fruit weight, and yield per plant. The
homozygous elF4E_1-3
F1 (G27-M36 × G247-M4464) and non-
Alignment of the amino acid sequences of non-edited and edited F1s. Red arrows indicate the Cas9-cleavage sites at the gRNA1 and gRNA2 (A).
Alignment of the nucleotide and amino acids sequences of edited and non-edited lines with chromatogram data. G27-M36 × G247-M4464 and
G27-254 × G247-M4591 amino acids sequences at gRNA2 positions were not given because of the early stop codon formation at the 122
of elF4E amino acid sequence (B).
Fidan et al. 10.3389/fpls.2023.1143813
Frontiers in Plant Science frontiersin.org05
edited F1 (G27-NE × G247-NE) were used for comparison at the
harvesting period and measured criteria were given in
Supplementary Table S5. There was no statistically signicant
difference (p= 0.05) in the compared groups in terms of
internode, leaf, and fruit lengths according to Welch Two Sample
t-test (Supplementary Table S6). We did not observe any yield
penalties in homozygous elF4E_1-3
F1 (Figure 7).
The climate crisis is having a signicant impact on plant health
and the spread of pathogens. Higher temperatures and changing
weather patterns make plants more vulnerable to infections that can
cause signicant damage to THE crop and natural vegetation. Viral
diseases can greatly reduce agricultural production, leading to
economic losses and food insecurity. Using traditional breeding
methods to develop virus-resistant plants is not sufcient in the face
of rapidly evolving viruses and the challenges posed by the climate
crisis. Classical methods can be time-consuming, imprecise, and
limited in generating new disease-resistant cultivars. Thus, CRISPR/
Cas9 and other gene-editing methods offer a solution to generate
new resistant plants to viral, bacterial, and fungal pathogens
(Karavolias et al., 2021).
In this study, we focused on the manipulation of elF4E-Vpg
interaction, a key requirement for potyvirus infection in host plants,
to generate the loss-of-elF4E function mutant for C. sativus. It has
been reported that manipulation of the elF4E-Vpg interaction
confers broad-spectrum potyvirus resistance in various plants,
including cucumber (Chandrasekaran et al., 2016;Macovei et al.,
2018;Gomez et al., 2019). Boosted potyvirus virus resistance was
also reported in various plants using CRISPR/Cas9 and
homozygous plants showed resistance after inoculation of tungro
spherical virus in rice or cassava brown streak virus in cassava
(Macovei et al., 2018;Gomez et al., 2019). Our aim was not only to
investigate the resistance mediated by loss of elF4E function in
cucumber but also to determine the positional and allelic effects of
elF4E mutations in cucumber. To this end, we constructed two
guide RNAs targeting exon 1 and exon 3 of C. sativus elF4E to
generate different types of mutants. Homozygous elF4E_1-3
Evaluation of zucchini yellow mosaic virus (ZYMV) inoculation in edited and non-edited F1 plants. Average scores of ZYMV-inoculated plants for
non-edited F1s, homozygous mutants, and heterozygous mutants. The same column color indicates that there is no statistical difference between
groups (A). ZYMV symptoms on homozygous elF4E_1-3
were absent, while restricted lesions were detected on 1-2 leaves of homozygous
, heterozygous elf4E_1
, and non-edited F1s displayed typical symptoms (B). ZYMV-associated symptoms on homozygous elF4E_1-3
and non-edited fruits (C). RT-PCR results showed that only homozygous elF4E_1-3
(lane 1) and homozygous elF4E_1
(lane 3) mutants were
negative. However, homozygouelF4E_3
(lane 5) was positive in RT-PCR, similarly non-edited (lane 7), and heterozygous mutants (D).
Fidan et al. 10.3389/fpls.2023.1143813
Frontiers in Plant Science frontiersin.org06
double mutants, homozygous elF4E_1
single mutant, and
single mutants were identied in the T1 generation
from transgene-free plants, and T2 seeds harvested after self-
crossing. We used the G27-M36, G247-M4464, G27-254, G247-
M398, G247-M4591, G27-MB7, G27-NE, and G247-NE inbred
lines in crossing experiments. Homozygous and heterozygous F1s
were produced for double and single elF4E mutations. We followed
the same path with previously reported research by Chandrasekaran
et al. (2016) and the gRNA2 was different by only one bp than their
study. However, their gRNA1 (exon 1 target) position did not hit
our genotypeselF4E and when we blast in Pyhtozome, results hit
with the Cucsa.300080. Hence, we have selected both gRNAs from
the sequence of Cucsa.212630.
WMV, ZYMV, and PRSV were inoculated onto each F1 plants,
including both edited and non-edited F1 genotypes, through both
mechanical and aphid transmission. The virus symptoms indicated
that homozygous elF4E_1-3
mutants are resistant to WMV, ZYMV, and PRSV. Previously,
elF4E-mediated resistance to ZYMV and PRSV is reported for
mechanical inoculation and aphid-transmitted viruses in
greenhouse conditions. However, elF4E-mediated WMV
resistance was not reported in C. sativus. Homozygous elF4E_1-
and homozygous elF4E_1
F1s showed resistance not only
to mechanically inoculated viruses but also to aphid-transmitted
ZYMV, WMV, and PRSV. While as the quite limited ZYMV and
WMV symptoms were detected on homozygous elF4E_3
plants, these symptoms were less severe than in non-edited F1s and
other heterozygous single and double mutants for elF4E.
Additionally, homozygous elF4E_3
mutant F1 plants are
positive in RT-PCR, even though fewer viral symptoms were
observed on their leaves. It could be claried with the viral load
of WMV, ZYMV, and PRSV. ELISA and qRT-PCR results showed
that the relative viral loads in homozygous elF4E_3
were higher
than in homozygous mutants, even if lower than in non-edited and
heterozygous mutants.
The elF4E-binding domain of VPgs in potyviruses has been
reported, and the function of this domain is thought to be
responsible for potyvirus replication. Identication of elF4E
function in lettuce has indicated elF4E interact with VPg or host
and pathogen factors and their mechanisms could involve
intracellular and cell-to-cell trafcking or encapsidation (German-
Retana et al., 2008). Also, elF4Es function was associated with the
potyvirus cell-to-cell movement in pea and pepper (Gao et al.,
2004). However, the binding target of VPg is not well understood in
C. sativus, but we speculate that the specic target of VPg may be
the second or third exon of elF4E. This is because mutations in the
rst exon that result in an amino acid alteration confer complete
resistance, as indicated by negative results in RT-PCR and ELISA
Evaluation of papaya ringspot virus (PRSV) resistance in edited and non-edited F1 plants. Disease scores on a 04 scale, the average scores were
presented with standard deviation bars, and the same colors are indicating that there is no signicant difference between evaluated groups (A). PRSV
symptoms on leaves and fruit were seen on non-edited and heterozygous mutant leaves and fruits, while there were no symptoms on homozygous
, homozygous elF4E_1
, and homozygous elF4E_1-3
(B, C). Homozygous elF4E_1-3
(lane 1) and homozygous elF4E_1
3) were negative in RT-PCR, while others (lanes 2, 4, 6, and 7) including homozygous elF4E_3
(lane 5) were positive (D).
Fidan et al. 10.3389/fpls.2023.1143813
Frontiers in Plant Science frontiersin.org07
Assessment of watermelon mosaic virus (WMV) inoculation in edited and non-edited F1 plants. Disease scores were rated according to the 05
scale, and the groups that have the same color column are indicating statistically indistinguishable in graphic (A). WMV symptoms on leaves were
observed 12 days after inoculation, and typical symptoms were detected on non-edited F1 fruit (B, C). Homozygous elF4E_3
mutant (lane 5) was
positive in RT-PCR, like heterozygous and non-edited F1s, even though there were no symptoms on homozygous elF4E_3
leaves or fruit.
Homozygous elF4E_1-3
(lane 1) and homozygous elF4E_1
(lane 3) were negative according to RT-PCR (D).
Determination of viral accumulation in edited and non-edited F1 plants according to 2
and absorbance at 405 nm, derived from quantitative
reverse transcription polymerase chain reaction (qRT-PCR) and DAS-ELISA, respectively. There were no signicant differences between the non-
edited and heterozygous plants for viral accumulation for each inoculated virus. However, lower viral accumulation was detected in homozygous
relative to non-edited and heterozygous mutants. Also, the accumulation of each virus was higher in homozygous elF4E_3
homozygous elF4E1_3
and homozygous elF4E1
(A). Absorbance values of samples were parallel to relative viral accumulation results derived
from the qRT-PCR for edited and non-edited F1s (B).
Fidan et al. 10.3389/fpls.2023.1143813
Frontiers in Plant Science frontiersin.org08
tests in these plants. These ndings are consistent with those
observed in homozygous elF4E_1-3
and elF4E_1
. However,
heterozygous mutations in gRNA1 and gRNA2 locations did not
result in resistance in F1 plants. These plants showed common
symptoms upon inoculation with mechanically or aphid
transmitted WMV, PRSV, and ZYMV, and the scores of these
inoculated heterozygous mutants are not different from those of
non-edited F1s. Also, WMV, ZYMV, and PRSV can multiply in the
host even when mutations occur in the third exon of elF4E,asin
homozygous elF4E_3
Furthermore, susceptibility was reduced in single elF4E_1
homozygous mutant F1s against WMV, ZYMV, and PRSV. qRT-
PCR and ELISA results indicated that WMV, ZYMV, and PRSV
cannot multiply in double elF4E_1-3
and single elF4E_1
mutant F1 plants, because there was no viral accumulation in
and single elF4E_1
. Although the viral loads and
virus-associated symptoms were lower in homozygous elF4E_3
than heterozygous mutants and non-edited plants, the viral
accumulations were detected for each virus with ELISA and qRT-
PCR in homozygous elF4E_3
. The results strongly suggest that
VPgs of potyviruses are associated with the rst or second exon of
the C.sativus elF4E. However, further experiments are required to
determine the function of the elF4E protein and its functional
domains in the cucumber for the understanding of the potyviruses-
plant interactions.
Elongation factors are also called cap-binding proteins, because
their regulator functions in cellular translation with the interaction
of mRNAs5-terminal cap and nuclear proteins (Sonenberg and
Gingras, 1998;Sonenberg and Dever, 2003). However, the loss of
elF4E function did not affect plant morphology or other yield
characteristics in homozygous elF4E_1-3
. Welch Two Sample
t-test showed that there was no signicant difference between edited
and non-edited F1 plants for plant length, leaf length, internode
length,fruitlength,yieldperplant, and single fruit weight.
Similarly, deletion mutations on elF4E and elF(iso)4E did not
change plant vigor in Arabidopsis thaliana,besidesdeletions
confer the resistance against TuMV (Pyott et al., 2016).
Additionally, naturally occurring mutants for elF4E or elF(iso)4E
were characterized for potyvirus resistance in various plants
(Gomez et al., 2009). Studies on the function of elF4E are mostly
related to its potyvirus interactions. Potyviruses have covalently
bonded VPg at 5of their RNAs, rather than a 7-methylguanosine
cap like in some eukaryotic and viral mRNAs. Therefore, elF4E or
elF(iso)4E interaction is essential for VPg to avoid RNA silencing
Comparison of morphological and yield characteristics between non-edited (G27-NE and G247-NE) and homozygous elF4E_1-3
(G27-M36 ×
G247-M4464) plants were given in graphs. No statistically signicant differences were observed between the two populations according to Welch
Two Sample t-test (p= 0.05). Boxplots are created using data, given in Supplementary Table S5.
Fidan et al. 10.3389/fpls.2023.1143813
Frontiers in Plant Science frontiersin.org09
and translation or stabilization of viral structures (Saha and
Mäkinen, 2020). The role of elF4E has also been reported for
lettuce mosaic virus infection cycle in lettuce (Lactuca sativa),
and it has been suggested that elF4E function could be different
from cellular mRNA translation (German-Retana et al., 2008).
Hence, controlled mutations to suppress the function of elF4E
function with novel gene-editing methods could be a reliable and
robust way to generate new potyvirus-resistant cultivars without
yield penalties.
Although CRISPR/Cas9 technology has been demonstrated as a
reliable means of precisely modifying plant genomes, there are
technical challenges that must be addressed to use it for the
commercial production of pathogen-resistant cultivars. It is
important to consider both scientic knowledge and commercial
production requirements to effectively utilize CRISPR-modied
plants in agriculture. Regeneration steps are essential for
developing gene-edited crops after the Agrobacterium-mediated
or particle bombardment transformation. The regeneration
process is one of the most entanglements for gene-editing
methods for non-model plants (Shan et al., 2020), because
efcient and successful regeneration is crucial for both transgenic
studies and the production of gene-edited crops by using CRISPR.
Poor regeneration and transformation rates can impede genetic
transformation efforts (Tan et al., 2022). The regeneration rate is
largely inuenced by the genotype and various factors such as the
source of explants, seedling stage (Chang et al., 2018), exogenous
hormones (Tang et al., 2010;Tang et al., 2011;Hiroshi et al., 2016),
Agrobacterium strains (Zheng, 2009), the pre-culture period (Wang
et al., 2014), and selection markers (Li et al., 2014). Therefore,
optimization of protocols for the G27 and G247 inbred lines was
performed before attempting the transformation and regeneration
of T0 plants. The most effective regeneration was observed in T29,
with 33.01 and 33.68 shoots induced for G27 and
G247, respectively.
The comparison of the transformation efciency for
Agrobacterium strains showed that EHA105-mediated
transformation is more effective than LBA4404-mediated
transformation in cotyledon and hypocotyl explants for both
genotypes. Hypocotyl explants did not induce shoot growth in
either LBA4404- or EHA105-mediated transformation in G27. The
efciency of various A. tumefaciens strains and explant sources has
been reported, and efciencies ranging were 0.89% to 21% for
EHA105 and 0.5% to 4.8% for LBA4404. In this study, EHA105
was found to have a higher transformant shooting rate than
LBA4404. The effect of seedling age on regeneration was also
evaluated, and it was found that using 1-day-old seedlings
cotyledons resulted in a higher number of regenerated shoots in
both G27 and G247, compared with using 5-day-old seedlings. This
nding is consistent with previous reports that younger cucumber
explants lead to higher regeneration efciency (Chang et al., 2018).
The optimal concentration of ABA and BAP for shoot induction
was found to be 1.5 mg l
of both ABA and BA for both G27 and
G247. Liu et al. (2018) also found that a regeneration frequency of
96.7% was achieved using 1.5 mg l
BAP and 1.0 mg l
ABA in
their genotypes (variety 9330). Following the optimization of
transformation and regeneration, T0 plants were obtained and
veried using PCR. T1 seeds were harvested from the regenerated
transgenic plants, and T2 populations were generated from
transgene-free T1 plants for each genotype. To avoid chimerism
and heterozygosity in the F1 generation, plants from T2 G27 and
G247 that have the same mutation at the gRNA target site were
selected. These plants were self-crossed and crossed with another
parental line to produce and store seeds of loss-of-function mutants.
The G27-M36 and G247-M4464 inbred lines were used in crossing
combinations, resulting in edited resistance F1 plants against
In conclusion, we have used CRISPR/Cas9 to reduce
susceptibility in G27 and G247 cucumber inbred lines by
suppressing elF4E gene, which is associated with potyviruses. The
positional and allelic effects of the elF4E mutations were
investigated in different heterozygous or homozygous and single
or double mutants. We found that stop-codon formation at the
second exon of elF4E and amino acid substitutions conferred
complete resistance to WMV, PRSV, and ZYMV without any
viral load. G27M36 and G247M4464, which have 4 and 2 bp
deletion mutations at elF4E, were crossed to produce F1 plants.
These edited F1 plants were compared with non-edited F1 plants,
and no signicant differences were observed in terms of yield,
quality, or morphology. In addition, we have also optimized
transformation and regeneration protocols for C. sativus G27 and
G247 inbred lines for large-scale production of the edited plants.
This study contributes the potential of using CRISPR/Cas9 to
generate resistant plants with knockout susceptibility genes for
suitable commercial agricultural production. This approach could
be used to generate a variety of resistant cultivars to manage viral,
fungal, and bacterial plant pathogens in future.
Materials and methods
Plant materials
G27 () and G247 () cucumber inbred lines were used in this
study as plant material. The non-edited F1 plants had the following
characteristics: plant length of 210225 cm, internode length of 8
10 cm, leaf length of 3032 cm, fruit length of 1719 cm, and single
fruit weight of 140150 g. These plants were not resistant to ZYMV,
WMV, and PRSV, as they did not possess specic R genes such as
zym and wmv02245.
gRNAs and vector construction
C sativus elF4Especic gRNA1 and gRNA2 were selected
(Supplementary Figure S1)fromtheCRISPORwebtool
(Concordet and Haeussler, 2018)andsynthesizedas
oligonucleotides. These gRNAs were then individually
consubstantiated in pTWIST Amp vector with the AtU6
promoter and scaffolds. XhoI : AtU6:gRNA1:scaffold-PacI and
PacI : AtU6:gRNA2:scaffold:XbaI constructs were obtained using
PCR to add the overhang of the restriction site. T4 DNA ligase was
used to linearly ligate the two constructs, and then the reaction
Fidan et al. 10.3389/fpls.2023.1143813
Frontiers in Plant Science frontiersin.org10
mixture was prepared with 1 µl of T4 DNA ligase, 2 µl of T4 DNA
ligase buffer (10×), 12.5 µl of ddH2O, and 150-ng construct. The
resulting ligated construct was assembled in the pFGC-pcoCas9
vector (Addgene: 52256) using restriction site cloning with XhoI
and XbaI.ThegRNAs construct that was cloned into pFGC-
pcoCas9 was conrmed with XhoI/XbaI restriction enzymes after
T4 ligation. The cloned plasmid (Supplementary Figure S1) was
then transferred to E. coli DH5acells via heat shock, as previously
described by Froger and Hall (2007). Transformant E. coli cells were
selected on kanamycin (50 ng µl
) containing Luria-Bertani Agar
(LBA), and the plasmid was isolated from E. coli DH5acells. The
isolated plasmid was then transferred to Agrobacterium tumefaciens
EHA105 electrocompetent cells using electroporation (Gene Pulser
Xcell Electroporation System, BioRad, California, USA), and the
cells were stored at -80°C after selection on kanamycin (50 ng µl
and rifampicin (15 ng µl
) in LBA.
Transformation and regeneration
Optimization trials were conducted to optimize regeneration
and transformation in G27 and G247. Specically, 50 different
culture media were tested for regeneration, and a medium
consisting of MS + 1.5 mg l
BA + 1.5 mg l
ABA was selected
(data not shown). For transformation, various factors including
genotype, bacterial strain, seedling age, explant type, pre-culture,
and antibiotic were optimized to maximize transformation
efciency. A total of 30 transformation optimization trials were
performed for each genotype, resulting in the identication of
optimal conditions for the nal eight transformations
(Supplementary Table S2).
To surface disinfect, the seeds of G27 and G247 were rst
immersed in 70% ethanol for 1 min, then treated with a solution of
15% commercial bleach containing 0.05% Tween 20 for 15 min.
Following this, the seeds were rinsed three times. These seeds were
then germinated in vitro on a medium composed of MS salts
(Murashige and Skoog, 1962), Nitsch and Nitsch vitamins (Nitsch
and Nitsch, 1969), 3% sucrose, and 0.7% agar (pH 5.8) in the dark at
28°C for 1 day. Cotyledon explants were obtained from 1-day-old
germinated seedlings by cutting their proximal regions using a
liquid regeneration medium (MS medium supplemented with 3%
sucrose and 200 µM acetosyringone). Meanwhile, to establish
bacterial colony cultures of the strain EHA105 carrying the
pFGC-pcoCas9 plasmid of Agrobacterium tumafaciens, bacterial
cells were streaked onto solid Luria Bertani (LB) medium (10 g l
NaCl, 5 g l
yeast extract, 10 g l
tryptophan, and 10 g l
agar) and
incubated at 28°C overnight. To propagate the liquid bacterial
culture, a single colony was taken from the bacterial culture using
a loop and transferred to 50 ml of liquid LB medium containing 50
mg l
kanamycin. This liquid bacterial culture was incubated
overnight at 28°C in a shaker at 200 rpm. The density of the
incubated bacterial solution was measured using a
spectrophotometer and adjusted to an optical density at 600 nm
) of 0.5. Acetosyringone was added to the bacterial solution
in LB medium at a concentration of 200 µM l
, and the culture was
incubated at 200 rpm for 3h to 4h. The explants were then
transferred to the prepared EHA105 culture for 20 min and
drained on sterile lter paper. Afterward, the explants were
incubated in a co-culture medium (consisting of MS medium
supplemented with 1 g l
MES, 1.5 mg l
BA, 1.5 mg l
200 µM acetosyringone, 3% sucrose, and 7-g plant agar) for 3 days
at 28°C. At the end of the 3-day co-culture period, the explants were
transferred to a selection medium (consisting of MS medium
supplemented with 1.5 mg l
BAP, 1.5 mg l
ABA, 1 mg l
phosphonitricine [PPT], 3% sucrose, and 7 g agar) and incubated at
26°C with a 16h photoperiod until shoot formation occurred. Also,
mediums were supplemented with 300 mg l
timentin to remove
EHA105 until acclimatization after the co-cultivation. The formed
shoots were separated from the explants and placed in a shoot
development medium (consisting of MS medium supplemented
with 1 mg l
, 100 mg l
timentin, 0.25 mg l
PPT, 3% sucrose,
and 7 g plant agar) to promote elongation. Plantlets displaying
healthy shoot growth were then rooted in a medium consisting of ½
MS medium containing 1 mg l
IBA, 50 mg l
timentin, 3%
sucrose, and 7 g plant agar. After the acclimatization process,
regenerated G27 and G247 were transferred to a greenhouse and
their DNAs were extracted using CTAB method.
Identication of elF4E mutations
The positions of gRNA in the G27 and G247 plants were
amplied using PCR and primers MC1F/R (521) and MC2F/R
(451 bp). The PCR reaction was prepared using Dream Taq
Master Mix and carried out with pre-denaturation at 95°C for 2 min,
35 cycles of denaturation at 95°C for 20 s, annealing at 56°C for 30 s,
and extension at 72°C for 1 min followed by a nal extension at 72°C
for 5 min. The Cas9 cleavage sites of the non-edited G27 and G247
elF4Es gRNA1 and gRNA2 positions contain MvaI and PsuI
restriction enzyme sites, respectively. These restriction enzymes
were used to cut only the non-edited elF4E fragment and detect
allelic mutations in T0, T1, and F1 generations. The restricted
fragments were visualized on a 2% agarose gel after electrophoresis.
The expected fragment sizes for non-edited plants were 293 and 228
bp for the elF4E-gRNA1 fragment and 295 and 156 bp for the elF4E-
gRNA2 fragment, while the non-restricted fragments (521 bp for
elF4E-gRNA1 and 451 bp for elF4E-gRNA2) were considered to
putative mutants due to loss of the restriction site as a result of NHEJ
after Cas9 cleavage. The gRNA targets of the mutant G27 and G247
plants were amplied using PCR and sequenced using the Sanger
method. The resulting data were analyzed using Geneious Prime to
identify mutations in the elF4E gene. The presence or absence of
introns and potential changes in amino acid sequence were also
analyzed. Homozygous mutants wereselected fromthe T0 generation
and self-pollinated to produce seeds. The presence of T-DNA was
conrmed using PCR in the T1 generation, and transgene-free
mutants were selected. Mutation analysis was also conducted in the
F1 generation after crossing T2 G27 and G247 mutants.
Fidan et al. 10.3389/fpls.2023.1143813
Frontiers in Plant Science frontiersin.org11
Crossing plots
T2 seeds were obtained from homozygous mutant parental lines
in the T1 generation that were free of transgenes. These seeds were
germinated, and silver nitrate was applied to the apical meristems of
both mutant G247 plants and non-edited G247 plants to induce
male ower formation. Male owers were collected, and their
stamens were used to pollinate the pistils of female owers from
the mutant G27 plant. Both mutant and non-edited G247 lines were
crossed with the mutant G27 line, with the different combinations
indicated in Table 1. These crossing combinations were designed to
produce F1 plants that were heterozygous or homozygous for the
mutation, as well as F1 plants that were not edited. Seeds were
harvested from these crosses and germinated for further analysis,
including virus inoculation, mutation testing, and
morphological characterization.
Virus inoculation
A phosphate buffer (pH 7.5) was prepared by mixing 2%
potassium phosphate (K
), 0.1% sodium sulte (Na
and 0.01% b-mercaptoethanol. This buffer was used to create an
inoculum for ZYMV, WMV, and PRSV for F1 plants. The viruses
were collected from infected cucumber plants in a greenhouse,
ground in a 0.02 M phosphate buffer, and used to mechanically
inoculate 20 plants, including ve wild-type control F1 plants. The
inoculation was carried out using a sponge pad soaked in the buffer
solution containing the virus, and the inoculated plants were
observed over time. Disease symptoms were evaluated at 710
days post-inoculation (dpi) for WMV and ZYMV and at 3 weeks
post-inoculation for PRSV. Inoculated plants were scored using
different scales: 0 to 4 (0 = no symptoms and 4 = severe mosaic on
many leaves) for PRSV (Wai and Grumet, 1995), 1 to 9 (1 = no
symptoms and 9 = plant dead) for ZYMV (Guner et al., 2018), and 0
to 5 (0 = no symptoms and 5 = severe mosaic and leaf distortion) for
WMV (Palomares-Rius et al., 2011). Vector-transmission
inoculation was carried out using Aphis gossypii,with
approximately 810 aphids per plant (Gal-On et al., 1992).
Detection of inoculated viruses and
viral loads
RT-PCR and qRT-PCR were performed to detect and quantify
the accumulation of ZYMV, WMV, and PRSV in non-edited and
mutant F1 plants. Total RNA was extracted from the leaf discs of
the plants using a Total RNA Isolation Kit (Thermo Fisher
Scientic, Karlsruhe, Germany) according to the manufacturers
protocol. The puried RNA was diluted in nuclease-free water, and
1 ng of RNA was used in the RT-PCR reaction. The reaction mix
was prepared with 1 µl of Verso Enzyme Mix, 25 µl of 1-Step
PCR ReddyMix, 2.5 µl of RT Enhancer, and 7 µl of nuclease-free
water. ZYMV-CP-285F_LK/ZYMV-CP-782R_LK (498 bp), PRSV-
F/R (~950 bp), and WMV-F/R (535 bp) were used to detect ZYMV,
PRSV, and WMV, respectively, and their sequences are in
Supplementary Table S7 (Valekunja et al., 2016;Kaldis et al.,
2018;de Souza Aguiar et al., 2019). The RT-PCR was performed
under the following conditions: cDNA synthesis at 50°C for 15 min,
RT inactivation at 95°C for 2 min, 35 cycles (denaturation at 95°C
for 20 s, annealing at 50°C [ZYMV], 52°C [WMV], and 58°C
[PRSV] for 30 s, extension at 72°C for 1 min), and nal extension at
72°C for 5 min. The RT-PCR products were analyzed by
electrophoresis on an agarose gel (1.5%) and visualized using a
UV transilluminator.
The detection of ZYMV, PRSV, and WMV in plant samples was
performed using DAS-ELISA kits from Agdia, which contained IgG
and conjugate antibodies, as well as positive and negative controls. A
mixture of 100 ml of coating buffer and 1 µl of IgG antibodies was
prepared according to the manufacturers instructions for a 1/100
dilution and distributed to the wells for a total of 10 samples. The
mixture was incubated at 37°C for 4h, washed with washing buffer,
and dried. Plant tissue samples for virus inoculation included six
mutant plants, one wild-type plant, and positive and negative
controls. According to the protocol, 100 mg of leaf tissue was taken
from each virus-infected plant sample, crushed with 600 µl of
extraction buffer in a mortar and pestle, and mixed with positive
and negative controls from Agdia (Elkhart, Indiana, USA). One
hundred of microliter of the crushed mixture was added to the
wells and incubated in the refrigerator at 4°C overnight. The next day,
after washing and drying with washing buffer, a mixture of conjugate
buffer and antibody was prepared in the same proportions as the IgG
antibodies and added to the wells. After incubating for 2h at 37°C, a
mixture of PNP and substrate buffer was prepared, and 100 µl was
added to the wells. The results were observed using an ELISA reader
and samples, with an absorbance value at 405 nm greater than two
times the negative control was considered positive and the results
were recorded for statistical analysis. This process was repeated three
times separately for each virus.
Isolated RNAs were used in qRT-PCR to determine the viral load
for each genotype. The iTaq Universal SYBR Green One-Step Kit
(Bio-Rad, California, USA) was used to prepare the reaction mixture
according to the manufacturers protocol, and qRT-PCR primers
were given in Supplementary Table S7. qRT-PCR was performed
under the following conditions: cDNA synthesis at 50°C for 10 min,
RT inactivation at 95°C for 1 min, 40 cycles of 95°C for 10 s, and 30°C
for 60 s. All reactions were done with three replicates for each sample
in CFX96 Touch (Bio-Rad). Relative viral accumulation was
determined for each genotype using the 2
Statistical analyses and
data visualization
The length of internodes, leaves, and fruits were measured in
mature plants, and the number of fruits and fruit weight was also
determined as yield criteria in mutant F1s (M36 × M4464) and non-
edited F1 plants. Quantitative data were collected from 10 edited and
non-edited F1 plants for each criterion (Supplementary Table S5). R
version 4.2.2 was used for Welch Two Sample t-test and constitution
Fidan et al. 10.3389/fpls.2023.1143813
Frontiers in Plant Science frontiersin.org12
of boxplots graphs with ggplot2package. tidyverse,dplyr,and
multcompViewpackages were used for the quantication of viral
accumulation determined by qRT-PCR and ELISA and data
visualization. Evaluation of inoculated ZYMV, WMV, and PRSV
symptoms data were performed in Microsoft Excel.
Data availability statement
The original contributions presented in the study are included
in the article/Supplementary Material. Further inquiries can be
directed to the corresponding author.
Author contributions
Conceptualization: HF and OC. Data curation: EA, HF, and
OC. Formal analysis: PS, AI, YO, and GF. Funding acquisition: HF
and AA. Methodology: HF, OC, EA, GF, and AI. Statistical analysis:
MT. Project administration: HF. Resources: HF and AA.
Supervision: HF, OC, and EA. Writing original draft: MT.
Writing review and editing: OC. All authors contributed to the
article and approved the submitted version.
This study is supported by Republic of Türkiye Ministry of
Agriculture and Forestry, General Directorate of Agricultural
Research and Policies with TAGEM/18/AR-GE/03 project number.
Authors would like to thank to Assoc. Prof Dr. Mehraj D. Shah
(Division of Plant Pathology, Khudwani, Sher-e-Kashmir
University of Agricultural Sciences and Technology of Kashmir,
Srinagar, Jammu and Kashmir, India) for suggestions.
Conict of interest
The authors declare that the research was conducted in the
absence of any commercial or nancial relationships that could be
construed as a potential conict of interest.
Publishers note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their afliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online
Cao, Y., Zhou, H., Zhou, X, and Li, F. (2020). Control of plant viruses by CRISPR/
Cas system-mediated adaptive immunity. Front. Microbiol. 11. doi: 10.3389/
Chandrasekaran, J., Brumin, M., Wolf, D., Leibman, D., Klap, C., Pearlsman, M., et al.
(2016). Development of broad virus resistance in non-transgenic cucumber using
CRISPR/Cas9 technology. Mol. Plant Pathol. 17 (7), 11401153. doi: 10.1111/mpp.12375
Chang, H. C., Ni, L., Sun, Y. D., and Luo, W. R. (2018). Factors inuencing
agrobacterium-mediated genetic transformation of cucumber. North. Hortic. 2, 914.
Concordet, J. P., and Haeussler, M. (2018). CRISPOR: intuitive guide selection for
CRISPR/Cas9 genome editing experiments and screens. Nucleic acids research 46(W1),
W242W245. doi: 10.1093/nar/gky354
de Almeida Engler, J., Favery, B., Engler, G., and Abad, P. (2005). Loss of
susceptibility as an alternative for nematode resistance. Curr. Opin. Biotechnol. 16
(2), 112117. doi: 10.1016/j.copbio.2005.01.009
de Souza Aguiar, R. W., Martins, A. R., Nascimento, V. L., Capone, A., Costa, L. T.
M., Campos, F. S., et al. (2019). Multiplex RT-PCR identication of ve viruses
associated with the watermelon crops in the Brazilian cerrado. Afr. J. Microbiol. Res.
13 (3), 6069. doi: 10.5897/AJMR2018.8976
Diaz-Pendon, J. A., Truniger, V., Nieto, C., Garcia-Mas, J., Bendahmane, A., and
Aranda, M. A. (2004). Advances in understanding recessive resistance to plant viruses.
Mol. Plant Pathol. 5, 223233. doi: 10.1111/j.1364-3703.2004.00223.x
Froger, A., and Hall, J. E. (2007).Transformation of plasmid DNA into E. coli using the
heat shock method. Journal of visualized experiments : JoVE (6), 253. doi: 10.3791/253
Gal-On, A., Antignus, Y., Rosner, A., and Raccah, B. (1992). A zucchini yellow mosaic
virus coat protein gene mutation restores aphid transmissibility but has no effect on
multiplication. J. Gen. Virol. 73 (Pt 9), 21832187. doi: 10.1099/0022-1317-73-9-2183
Gao, Z., Johansen, E., Eyers, S., Thomas, C. L., Noel Ellis, T. H., and Maule, A. J.
(2004). The potyvirus recessive resistance gene, sbm1, identies a novel role for
translation initiation factor eIF4E in cell-to-cell trafcking. Plant J. 40(3), 376385.
doi: 10.1111/j.1365-313X.2004.02215.x
German-Retana, S., Walter, J., Doublet, B., Roudet-Tavert, G., Nicaise, V.,
Lecampion, C., et al. (2008). Mutational analysis of plant cap-binding protein eIF4E
reveals key amino acids involved in biochemical functions and potyvirus infection. J.
Virol. 82 (15), 76017612. doi: 10.1128/JVI.00209-08
Gomez, M. A., Lin, Z. D., Moll, T., Chauhan, R. D., Hayden, L., Renninger, K., et al.
(2019). Simultaneous CRISPR/Cas9-mediated editing of cassava EIF4E isoforms
NCBP-1 and NCBP-2 reduces cassava brown streak disease symptom severity and
incidence. Plant Biotechnol. J. 17, 421434. doi: 10.1111/pbi.12987
Gomez,P., Rodrı
guez-Hernandez, A. M., Moury, B., and Aranda, M. A. (2009). Genetic
resistance for the sustainable control of plant virus diseases: breeding, mechanisms and
durability. Eur. J. Plant Pathol. 125, 122. doi: 10.1007/s10658-009-9468-5
Guner, N., Rivera-Burgos, L. A., and Wehner, T. C. (2018). Inheritance of resistance
to zucchini yellow mosaic virus in watermelon. HortScience horts 53(8), 11151118.
doi: 10.21273/HORTSCI13169-18
Guner, N., Strange, E. B., Wehner, T. C., and Pesic-VanEsbroeck, Z. (2002). Methods
for screening watermelon for resistance to Papaya ringspot virus type-W. Scientia
horticulturae 94(3-4), 297307. doi: 10.1016/S0304-4238(02)00007-9
Hiroshi, H., Takakazu, M., Izumi, C. M., Miki, Y., and Kazuhiro, S. (2016). Endogenous
hormone levels affect the regeneration ability of callus derived from different organs in
barley. Plant Physiol. Biochem. 99, 6672. doi: 10.1016/j.plaphy.2015.12.005
Kaldis, A., Berbati, M., Melita, O., Reppa, C., Holeva, M., Otten, P., et al. (2018).
Exogenously applied dsRNA molecules deriving from the zucchini yellow mosaic virus
(ZYMV) genome move systemically and protect cucurbits against ZYMV. Mol. Plant
Pathol. 19 (4), 883895. doi: 10.1111/mpp.12572
Kanyuka, K., Druka, A., Caldwell, D. G., Tymon, A., McCallum, N., Waugh, R., et al.
(2005). Evidence that the recessive bymovirus resistance locus rym4 in barley
Fidan et al. 10.3389/fpls.2023.1143813
Frontiers in Plant Science frontiersin.org13
corresponds to the eukaryotic translation initiation factor 4E gene. Mol. Plant Pathol. 6
(4), 449458. doi: 10.1111/j.1364-3703.2005.00294.x
Karavolias, N. G., Horner, W., Abugu, M. N., and Evanega, S. N. (2021). Application
of gene editing for climate change in agriculture. Front. Sustain. Food Syst. 5.
doi: 10.3389/fsufs.2021.685801
Le, N. T., Tran, H. T., Bui, T. P., Nguyen, G. T., Van Nguyen, D., Ta, D. T., et al.
(2022). Simultaneously induced mutations in eIF4E genes by CRISPR/Cas9 enhance
PVY resistance in tobacco. Sci. Rep. 12 (1), 14627. doi: 10.1038/s41598-022-18923-0
Leonard, S., Plante, D., Wittmann, S., Daigneault, N., Fortin, M. G., and Laliberte, J.-
F. (2000). Complex formation between potyvirus VPg and translation eukaryotic
initiation factor 4E correlates with virus infectivity. J. Virol. 74, 77307737. doi:
Li, Y., Zhao, J., Yang, X., Yang, J., Pan, J., He, H., and Cai, R. (2014). Optimization of
genetic transformation system in cucumber (Cucumis sativus L.) and transformation of
cucumber trichome gene Gl. Journal of Shanghai Jiaotong University-Agricultural
Science 32(6), 777.
Liu, X. F., Ning, K., Che, G., Yan, S. S., Han, L. G., Gu, R., et al. (2018). CsSPL
functions as an adaptor between HD-ZIP III and CsWUS transcription factors
regulating anther and ovule development in cucumis sativus (cucumber). Plant J. 94,
535547. doi: 10.1111/tpj.13877
Macovei, A., Sevilla, N. R., Cantos, C., Jonson, G. B., Slamet-Loedin, I., Cermak, T.,
et al. (2018). Novel alleles of rice EIF4G generated by CRISPR/Cas9-targeted
mutagenesis confer resistance to rice tungro spherical virus. Plant Biotechnol. J. 16,
19181927. doi: 10.1111/pbi.12927
Mazier, M., Flamain, F., Nicolaï, M., Sarnette, V., and Caranta, C. (2011). Knock-
down of both eIF4E1 and eIF4E2 genes confers broad-spectrum resistance against
potyviruses in tomato. PloS One 6 (12), e29595. doi: 10.1371/journal.pone.0029595
Murashige, T., and Skoog, F. (1962). A revised medium for rapid growth and
bioassays with tobacco tissue cultures. Physiol. Plant 15, 473497. doi: 10.1111/j.1399-
Murphy, J. F., Klein, P. G., Hunt, A. G., and Shaw, J. G. (1996). Replacement of the
tyrosine residue that links a potyviral VPg to the viral RNA is lethal. Virology 220, 535
538. doi: 10.1006/viro.1996.0344
Nicaise, V., German-Retana, S., Sanjuan,R.,Dubrana,M.P.,Mazier,M.,
Maisonneuve, B., et al. (2003). The eukaryotic translation initiation factor 4E
controls lettuce susceptibility to the potyvirus lettuce mosaic virus. Plant Physiol. 132
(3), 12721282. doi: 10.1104/pp.102.017855
Nitsch, J., and Nitsch, C. (1969). Haploid plants from pollen grains. Science 163, 85
87. doi: 10.1126/science.163.3862.85
Palomares-Rius, F. J., Viruel, M. A., Yuste-Lisbona, F. J., Lopez-Sese, A. I., and
Gomez-Guillamon, M. L. (2011). Simple sequence repeat markers linked to QTL for
resistance to watermelon mosaic virus in melon. TAG. Theor. Appl. Genet. Theoretische
und angewandte Genetik 123 (7), 12071214. doi: 10.1007/s00122-011-1660-2
Pyott, D. E., Sheehan, E., and Molnar, A. (2016). Engineering of CRISPR/Cas9-
mediated potyvirus resistance in transgene-free Arabidopsis plants. Molecular Plant
Pathology 17(8), 12761288. doi: 10.1111/mpp.12417
Revers, F., and Garcia, J. A. (2015). Chapter threemolecular biology of
potyviruses,in Advances in virus research.Eds.K.MaramoroschandT.C.
Mettenleiter (New York: Academic Press), 101199.
Ruffel, S., Dussault, M. H., Palloix, A., Moury, B., Bendahmane, A., Robaglia, C., et al.
(2002). A natural recessive resistance gene against potato virus y in pepper corresponds
to the eukaryotic initiation factor 4E (eIF4E). Plant J. 32, 10671075. doi: 10.1046/
Ruffel, S., Gallois, J. L., Moury, B., Robaglia, C., Palloix, A., and Caranta, C. (2006).
Simultaneous mutations in translation initiation factors eIF4E and eIF(iso)4E are
required to prevent pepper veinal mottle virus infection of pepper. J. Gen. Virol. 87,
20892098. doi: 10.1099/vir.0.81817-0
Saha, S., and Mäkinen, K. (2020). Insights into the functions of eIF4E-biding motif of
VPg in potato virus a infection. Viruses 12 (2), 197. doi: 10.3390/v12020197
Shan, S., Soltis, P. S., Soltis, D. E., and Yang, B. (2020). Considerations in adapting
CRISPR/Cas9 in nongenetic model plant systems. Appl. Plant Sci. 8 (1), e11314.
doi: 10.1002/aps3.11314
Sonenberg, N., and Dever, T. E. (2003). Eukaryotic translation initiation factors and
regulators. Curr. Opin. Struct. Biol. 13, 5663. doi: 10.1016/S0959-440X(03)00009-5
Sonenberg, N., and Gingras, A. C. (1998). The mRNA capbinding protein eIF4E and
control of cell growth. Curr. Opin. Cell Biol. 10, 268275. doi: 10.1016/S0955-0674(98)80150-6
Tan, J., Lin, L., Luo, H., Zhou, S., Zhu, Y., Wang, X., et al. (2022). Recent progress in
the regeneration and genetic transformation system of cucumber. Appl. Sci. 12, 7180.
doi: 10.3390/app12147180
Tang, Y., Liu, J., Li, J., Li, X. M., and Liu, B. (2011). The inuence of endogenous
hormones on the formation of buds from stems of bitter melon (Momordıca charantıa
l.). Afr. J. Biotechnol. 10, 58565860. doi: 10.5897/AJB11.719
Tang, Y., Liu, J., Liu, B., Li, X. M., Li, J., and Li, H. X. (2010). Endogenous hormone
concentrations in explants and calluses of bitter melon (Momordica charantia l. ).
Interciencia 35, 680683.
Truniger, V., and Aranda, M. A. (2009). Recessive resistance to plant viruses,in
Advances in virus research. Eds. G. Loebenstein and J.P Carr (Wymondham, Norfolk:
Caister Academic Press), 119231.
Valekunja, R. B., Kamakoti, V., Peter, A., Phadnis, S., Prasad, S., and Nagaraj, V. J.
(2016). The detection of papaya ringspot virus coat protein using an electrochemical
immunosensor. Analytical Methods 8 (48), 85348541. doi: 10.1039/C6AY02201D
van Schie, C. C., and Takken, F. L. (2014). Susceptibility genes 101: how to be a good
host. Annu. Rev. Phytopathol. 52, 551581. doi: 10.1146/annurev-phyto-102313-
Wai, T., and Grumet, R. (1995). Inheritance of resistance to the watermelon strain of
papaya ringspot virus in the cucumber line TMG-1. HortScience HortSci. 30(2), 338
340. doi: 10.21273/HORTSCI.30.2.338
Wang, Y., Gu, X. F., Zhang, S. P., Miao, H., Chen, G. H., and Xie, B. Y. (2014).
Transformation of RNAi vector in cucumber (Cucumis sativus) in vitro by
agrobacterium tumefaciens-mediated transfection. Chin. Bull. Bot. 49, 183189. doi:
Wang, A., and Krishnaswamy, S. (2012). Eukaryotic translation initiation factor
4Emediated recessive resistance to plant viruses and its utility in crop improvement.
Mol. Plant Pathol. 13, 795803. doi: 10.1111/j.1364-3703.2012.00791.x
Wittmann, S., Chatel, H., Fortin, M. G., and Laliberte, J.-F. (1997). Interaction of the
viral protein genome linked of turnip mosaic potyvirus with the translational
eukaryotic initiation factor (iso)4E of arabidopsis thaliana using the yeast two-hybrid
system. Virology 234, 8492. doi: 10.1006/viro.1997.8634
Zheng, L. J. (2009). Study on regeneration and agrobacterium-mediated genetic
transformation of cucumber (Cucumis sativus l.) (Yangzhou, China: Yangzhou
University). Masters Thesis.
Fidan et al. 10.3389/fpls.2023.1143813
Frontiers in Plant Science frontiersin.org14
... CRISPR/Cas9 is a robust and effective gene-editing technology for plant breeding, 63 which can precisely and efficiently modify a genome (Fidan et al., 2023). CRISPR/Cas 64 system was initially found to recognize and cleave the invading viruses or phages' ...
Full-text available
CRISPR/Cas9 system has emerged as a powerful tool in genome editing; however, generation of CRISPR-edited DNA-free plants is still challenging. In this study, Betula platyphylla (birch) was used to build a method to generate CRISPR-edited plant without foreign DNA integration using Agrobacterium-mediated transformation (CPDAT method). This technique utilizes transient genetic transformation to introduce T-DNA coding gRNA and Cas9 into birch cells, and T-DNA will express to synthesize gRNA and Cas9 protein, which will form a complex to cleave the target DNA site. The genome may be mutated due to DNA repair, and these mutations will be preserved and accumulated not dependent on whether T-DNA is integrated into the genome or not. After transient transformation, birch plants were cut into explants to induce adventitious buds without antibiotic selection pressure. Each adventitious bud can be considered as an independent potentially CRISPR-edited line for mutation detection. CRISPR-edited birch plants without foreign DNA integration are further selected by screening CRISPR-edited lines without T-DNA integration. Among 65 randomly chosen independent lines, the mutation rate was 80.00% including 40.00% of lines with both alleles mutated. In addition, 5 lines out of 65 studied lines (7.69%) were CRISPR-edited birch plants without DNA integration. In conclusion, this innovative method presents a novel strategy for generating CRISPR-edited birch plants, thereby significantly enhancing the efficiency of generating common CRISPR-edited plants. These findings offer considerable potential to develop plant genome editing techniques further.
Full-text available
Tobacco is an important commercial crop and a rich source of alkaloids for pharmaceutical and agricultural applications. However, its yield can be reduced by up to 70% due to virus infections, especially by a potyvirus Potato virus Y (PVY). The replication of PVY relies on host factors, and eukaryotic translation initiation factor 4Es (eIF4Es) have already been identified as recessive resistance genes against potyviruses in many plant species. To investigate the molecular basis of PVY resistance in the widely cultivated allotetraploid tobacco variety K326, we developed a dual guide RNA CRISPR/Cas9 system for combinatorial gene editing of two clades, eIF4E1 (eIF4E1-S and eIF4E1-T) and eIF4E2 (eIF4E2-S and eIF4E2-T) in the eIF4E gene family comprising six members in tobacco. We screened for CRISPR/Cas9-induced mutations by heteroduplex analysis and Sanger sequencing, and monitored PVYO accumulation in virus challenged regenerated plants by DAS-ELISA both in T0 and T1 generations. We found that all T0 lines carrying targeted mutations in the eIF4E1-S gene displayed enhanced resistance to PVYO confirming previous reports. More importantly, our combinatorial approach revealed that eIF4E1-S is necessary but not sufficient for complete PVY resistance. Only the quadruple mutants harboring loss-of-function mutations in eIF4E1-S, eIF4E1-T, eIF4E2-S and eIF4E2-T showed heritable high-level resistance to PVYO in tobacco. Our work highlights the importance of understanding host factor redundancy in virus replication and provides a roadmap to generate virus resistance by combinatorial CRISPR/Cas9-mediated editing in non-model crop plants with complex genomes.
Full-text available
Cucumber (Cucumis sativus L.), belonging to the gourd family (Cucurbitaceae), is one of the major vegetable crops in China. Conventional genetic breeding methods are ineffective for improving the tolerance of cucumber to various environmental stresses, diseases, and pests in the short term, but bio-engineering technologies can be applied to cucumber breeding to produce new cultivars with high yield and quality. Regeneration and genetic transformation systems are key technologies in modern cucumber breeding. Compared with regeneration systems, genetic transformation systems are not yet fully effective, and the low efficiency of genetic transformation is a bottleneck in cucumber cultivation. Here, we systematically review the key factors influencing the regeneration and genetic transformation of cucumber plants, including the selection of genotype, source of explants and forms of exogenous hormones added to the medium, the methods of transgene introduction and co-cultivation, and selection methods. In addition, we also focus on recent advances in the study of molecular mechanisms underlying important agronomic traits using genetic transformation technology, such as fruit length, fruit warts, and floral development. This review provides reference information for future research on improvements in cucumber varieties.
Full-text available
Climate change imposes a severe threat to agricultural systems, food security, and human nutrition. Meanwhile, efforts in crop and livestock gene editing have been undertaken to improve performance across a range of traits. Many of the targeted phenotypes include attributes that could be beneficial for climate change adaptation. Here, we present examples of emerging gene editing applications and research initiatives that are aimed at the improvement of crops and livestock in response to climate change, and discuss technical limitations and opportunities therein. While only few applications of gene editing have been translated to agricultural production thus far, numerous studies in research settings have demonstrated the potential for potent applications to address climate change in the near future.
Full-text available
Plant diseases caused by invading plant viruses pose serious threats to agricultural production in the world, and the antiviral engineering initiated by molecular biotechnology has been an effective strategy to prevent and control plant viruses. Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system-mediated DNA or RNA editing/interference in plants make them very attractive tools applicable to the plant protection field. Here, we review the development of CRISPR/Cas systems and summarize their applications in controlling different plant viruses by targeting viral sequences or host susceptibility genes. We list some potential recessive resistance genes that can be utilized in antiviral breeding and emphasize the importance and promise of recessive resistance gene-based antiviral breeding to generate transgene-free plants without developmental defects. Finally, we discuss the challenges and opportunities for the application of CRISPR/Cas techniques in the prevention and control of plant viruses in the field.
Full-text available
The interaction between the viral protein genome-linked (VPg) and eukaryotic initiation factor 4E (eIF4E) or eIF(iso)4E of the host plays a crucial role in potyvirus infection. The VPg of potato virus A (PVA) contains the Tyr-X-X-X-X-Leu-phi (YXXXLΦ) binding motif for eIF(iso)4E. In order to investigate its role in PVA infection, we substituted the conserved tyrosine and leucine residues of the motif with alanine residues in the infectious cDNA of PVA (PVAVPgmut). PVAVPgmut RNA replicated in infiltrated leaves, but RNA accumulation remained low. Systemic infection occurred only if a reversion to wild type PVA occurred. VPg was able to stabilize PVA RNA and enhance the expression of Renilla luciferase (3’RLUC) from the 3’ end of the PVA genome. VPgmut could not support either PVA RNA stabilization or enhanced 3’RLUC expression. The RNA silencing suppressor helper-component proteinase (HCPro) is responsible for the formation of PVA-induced RNA granules (PGs) during infection. While VPgmut increased the number of PG-like foci, the percentage of PVA RNA co-localization with PGs was reduced from 86% to 20%. A testable hypothesis for future studies based on these results is that the binding of eIF(iso)4E to PVA VPg via the YXXXLΦ motif is required for PVA RNA stabilization, as well as the transfer to the RNA silencing suppression pathway and, further, to polysomes for viral protein synthesis.
Full-text available
The past six years have seen the rapid growth of studies of CRISPR/Cas9 in plant genome editing, a method that enormously facilitates both basic research and practical applications. Most studies have focused on genetic model species, but plant species that are not genetic models may also be economically important or biologically significant, or both. However, developing the CRISPR/Cas9 system in a nongenetic model is challenging. Here, we summarize CRISPR/Cas9 applications in 45 plant genera across 24 families and provide a reference for practical application of CRISPR in nongenetic model plant systems. Suggestions for selecting plant species and target genes are given for proof‐of‐principle CRISPR studies, and the processes of vector construction are reviewed. We recommend using transient assays to identify a desired CRISPR/Cas9 system in a nongenetic model. We then review methods of plant transformation and describe approaches, using regenerated transgenic plants, for evaluating CRISPR editing results. Lastly, potential future applications of CRISPR in nongenetic model plant species are discussed. This review provides a road map for developing CRISPR in nongenetic models, an application that holds enormous potential in plant biology.
Full-text available
Watermelon plants can be naturally infected by several viruses in single or mixed infections, of which the diagnosis is difficult and require specific techniques. This study aims to detect and to verify the presence of Cucumber mosaic virus (CMV), Papaya ringspot virus type W (PRSV-W), Watermelon mosaic virus (WMV), Zucchini lethal chlorosis virus (ZLCV), Zucchini yellow mosaic virus (ZYMV), and the main cucurbit viruses in Brazil using multiplex reverse transcriptase polymerase chain reaction (RTPCR) assay. Oligonucleotides were designed towards the conserved regions of the virus genomes. In the duplex-PCR, it was possible to detect all the virus combinations, except ZLCV with ZYMV. The amplified product sizes were 644 bp (CMV), 535 bp (WMV-2), 398 bp (PRSV-W), 244 bp (ZLCV), and 214 bp (ZYMV). To test the efficacy of the methodology, we analyzed plants with virus symptoms from four municipalities in the state of Tocantins, located at Brazilian Cerrado. Mixed infections were detected in 80% of samples in all the municipalities. The multiplex RT-PCR assay can be used to detect and differentiate watermelon viruses that affect crops in the state of Tocantins
Full-text available
Sources of resistance to the Zucchini yellow mosaic virus-Florida strain (ZYMVFL) have been identified within the Citrullus genus. Inheritance of resistance to ZYMVFL was studied in PI 595203 (Citrullus mucosospermus), a resistant watermelon accession. The F1, F2, and BC1 progenies derived from the cross ‘Calhoun Gray’ (CHG) × PI 595203 and ‘New Hampshire Midget’ (NHM) 3 PI 595203 were used to study the inheritance of resistance to ZYMV-FL. Seedlings were inoculated with a severe isolate of ZYMV-FL at the first true leaf stage and rated weekly for at least 6 weeks on a scale of 1 to 9 on the basis of severity of viral symptoms. A single recessive gene (zym- FL) was found to control the high level of resistance to ZYMV-FL in PI 595203. © 2018, American Society for Horticultural Science. All rights reserved.
Full-text available
Cassava brown streak disease (CBSD) is a major constraint on cassava yields in East and Central Africa and threatens production in West Africa. CBSD is caused by two species of positive sense RNA viruses belonging to the family Potyviridae, genus Ipomovirus: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Diseases caused by the family Potyviridae require the interaction of viral genome‐linked protein (VPg) and host eukaryotic translation initiation factor 4E (eIF4E) isoforms. Cassava encodes five eIF4E proteins: eIF4E, eIF(iso)4E‐1, eIF(iso)4E‐2, novel cap‐binding protein‐1 (nCBP‐1), and nCBP‐2. Protein‐protein interaction experiments consistently found that VPg proteins associate with cassava nCBPs. CRISPR/Cas9‐mediated genome editing was employed to generate ncbp‐1, ncbp‐2, and ncbp‐1/ncbp‐2 mutants in cassava cultivar 60444. Challenge with CBSV showed that ncbp‐1/ncbp‐2 mutants displayed delayed and attenuated CBSD aerial symptoms, as well as reduced severity and incidence of storage root necrosis. Suppressed disease symptoms were correlated with reduced virus titer in storage roots relative to wild‐type controls. Our results demonstrate the ability to modify multiple genes simultaneously in cassava to achieve tolerance to CBSD. Future studies will investigate the contribution of remaining eIF4E isoforms on CBSD and translate this knowledge into an optimized strategy for protecting cassava from disease. This article is protected by copyright. All rights reserved
Full-text available is a web tool for genome editing experiments with the CRISPR-Cas9 system. It finds guide RNAs in an input sequence and ranks them according to different scores that evaluate potential off-targets in the genome of interest and predict on-target activity. The list of genomes is continuously expanded, with more 150 genomes added in the last two years. CRISPOR tries to provide a comprehensive solution from selection, cloning and expression of guide RNA as well as providing primers needed for testing guide activity and potential off-targets. Recent developments include batch design for genome-wide CRISPR and saturation screens, creating custom oligonucleotides for guide cloning and the design of next generation sequencing primers to test for off-target mutations. CRISPOR is available from, including the full source code of the website and a stand-alone, command-line version.