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Knockout of elF4E using CRISPR/
Cas9 for large-scale production
of resistant cucumber cultivar
against WMV, ZYMV, and PRSV
Hakan Fidan
1
, Ozer Calis
1
*, Esin Ari
2
, Aydin Atasayar
3
,
Pelin Sarikaya
1,3
, Mumin Ibrahim Tek
1
, Ahmet Izmirli
2
,
Yasemin Oz
2
and Gulsah Firat
3
1
Plant Protection Department Faculty of Agriculture Akdeniz University, Antalya, Türkiye,
2
Agricultural
Biotechnology Department, Faculty of Agriculture, Akdeniz University, Antalya, Türkiye,
3
Research and
Development Department AD ROSSEN Seeds, Antalya, Türkiye
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_1
DEL
or
elF4E_3
DEL
) and double (elF4E_1-3
DEL
) 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
DEL
and
elF4E_1
DEL
mutants. However, homozygous elF4E_3
DEL
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_3
DEL
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
OPEN ACCESS
EDITED BY
Rajib Roychowdhury,
Volcani Center, Israel
REVIEWED BY
Ashish Prasad,
Kurukshetra University, India
Umesh K. Reddy,
West Virginia State University, United States
*CORRESPONDENCE
Ozer Calis
ozercalis@akdeniz.edu.tr
SPECIALTY SECTION
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
CITATION
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
COPYRIGHT
©2023Fidan,Calis,Ari,Atasayar,Sarikaya,
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.
KEYWORDS
cucumber,CRISPR/Cas9,virusresistance,WMV,ZYMV,PRSV,gene-editing,
tissue culture
Introduction
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 host–virus
interactions (Diaz-Pendon et al., 2004).
Eukaryotic translation initiation factors (eIFs) such as elF4E and
elF4G have been extensively studied for their role in host–virus
interactions in various plant species over the past two decades. It
has been established that elF4E plays a key role in determining a
host’s 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 mRNA’s5’-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 host’s 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 identified 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, identified 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 host–virus 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 plant–virus 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-gene–mediated 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 efficiencies 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.
Results
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
-1
timentin antibiotic in medium to suppress bacterial
growth. Additionally, 1.5 mg l
-1
BAP (6- benzylaminopurine) and
1.0 mg l
-1
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 efficiency 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 confirm T-DNA insertion into
G27 and G247 inbred lines. Among these, 34 G27 and 22 G247
D
A
B
E
FG
C
FIGURE 1
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 fit 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 amplified 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 five 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
elF4E_1-3
DEL
F1 populations, while as G27-M36 was crossed with
non-edited G247 to obtain heterozygous elF4E_1-3
DEL
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
DEL
F1
plants, and G27-254 was used in crossing combinations with non-
edited G247 for heterozygous elF4E_1
DEL
genotype. Similarly, G27-
MB7 and G247-M398 that had two deletions at the gRNA2 position
were used to generate homozygous elF4E_3
DEL
F1 populations, and
G27-MB7 was crossed with non-edited G247 for heterozygous
elF4E_3
DEL
. Their F1 genotypes were confirmed with MvaI and
PsuI restriction enzymes after amplification 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 F1’s 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
DEL
Heterozygous elF4E_1-3
DEL
G27-254 × G247-M4591 G27-254 × G247-NE
gRNA1 2 del 2 del 2 del WT
gRNA2 WT WT WT WT
F1 Homozygous elF4E_1
DEL
Heterozygous elF4E_1
DEL
G27-MB7 × G247-M398 G27-MB7 × G247-NE
gRNA1 WT WT WT WT
gRNA2 2 del 2 del 2 del WT
F1 Homozygous elF4E_3
DEL
Heterozygous elF4E_3
DEL
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
th
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
th
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
st
amino acid position, after
the altered amino acids “IWAG”rather than “RSGQ”(Figure 2).
Homozygous elF4E_1-3
DEL
, elF4E_1
DEL
,
and elF4E_3
DEL
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 0–5, 1–9, and 0–4
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
elF4E_1-3
DEL
,elF4E_1
DEL
, and elF4E_3
DEL
F1 plants. Their scores
were WMV = 4.24, ZYMV = 6.64, and PRSV = 3.4 for heterozygous
elF4E_1-3
DEL
; WMV = 4.24, ZYMV = 7.12, and PRSV = 3.24 for
heterozygous elF4E_1
DEL
; WMV = 4.32, ZYMV = 7.28, and PRSV =
3.32 for heterozygous elF4E_3
DEL
. We did not find any resistance
reaction in heterozygous mutations in these genotypes. However, the
lowest scores were measured in homozygous mutants for elF4E_1-
3
DEL
, elF4E_1
DEL
, and elF4E_3
DEL
. There were no symptoms in
homozygous mutants elF4E_1-3
DEL
and elF4E_1
DEL
F1 plants for
each inoculated virus, while as restricted small lesions associated with
ZYMV were detected on 1-2 leaves of homozygous elF4E_3
DEL
F1s.
Homozygous elF4E_1-3
DEL
and elF4E_1
DEL
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
purified 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
DEL
and elF4E_1
DEL
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
DEL
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
DEL
, and only
homozygous elF4E_1-3
DEL
and elF4E_1
DEL
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
elF4E_3
DEL
was less than heterozygous and non-edited F1s. We did
not detect any viral loads in homozygous elF4E_1-3
DEL
and
elF4E_1
DEL
plants according to qRT-PCR. However, relative
ZYMV, WMV, and PRSV loads were higher in homozygous
elF4E_3
DEL
than homozygous elF4E_1-3
DEL
and elF4E_1
DEL
plants,
even if viral load of homozygous elF4E_3
DEL
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
DEL
F1 (G27-M36 × G247-M4464) and non-
A
B
FIGURE 2
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
nd
position
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 significant
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
DEL
F1 (Figure 7).
Discussion
The climate crisis is having a significant impact on plant health
and the spread of pathogens. Higher temperatures and changing
weather patterns make plants more vulnerable to infections that can
cause significant 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 sufficient 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
DEL
D
A
B
C
FIGURE 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
DEL
were absent, while restricted lesions were detected on 1-2 leaves of homozygous
elF4E_3
DEL
, heterozygous elf4E_1
DEL
, and non-edited F1s displayed typical symptoms (B). ZYMV-associated symptoms on homozygous elF4E_1-3
DEL
and non-edited fruits (C). RT-PCR results showed that only homozygous elF4E_1-3
DEL
(lane 1) and homozygous elF4E_1
DEL
(lane 3) mutants were
negative. However, homozygouelF4E_3
DEL
(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
DEL
single mutant, and
elF4E_3
DEL
single mutants were identified 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 genotypes’elF4E 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
DEL
,elF4E_1
DEL
,andelF4E_3
DEL
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-
3
DEL
and homozygous elF4E_1
DEL
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
DEL
F1
plants, these symptoms were less severe than in non-edited F1s and
other heterozygous single and double mutants for elF4E.
Additionally, homozygous elF4E_3
DEL
mutant F1 plants are
positive in RT-PCR, even though fewer viral symptoms were
observed on their leaves. It could be clarified with the viral load
of WMV, ZYMV, and PRSV. ELISA and qRT-PCR results showed
that the relative viral loads in homozygous elF4E_3
DEL
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. Identification 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 trafficking or encapsidation (German-
Retana et al., 2008). Also, elF4E’s 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 specific target of VPg may be
the second or third exon of elF4E. This is because mutations in the
first exon that result in an amino acid alteration confer complete
resistance, as indicated by negative results in RT-PCR and ELISA
D
A
B
C
FIGURE 4
Evaluation of papaya ringspot virus (PRSV) resistance in edited and non-edited F1 plants. Disease scores on a 0–4 scale, the average scores were
presented with standard deviation bars, and the same colors are indicating that there is no significant 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
elF4E_1-3
DEL
, homozygous elF4E_1
DEL
, and homozygous elF4E_1-3
DEL
(B, C). Homozygous elF4E_1-3
DEL
(lane 1) and homozygous elF4E_1
DEL
(lane
3) were negative in RT-PCR, while others (lanes 2, 4, 6, and 7) including homozygous elF4E_3
DEL
(lane 5) were positive (D).
Fidan et al. 10.3389/fpls.2023.1143813
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D
A
B
C
FIGURE 5
Assessment of watermelon mosaic virus (WMV) inoculation in edited and non-edited F1 plants. Disease scores were rated according to the 0–5
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
DEL
mutant (lane 5) was
positive in RT-PCR, like heterozygous and non-edited F1s, even though there were no symptoms on homozygous elF4E_3
DEL
leaves or fruit.
Homozygous elF4E_1-3
DEL
(lane 1) and homozygous elF4E_1
DEL
(lane 3) were negative according to RT-PCR (D).
A
B
FIGURE 6
Determination of viral accumulation in edited and non-edited F1 plants according to 2
−DDCT
and absorbance at 405 nm, derived from quantitative
reverse transcription polymerase chain reaction (qRT-PCR) and DAS-ELISA, respectively. There were no significant differences between the non-
edited and heterozygous plants for viral accumulation for each inoculated virus. However, lower viral accumulation was detected in homozygous
elF4E_3
DEL
relative to non-edited and heterozygous mutants. Also, the accumulation of each virus was higher in homozygous elF4E_3
DEL
than
homozygous elF4E1_3
DEL
and homozygous elF4E1
DEL
(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
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tests in these plants. These findings are consistent with those
observed in homozygous elF4E_1-3
DEL
and elF4E_1
DEL
. 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
DEL
.
Furthermore, susceptibility was reduced in single elF4E_1
DEL
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
DEL
and single elF4E_1
DEL
mutant F1 plants, because there was no viral accumulation in
elF4E_1-3
DEL
and single elF4E_1
DEL
. Although the viral loads and
virus-associated symptoms were lower in homozygous elF4E_3
DEL
than heterozygous mutants and non-edited plants, the viral
accumulations were detected for each virus with ELISA and qRT-
PCR in homozygous elF4E_3
DEL
. The results strongly suggest that
VPgs of potyviruses are associated with the first 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 mRNA’s5’-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
DEL
. Welch Two Sample
t-test showed that there was no significant 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 5′of 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
FIGURE 7
Comparison of morphological and yield characteristics between non-edited (G27-NE and G247-NE) and homozygous elF4E_1-3
DEL
(G27-M36 ×
G247-M4464) plants were given in graphs. No statistically significant 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 scientific knowledge and commercial
production requirements to effectively utilize CRISPR-modified
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
efficient 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 influenced 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 efficiency 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
efficiency of various A. tumefaciens strains and explant sources has
been reported, and efficiencies 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
finding is consistent with previous reports that younger cucumber
explants lead to higher regeneration efficiency (Chang et al., 2018).
The optimal concentration of ABA and BAP for shoot induction
was found to be 1.5 mg l
-1
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
-1
BAP and 1.0 mg l
-1
ABA in
their genotypes (variety 9330). Following the optimization of
transformation and regeneration, T0 plants were obtained and
verified 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
WMV, ZYMV, and PRSV.
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 significant 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 210–225 cm, internode length of 8–
10 cm, leaf length of 30–32 cm, fruit length of 17–19 cm, and single
fruit weight of 140–150 g. These plants were not resistant to ZYMV,
WMV, and PRSV, as they did not possess specific R genes such as
zym and wmv02245.
gRNAs and vector construction
C sativus elF4E–specific 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.ThegRNA’s construct that was cloned into pFGC-
pcoCas9 was confirmed 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
-1
) 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
-1
),
and rifampicin (15 ng µl
-1
) in LBA.
Transformation and regeneration
Optimization trials were conducted to optimize regeneration
and transformation in G27 and G247. Specifically, 50 different
culture media were tested for regeneration, and a medium
consisting of MS + 1.5 mg l
-1
BA + 1.5 mg l
-1
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
efficiency. A total of 30 transformation optimization trials were
performed for each genotype, resulting in the identification of
optimal conditions for the final eight transformations
(Supplementary Table S2).
To surface disinfect, the seeds of G27 and G247 were first
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
-1
NaCl, 5 g l
-1
yeast extract, 10 g l
-1
tryptophan, and 10 g l
-1
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
-1
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
(OD
600
) of 0.5. Acetosyringone was added to the bacterial solution
in LB medium at a concentration of 200 µM l
-1
, 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 filter paper. Afterward, the explants were
incubated in a co-culture medium (consisting of MS medium
supplemented with 1 g l
-1
MES, 1.5 mg l
-1
BA, 1.5 mg l
-1
ABA,
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
-1
BAP, 1.5 mg l
-1
ABA, 1 mg l
-1
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
-1
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
-1
GA
3
, 100 mg l
-1
timentin, 0.25 mg l
-1
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
-1
IBA, 50 mg l
-1
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.
Identification of elF4E mutations
The positions of gRNA in the G27 and G247 plants were
amplified using PCR and primers MC1F/R (521) and MC2F/R
(451 bp). The PCR reaction was prepared using Dream Taq 2×
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 final extension at 72°C
for 5 min. The Cas9 cleavage sites of the non-edited G27 and G247
elF4E’s 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 amplified 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
confirmed 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.
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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 flower formation. Male flowers were collected, and their
stamens were used to pollinate the pistils of female flowers 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
2
HPO
4
), 0.1% sodium sulfite (Na
2
SO
3
),
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 five 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 7–10
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 8–10 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
Scientific, Karlsruhe, Germany) according to the manufacturer’s
protocol. The purified 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 2× 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 final 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 manufacturer’s 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 manufacturer’s 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
−DDCT
method.
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 “ggplot2”package. “tidyverse”,“dplyr”,and
“multcompView”packages were used for the quantification 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.
Funding
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.
Acknowledgments
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.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
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
at: https://www.frontiersin.org/articles/10.3389/fpls.2023.1143813/
full#supplementary-material
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