(AGENPARL) – Mon 15 September 2025 The Plant Journal (2025) 122, e17204
doi: 10.1111/tpj.17204
CRISPR/Cas9-driven double modification of grapevine
MLO6-7 imparts powdery mildew resistance, while editing of
NPR3 augments powdery and downy mildew tolerance
Loredana Moffa1,†, Giuseppe Mannino2,†, Ivan Bevilacqua1,3,†, Giorgio Gambino4, Irene Perrone4, Chiara Pagliarani4,
Cinzia Margherita Bertea2, Alberto Spada1,3, Anna Narduzzo1,3, Elisa Zizzamia1, Riccardo Velasco1, Walter Chitarra1,4,*,‡
and Luca Nerva1,4,*,‡
Council for Agricultural Research and Economics – Research Centre for Viticulture and Enology, Via XXVIII Aprile 26, 31015
Conegliano, TV, Italy,
Department of Life Sciences and Systems Biology, Plant Physiology Unit, University of Turin, Via Quarello 15/A, 10135
Turin, Italy,
Department of Agronomy, Food, Natural resources, Animals and Environment, University of Padova, Via dell’Universita 16,
35020 Legnaro, PD, Italy, and
Institute for Sustainable Plant Protection, National Research Council, Strada delle Cacce 73, 10135 Torino, Italy
Received 16 July 2024; revised 27 October 2024; accepted 28 November 2024.
These authors equally contributed as first authors.
These authors equally contributed as senior authors.
SUMMARY
The implementation of genome editing strategies in grapevine is the easiest way to improve sustainability
and resilience while preserving the original genotype. Among others, the Mildew Locus-O (MLO) genes have
already been reported as good candidates to develop powdery mildew-immune plants. A never-explored
grapevine target is NPR3, a negative regulator of the systemic acquired resistance. We report the exploitation of a cisgenic approach with the Cre-lox recombinase technology to generate grapevine-edited plants
with the potential to be transgene-free while preserving their original genetic background. The characterization of three edited lines for each target demonstrated immunity development against Erysiphe necator in
MLO6-7-edited plants. Concomitantly, a significant improvement of resilience, associated with increased
leaf thickness and specific biochemical responses, was observed in defective NPR3 lines against E. necator
and Plasmopara viticola. Transcriptomic analysis revealed that both MLO6-7 and NPR3 defective lines modulated their gene expression profiles, pointing to distinct though partially overlapping responses. Furthermore, targeted metabolite analysis highlighted an overaccumulation of stilbenes coupled with an improved
oxidative scavenging potential in both editing targets, likely protecting the MLO6-7 mutants from detrimental pleiotropic effects. Finally, the Cre-loxP approach allowed the recovery of one MLO6-7 edited plant with
the complete removal of transgene. Taken together, our achievements provide a comprehensive understanding of the molecular and biochemical adjustments occurring in double MLO-defective grape plants. In
parallel, the potential of NPR3 mutants for multiple purposes has been demonstrated, raising new questions
on its wide role in orchestrating biotic stress responses.
Keywords: Vitis vinifera, Erysiphe necator, stilbenes, New Plant Breeding Techniques (NPBTs), Plasmopara
viticola.
INTRODUCTION
Climate change, due to its detrimental effects on agriculture and consequently on food production and the global
economy, stands as one of the greatest challenges of the
century (Malhi et al., 2021). It causes significant shifts in
temperature, precipitation, and corresponding alterations
in soil characteristics, adversely affecting crop performances (Anderson et al., 2020). Furthermore, these
changes may exacerbate the proliferation of pests and
pathogens, altering their interactions with plants (Singh
et al., 2023). To counteract the development of pathogens,
numerous agrochemicals are used, even if their excessive
Ó 2024 The Author(s).
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This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License,
which permits use, distribution and reproduction in any medium, provided the original work is properly cited and
is not used for commercial purposes.
1 of 20
application revealed negative impacts on the environment
and human health (Kim et al., 2017). To mitigate pathogen
infections and preserve environmental biodiversity and
integrity, alternative strategies must be identified. This
necessity is driving researchers to explore innovative solutions, including the development of resilient plant varieties
through several approaches. Among other techniques,
plant breeding, a historically successful method for crop
development, has advanced significantly with the advent
of New Plant Breeding Techniques (NPBTs) (Giudice et al.,
2021).
The identification of candidate genes is the first critical step for the application of NPBTs (Nerva et al., 2023).
Indeed, the development of resistant varieties through
conventional breeding relies on harnessing resistance (R)
genes to trigger the plant immune system and act as the
primary defense barrier against pathogens. Upon detecting effectors, plants induce the effector-triggered immunity (ETI) pathway that typically results in localized cell
death or in a hypersensitive response (Yuan et al., 2021;
Zaidi et al., 2018). An alternative approach for developing
disease-resilient crops involves susceptibility (S ) genes,
which play a pivotal role in pathogen-induced diseases,
facilitating infection and supporting compatibility between
a pathogen and its plant host (Zaidi et al., 2018). In contrast to R genes, mutation or loss of function of S-genes
confers resistance to pathogens (van Schie & Takken, 2014). Mildew Locus-O (MLO) genes are acknowledged S-genes, prominently associated with powdery
mildew (PM) susceptibility. They were initially character€ schges et al., 1997; Piffanelli et al., 2002),
ized in barley (Bu
then their role in the regulation of PM susceptibility has
been extensively corroborated across a broad spectrum of
plant
species,
including
Arabidopsis
(Consonni
et al., 2006, 2010), tomato (Zheng et al., 2013), wheat (Li
et al., 2022), apple (Pessina, Angeli, et al., 2016), pea
(Humphry et al., 2011), and grapevine (Pessina, Lenzi,
et al., 2016; Wan et al., 2020). MLO genes encode proteins
characterized by a seven-transmembrane domain (including a CaM-binding domain) structure localized on the
plasma membrane and acting as Ca2+ channels (Gao
et al., 2022; Kim et al., 2002; Reddy et al., 2003). The MLO
family is divided into seven phylogenetic clades, among
these only two encompass S-genes: clade IV, housing all
monocot S-genes, and clade V, containing all dicot
S-genes (Acevedo-Garcia et al., 2014). While not all members of clades IV and V exhibit S-gene features, potential
candidates can be discerned during early stages of PM
infection due to heightened expression levels. For
instance, in grapevine, three out of four clade V-MLO
genes (VvMLO7, VvMLO11, and VvMLO13) show an early
increased expression after PM infection (Goyal et al.,
2021). Consequently, exploiting RNA interference, the
simultaneous knockdown of the genes VvMLO7 with
VvMLO6 and VvMLO11 led to a substantial decrease in
PM severity (Pessina, Lenzi, et al., 2016). These findings
suggest that the knockdown of VvMLO7, in combination
with VvMLO6 and VvMLO11, plays a crucial role in
enhancing PM resilience, whereas the contribution of
VvMLO13 appears less significant. Recent studies have
demonstrated the successful application of CRISPR/Cas9
technology to achieve targeted mutagenesis of VvMLO3
and VvMLO4 genes in grapevine (Wan et al., 2020). The
edited lines exhibited varying phenotypes: while some
displayed normal growth patterns, others showed
senescence-like chlorosis and necrosis damages. These
observations highlight that pleiotropic effects can arise in
MLO-edited lines. Notably, four of the VvMLO3-edited
lines showed increased resistance to PM, a resistance
mechanism based on host cell death, cell wall apposition,
and the accumulation of H2O2.
Systemic Acquired Resistance (SAR) is a key component of plant immunity (Vlot et al., 2021) and salicylic acid
(SA) is the key phytohormone accumulated in tissues
upon pathogen infection and orchestrating SAR responses
(Peng et al., 2021). The Nonexpressor of PathogenesisRelated (NPR) gene class is a key regulator of
SA-mediated signal transduction triggered after SA binding. In Arabidopsis, this protein family comprises numerous genes, including NPR1, NPR2, NPR3, and NPR4.
According to the literature, NPR1 and NPR2 positively regulate SA association with WRKY and TGA (TGACGbinding) transcription factors in the nucleus, enhancing
the expression of Pathogenesis-Related (PR) genes by
binding to their promoter sequences (Liu et al., 2020).
Many studies in various species, such as Arabidopsis
(Wang et al., 2020; Wu et al., 2012), periwinkle (Sung
mez-Mun
et al., 2019), and citrus (Dutt et al., 2015; Go
et al., 2017), confirmed the pivotal role of NPR1 in
inducing PR gene expression and pathogen tolerance.
Conversely, NPR3 and NPR4, despite being paralogues
of NPR1, exhibit an antagonistic role, functioning as
transcriptional co-repressors of key immune regulators
such as SARD1 and WRKY70, as well as adaptors of
Cullin3 (CUL3)-based E3 ligase, which mediates the degradation of NPR1 (Chang et al., 2019; Liu et al., 2020).
This opposite role was demonstrated in previous experiments performed on Theobroma cacao in which the
knock-out of TcNPR3 resulted in enhanced resilience to
Phytophthora capsici and Phytophthora tropicalis,
accompanied by elevated expression of downstream
defense genes (Fister et al., 2018; Shi et al., 2013). We
chose a similar approach in grapevine with the aim of
reducing the susceptibility against different fungal pathogens. A similar approach in grapevine would potentially
allow reducing susceptibility against not only PM but
also Plasmopara viticola, the causative agent of downy
mildew (DM).
Ó 2024 The Author(s).
The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.,
The Plant Journal, (2025), 122, e17204
2 of 20 Loredana Moffa et al.
CRISPR/Cas9 stands out among the NPBTs as a powerful and precise tool, which can be used to enhance tolerance
to biotic and abiotic stresses in plants and also to functionally characterize putative genes involved in plant immunity
and environmental adaptation (Giudice et al., 2021; Zhu
et al., 2020). Despite the global economic significance of
grapevines, there are currently few studies on
CRISPR/Cas9-mediated genome editing (Li et al., 2020; Scintilla et al., 2022; Wan et al., 2020). This is due to a long and
challenging process that encompasses many difficult steps,
like the necessity to develop embryogenic calli, the recalcitrance of such tissue to regenerate embryos and
the following regeneration of plants from the developed
embryos (Nuzzo et al., 2022). Furthermore, the difficulties in
developing marker-free edited grape plants limit the application of genome editing. Indeed, conventional transformation
approaches based on Agrobacterium spp. rely on the presence of foreign genes (i.e., the Cas9 protein, the gRNAs, and
the selection marker), which then turn the plants into genetically modified organisms (GMOs) (Nerva et al., 2023). As
such, GMOs undergo specific regulations (very restrictive in
Europe) and meet resistance from both the society and politicians (Ceasar & Ignacimuthu, 2023). For this reason, the
exploitation of transgene-free approaches is the only way to
avoid the GMOs regulation and to develop marketable products. In such context the exploitation of DNA-free genome
editing (Gambino et al., 2024) or the cisgenic-like approach
(Nerva et al., 2023) is the only two available strategies for
preserving the original genotype avoiding the generation of
GMOs. In specific, the cisgenic-like approach relies on the
exploitation of recombinase systems able to remove
the transgenes once the editing event is achieved (Dalla
Costa et al., 2016; Ye et al., 2023). Among the different
recombinase systems, the Cre/loxP recombination system
has been extensively documented for its use in eliminating
selectable marker genes across various species (Gilbertson, 2003; Ye et al., 2023). The expression of Cre recombinase can be controlled by inducible promoters that respond
et al., 2018;
to heat, cold, drought, or chemical stimuli (Eva
Khattri et al., 2011; Wang et al., 2005; Zuo et al., 2001).
This study reports an efficient approach to overcome
these difficulties and provides novel insights into the biological function of genes involved in pathogen-resilience
through application of the CRISPR/Cas9 technique. Notably, knock-out grapevine plants with double mutation of
VvMLO6 and VvMLO7 and VvNPR3 mutants exhibited significantly enhanced tolerance against PM, with the double
mutants resulting almost immune. Interestingly, only
NPR3 mutants exhibited also an improved resilience
against downy mildew (DM), disease caused by the fungal
pathogen P. viticola. Finally, the Cre-loxP system for transgene excision displayed a good efficiency in reducing the
copy number and allowed to recover a MLO6-7 edited
plant without the presence of transgenes.
RESULTS
Cre-lox recombinase system allows the transgene copy
number to be lowered up to ten times
Phylogenetic placement of VvMLO6 and VvMLO7 protein
sequences confirmed their belonging to MLO Clade V
(Figure S1). Similarly, the VvNPR3 protein grouped within
the clade of NPR3/4 from Arabidopsis and very close to
NPR3 from T. cacao (Figure S2). Employing a conventional
Agrobacterium-mediated transformation protocol, we
induced the differentiation of somatic embryos of Vitis
vinifera cv Chardonnay and 18 putatively transgenic
embryos were obtained. We successfully regenerated 17
plants harboring the MLO6/MLO7 silencing construct, all of
which exhibited amplification with Cas9 primers. Evaluation of editing efficiency was assessed on PCR amplicons
using TIDE software, revealing successful events in 13 (out
of 15) of the selected plants. Concurrently, for the NPR3
construct, 129 somatic embryos of “Chardonnay” were
obtained after Agrobacterium-mediated transformation.
Forty-eight plants were regenerated from embryos, 53 of
which exhibited successful transformation (positive to
Cas9) and 33 displayed targeted editing. Subsequent
screening of the target regions of gRNAs VvNPR3g2,
VvMLO6g7, and VvMLO7g25 via high-throughput sequencing unveiled some editing frequencies exceeding 90% (Figure 1). Three lines for each construct were selected (editing
characteristics of each line are reported in Figure 1) and
subsequently exploited for further analyses. Specifically,
for NPR3g2, the lines NPR3#4, NPR3#9, NPR3#15 exhibited
editing frequencies of 88.71, 89.82, and 90.35% respectively
(Figure 1E). Regarding mutations in the MLO genes, the
percentage of modified reads around VvMLO6g7 was consistently higher than 85% across all lines (Figure 1A). A
larger variation in editing frequencies was observed for
MLO7g25, as illustrated in Figure 1(C). The mutations associated with the lines selected for further analyses and
experiments are illustrated in Figure 1(B,D,F). Off-target
analysis using Sanger sequencing revealed that exclusively
the target sequence has been mutated for each gRNA.
After confirming successful genome editing, selected edited lines (MLO#1, #3, #9 and NPR#4, #9, #15) and wild-type
(WT) plants underwent micropropagation and acclimatization (25 plants for each) for further analysis. No phenotypic
differences of MLO6-7- and NPR3-edited lines in comparison with WT have been observed (representative plants
are shown in Figure S3).
Edited plants were subjected to high temperature in
order to activate the excision Cre-loxP system. To evaluate
the copy number variation (CNV) in acclimatized plants of
NPR3- and MLO6/7-edited lines compared to the original
regenerated ones (still in vitro), qPCR was performed on at
least 10 plants for each line. Interestingly, following the
heat treatments, some plants displayed a copy number up
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The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.,
The Plant Journal, (2025), 122, e17204
Characterization of grapevine MLO6/7 and NPR3 genes 3 of 20
Ó 2024 The Author(s).
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The Plant Journal, (2025), 122, e17204
4 of 20 Loredana Moffa et al.
Figure 1. Alignment and editing frequencies.
(A, C, E) Frequencies of editing percentages for each guide RNA (gRNA) and target gene. Numbers within the pie charts represent the number of plants belonging to the specific class.
(B, D, F) For each target gene, three lines have been selected for subsequent experiments. The editing event for each line is reported for MLO6g7 (B), MLO7g25
(D), and NPR3g2 (F).
Table 1 Copy number variation (CNV)
Target—VvMLO6 and VvMLO7
Plant
MLO6-7#1
MLO6-7#3
MLO6-7#9
Target—NPR3-like
Reduction (%)
Plant
NPR3#4
NPR3#9
NPR3#15
Reduction (%)
Plants subjected to heat treatments were analyzed to determine the final copy number and relative percentage reduction. CN, copy number.
The original copy number is reported beside the line name.
to 10 times lower than the original plant from which they
derived (Table 1). Later in the season, once the plants were
acclimatized, the CNV was checked again, and one MLO67#3 plant resulted with a copy number of 0. To further
confirm the absence of transgene PCR reactions at 35, 40,
and 45 cycles have been run (Figure S4). The plant was
checked again with the Sanger sequencing method and
the editing status was confirmed.
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Characterization of grapevine MLO6/7 and NPR3 genes 5 of 20
Edited plants displayed from increased tolerance to
immunity to Erysiphe necator, depending on the edited
target
Initial signs of spontaneous PM infection were observed in
WT plants at the end of August 2023 (Figure 2). When all
WT plants exhibited symptoms on at least three leaves,
disease indices were assessed. WT plants displayed the
highest susceptibility to PM, with an incidence of 84% and
a severity of 13% (Figure 2A,B). In contrast, the edited
lines, particularly the MLO-edited ones, showed significantly reduced infection rates. Specifically, the MLO6-7#1,
MLO6-7#3, and MLO6-7#9 lines exhibited markedly lower
incidences of 0.5, 0, and 4.5%, respectively, all statistically
different from the WT (Figure 2A). Correspondingly, these
MLO-edited lines demonstrated significantly lower disease
severities, ranging from 0 to 0.2% (Figure 2B). Similarly,
the NPR3-edited lines also exhibited reduced infection,
though to a lesser extent than the MLO-edited lines. Notably, NPR3#4 and NPR3#9 lines showed a significantly
lower incidence than the WT, while NPR3#15 showed an
incidence of 75.3%, not significantly different from the WT
plants (Figure 2A). However, all NPR3-edited lines displayed significantly lower severities of 3.1, 1.7, and 6.7%,
respectively, compared to the WT plants (Figure 2B). Representative examples of leaf infections are reported in Figure 2(C–E).
Evaluation of leaf ecophysiological parameters,
including measurement of the chlorophyll content index
(CCI), maximum quantum efficiency of photosystem II by
determination of the Fv/Fm ratio and leaf thickness, was
conducted for both WT and edited plants (Figure 2F–H).
CCI analysis (Figure 2F) indicated a slightly lower, though
not statistically significant, value in NPR3 edited lines in
comparison with both WT and MLO6-7 mutated plants.
Fv/Fm values did not significantly vary across edited and
WT plants (Figure 2G), likely suggesting that PSII efficiency
was not altered. Conversely, leaf thickness showed a significant increase only in NPR3 edited plants, while no
significant differences were recorded between MLO6-7 edited lines and WT plants (Figure 2H).
Only NPR3-edited plants displayed increased tolerance to
Plasmopara viticola
Considering the improved resilience observed while the
spontaneous infection of PM was occurring, a controlled
experiment using DM was initiated during the summer
2024. Once confirmed that the non-inoculated leaves did
not show any sign of infection after 7 days of incubation,
we proceeded to evaluate the inoculated leaves following
the OIV descriptor 452-1. The experiment was repeated
twice in order to confirm the results with two independent
replications. Results showed that WT- and MLO-edited
lines displayed a similar distribution across the disease
resistance classes in both experiments (Figure 3A,B). In
fact, widespread visible signs of infection were observed
both in WT and MLO-edited lines by the emergence of P.
viticola from the abaxial surface of the leaves (Figure 3C).
On the contrary, both the independent replication
confirmed a higher disease resistance score for the
NPR3-edited lines (Figure 3A,B), with a highly significant
difference from the WT (P-value 1) in comparison
with the WT plants (Table S3). The majority of the genes
(211 over 376) show downregulation, with an overrepresentation of genes belonging to the energy metabolism
cluster, photosynthesis, and transport (Figure S5A–C).
Instead, all the MLO6-7 edited lines displayed an upregulation of genes belonging to the carbohydrate metabolism
(Figure S5A–C).
When considering the NPR3-like edited lines, the number of significantly differentially regulated genes (FC >1) in
comparison with WT plants was 1216 (Table S4). Contrary
to what was observed in MLO6-7 edited plants, a slight
majority of genes were upregulated (646 over 1216).
Among them we observed an overrepresentation of genes
belonging to the metabolic process cluster (including three
polyphenol oxidases—VIT_00s0480g00060, VIT_00s0480g
00070, VIT_10s0116g00560), particularly those involved in
maltose, starch, polysaccharide and thiamine-containing
compound metabolism (Figure S6D–F). Similarly, NPR3like edited lines displayed a downregulation of genes
belonging to energy metabolism, photosynthesis, and
transport (Figure S5D–F). Genes belonging to the alphaexpansin group (VIT_17s0053g00990, VIT_06s0004g
07970, VIT_14s0108g01020, VIT_13s0067g02930) as well as
auxin-responsive genes (VIT_03s0038g01120, VIT_03s0038
g00950, VIT_05s0062g00850, VIT_11s0016g00640) were
strongly downregulated. Interestingly, two isoforms of
MLO genes (namely MLO1 and MLO7) were downregulated in NPR3-edited plants (Table S4).
Finally, a comparison between MLO6-7 and NPR3-like
edited lines revealed 153 genes with a significant
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Figure 4. Analysis of the leaf-associated fungal
population in edited and WT plants.
Fungal genera were identified through the qiime2ITSexpress pipeline and compared among samples
(n = 4). Only the top 20 representative genera
among the total classified amplicons were retained
in the graphic representation.
differential expression pattern between the two conditions
(Table S5). Among them, 62 displayed exclusively downregulation, while 91 displayed a stronger upregulation in
MLO6-7-edited compared to NPR3-edited plants. Looking
at the processes downregulated in MLO6-7-edited lines, an
enrichment of genes involved in transport (e.g., anion
transport and inorganic anion transport) and response to
abiotic stimuli (e.g., spermine and polyol metabolism) was
observed (Figure S5G–I). In parallel, an upregulation of
genes related to peculiar processes in MLO6-7-edited lines
was observed, specifically those involved in maltose and
starch metabolism and cell homeostasis (iron homeostasis
and RNA/mRNA modification) (Figure S5G–I).
Stilbenes are differentially accumulated in MLO6/7 and
NPR3-edited plants
In order to assess alterations in key target defense-related
metabolites (i.e., stilbenes), HPLC-DAD-MS/MS analysis
was conducted on edited MLO and NPR lines compared
with WT plants. HPLC-MS/MS analyses revealed the presence of t-resveratrol (Figure 5A) and its glycosylated form
(piceid) (Figure 5B), along with two different oxodimers of
resveratrol (d-viniferin and e-viniferin) (Figure 5C,D). Looking at the whole dataset, our values were similar to those
measured in the leaves of different grape cultivars
(Mohammadparast et al., 2024). As a general trend, the
lowest levels were found in WT conditions, while the
MLO6-7 and NPR3 mutant lines exhibited slightly higher
contents. On average, the leaves of plants mutated for
these genes showed an increase in total stilbene content
compared to the WT (Figure 5A), of +1.87 and +1.60,
respectively. Specifically, while the rise in t-resveratrol content was similar between MLO6-7 and NPR3 plants (about
+1.4), edited lines resulted in different profiles for the more
complex forms of stilbenes. Specifically, the MLO6-7
mutant plants showed a substantial increase in the polymeric forms of t-resveratrol, with increments of +2.3 and
+1.73 for d-viniferin and e-viniferin (Figure 5C,D), respectively. Instead, the NPR3 mutant plants, despite an increase
in d-viniferin (+1.91), exhibited a significant increase in
piceid, with an increment of +1.65 (+1.71) (Figure 5B).
To comprehensively analyze the oxidative profile of
WT, MLO6-7, and NPR3 leaves, the total peroxide content
(Figure S7A), total polyphenol content (Figure S7B), and
the enzymatic activity of superoxide dismutase (SOD)
and catalase (CAT) (Figure S7C,D) were determined. Under
our experimental conditions, a significant reduction in total
peroxide content was observed in the mutated lines.
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Characterization of grapevine MLO6/7 and NPR3 genes 9 of 20
Figure 5. Quantification of leaf stilbenes in edited
and WT plants.
(A–D) Contents of trans-resveratrol (A), piceid (B), dviniferin (C), and e-viniferin (D) quantified in leaf
samples of Vitis vinifera wild type (WT), MLO6-7edited, or NPR3-like plants (n = 3 for each line and
compound). Data are expressed as lmol g 1 of dry
weight (DW). Lowercase letters above bars indicate
significant differences among the samples, as
determined by ANOVA followed by Tukey post hoc
test. All data in bar charts represent mean values
standard deviation bars.
Specifically, values decreased from 1.55 0.22 lmol of
H2O2 to approximately 1.04 0.09 and 0.89 0.05, for
MLO6-7 and NPR3 plants, respectively (Figure S3A). In contrast, a complementary trend was observed for the total
polyphenol content (TPC) measured through the molybdenum and tungsten salt reduction assay (Figure S7B). TPC
ranged from 165.80 10.5 (WT) to 446.46 19.22
(NPR3#9). Although variations were evident among the
mutated plant lines, WT plants exhibited a significantly
lower polyphenol content, approximately 2.05 (in comparison with MLO6-7) and 2.55 (in comparison with
NPR3) times lower. From the enzymatic point of view, both
SOD and CAT activities were significantly reduced in WT
plants compared to the edited lines (Figure S7C,D). However, while SOD appeared to be more active in the MLO6-7
edited lines (Figure S7C), the NPR3 lines exhibited higher
CAT levels (approximately +2.05) compared to WT
(Figure S7D).
DISCUSSION
The development of gene-edited plant varieties able to
cope with environmental stresses represents a promising
tool to improve agricultural sustainability (Giudice
et al., 2021). Considering the specific case of grapevine,
implementing genome editing strategies emerges as the
most straightforward method for enhancing sustainability
and resilience while maintaining the original genotype
(Giudice et al., 2021). Gene editing allows to disrupt as little as possible the genome architecture and the gene pool
of “elite” varieties while improving their stress resilience
features (Sheridan, 2024).
Despite the great potential of genome editing, its
application in grapevine is still limited, mainly due to three
bottlenecks. The first issue is related to the knowledge of
gene functions. Even though the grapevine genome has
been available for about 20 years (Jaillon et al., 2007;
Velasco et al., 2007; Wang et al., 2024) most genes are still
annotated only in-silico, limiting the available pool of targets (Nerva et al., 2023). A second limitation concerns the
development of embryogenic calli, the explant required as
a starting point to perform either Agrobacterium-mediated
transformation or DNA-free transfection of protoplasts
(Gambino et al., 2024; Najafi et al., 2023; Scintilla
et al., 2022; Tricoli & Debernardi, 2024). The last limitation,
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10 of 20 Loredana Moffa et al.
but equally important, is the ability of the calli to regenerate (Nuzzo et al., 2022). The latter two limitations are primarily associated with genetic features, meaning that each
specific variety possesses its own ability to produce
embryogenic calli and its own regeneration efficiency (Gribaudo et al., 2017; Nuzzo et al., 2022). Collectively, these
limitations led to the application of editing approaches predominantly in easy-to-work genotypes, like, for example,
table grape varieties [e.g., Crimson seedless or Sugraone
(Clemens et al., 2022; Scintilla et al., 2022)] or raisin grapes
[e.g., Thompson seedless (Najafi et al., 2023; Olivares
et al., 2021)], which represent only a small area of cultivated lands. A few examples of gene editing in elite wine
grape cultivars are beginning to emerge in the literature
(Gambino et al., 2024; Tricoli & Debernardi, 2024), and here
we report its application in the worldwide cultivated Chardonnay cultivar.
To preserve the original genetic background and avoid
the issues related to DNA-free or stable transformation
approaches (Nerva et al., 2023), we decided to exploit a
recombinase mechanism based on the Cre-loxP system
(Chong-Perez et al., 2012; Gilbertson, 2003). The recovered
data showed that we reduced the transgene copy number
by 90%, applying a single cycle of heat-shock, and that
lately in the season a complete transgene removal was
achieved at least in one MLO6-7#3 plant. This result is in
contrast to what previously observed in grapevine using
flp-frt recombination, which did not result in a significant
reduction of copy number in transgenic grape plants (Dalla
Costa et al., 2020), suggesting a more efficient activity of
the Cre-loxP system. This result is very encouraging since,
as previously mentioned, the editing of grapevine protoplasts, and the subsequent regeneration of plants, is still
very challenging. Thus, by avoiding such a step we will be
able to improve the number of exploitable genotypes while
reducing the time for generation of edited grapevine
plants.
Considering the limited gene function information, we
opted to exploit already available information on VvMLO6
and VvMLO7 to investigate the effects of a double mutation. Indeed, previous studies took into consideration the
role of the different MLO genes in grapevine (Feechan
et al., 2008) confirming that MLO6 and MLO7 were the two
most promising targets (Pessina, Angeli, et al., 2016). Our
results highlight that the double mutation led to the almost
complete loss of susceptibility to PM. Indeed, both disease
indices (incidence and severity) remained close to zero,
with just one MLO6-7-edited line showing very mild infection symptoms. It is worth noting that, even if MLO
mutants were already generated in transgenic grapevines
using RNA interference (Pessina, Lenzi, et al., 2016), no
studies have looked at the metabolic shift induced by the
disruption of these genes. Indeed, the understanding that
mutations in MLOs can elicit broader effects beyond
conferring resistance to PM has long been reported (Wolter
et al., 1993). In particular, MLO mutants of herbaceous species present a premature decay of photosynthesis associated with early decline in photosynthetic performance, an
altered transcriptional profile, and abnormal levels of secondary metabolites derived by tryptophan (Consonni
et al., 2010). All these modifications led to spontaneous
mesophyll cell death, probably reflecting an uncontrolled
senescence program (Piffanelli et al., 2002). Additionally, a
recent work conducted on Arabidopsis linked the emergence of pleiotropic effects to the nitrogen deficiency,
pointing out that also external factors can alter the establishment of deleterious effects (Freh et al., 2024). Our findings show that grapevine mutants for MLO6 and MLO7
display a decline of molecular processes related to photosynthesis process, partially overlapping to what was
observed in Arabidopsis (Consonni et al., 2010). Nonetheless, MLO grape mutants displayed a transcriptional reprogramming of genes involved in carbohydrate metabolism,
a response usually linked to plant immunity (Trouvelot
et al., 2014), suggesting a possible trade-off toward
growth-defense. Additionally, the biochemical profiling
revealed an overaccumulation of stilbenes, which have
been consistently identified as primary defense responses
to pathogen attacks (including PM) (Schnee et al., 2008),
and largely recognized as markers for disease resistance in
grape plants (Viret et al., 2018). Collectively, these findings
suggest that also in grapevine MLO mutants, as in other
species where MLOs were studied, an enhancement of
some defense responses was established, at least considering changes in the stilbene profile. Further investigation
on this subject is definitely needed to better elucidate the
whole metabolomics changes controlling such defensive
mechanisms.
In parallel to mutations of MLOs, we decided to
explore the effects of the loss of function mutants of NPR3like. This gene belongs to the plant-conserved Nonexpresser of Pathogenesis-Related (NPR) protein family and is
classified within clade II, which, together with members of
clade I, plays prominent roles in orchestrating plant
defense responses (Zhou et al., 2023). Their activity is
mediated by the ability to bind salicylic acid, but with an
opposite outcome. While members of clade I are essential
for activating the SAR responses (Glazebrook et al., 1996),
members of clade II impair the activation of defense
responses (Ding et al., 2018). In fact, loss of function
mutants for NRP3 and NPR4 in Arabidopsis showed an
increased activation of the immune system and a concomitant upregulation of PR proteins, leading to an increased
tolerance to pathogen attacks (Fu et al., 2012). Similarly,
the transient knock-out of NPR3 in T. cacao leaves limited
the spread of P. tropicalis (Fister et al., 2018). Considering
this information, and that in grapevine there is only one
member of clade I (VvNPR1) (Le Henanff et al., 2009) and
Ó 2024 The Author(s).
The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.,
The Plant Journal, (2025), 122, e17204
Characterization of grapevine MLO6/7 and NPR3 genes 11 of 20
one member of clade II (NPR3) (Zhou et al., 2023), we
opted to evaluate an editing approach targeting the NPR3
gene. From the susceptibility point of view, the
NPR3-edited lines displayed a significant improvement in
terms of resilience against PM but to a lesser extent if compared to MLO mutants. In fact, based on disease incidence,
only two out of three NPR3-like mutant lines (i.e., NPR3#4
and NPR3#9) displayed a significant difference when compared to WT plants. Conversely, the disease severity index
was always significantly lower than in WT. These findings
are in line with our expectations since in MLO-edited
plants we disrupted the production of proteins involved in
recognition between the fungus and its host, thereby inhibiting fungal growth and tissues colonization. On the other
hand, in edited NPR3-like plants, we blocked the production of a protein that negatively regulates the plant’s
defense responses, but which is not involved in the early
interaction stages between the pathogen and its host.
Nonetheless, a significant downregulation of MLO1 and
MLO7 was observed, suggesting that NPR3 downregulation can indirectly impair the transcription of MLO genes.
The exploitation of a pathogen-independent defense mechanism allowed the NPR3-edited lines to show, besides
improved tolerance to PM, an improved resilience against
P. viticola, strongly suggesting a much wider resilience to
biotic stresses. This feature makes this approach more suitable for generating a broad-spectrum disease tolerance, as
also suggested by the metabarcoding analyses which
highlighted a significant reduction in the relative abundance of potential pathogens. In addition to the reported
molecular and biochemical adjustments, we observed an
improvement in leaf thickness probably related to the
molecular adjustment (i.e., downregulation of genes
encoding alpha-expansins and auxin-responsive factors)
involving more complex metabolic processes. Such a trait
was already associated with disease tolerance in other species, including woody plants such as Populus clones,
where those with greater leaf thickness were more tolerant
to rust infections (Fernandez-Martınez et al., 2013). For the
first time in genome editing studies, we demonstrated here
that the outcomes of a lower infection rate can be confirmed by ITS metabarcoding. Indeed, this approach
highlighted a significantly lower presence of E. necator on
the leaves of both NPR3-like and MLO-edited plants. In parallel, the relative abundance of Ampelomyces was reduced
in the MLO6/7 mutants but not in NPR3-edited lines. It is
worth noting that Ampelomyces represent a natural biocontrol agent of PM, able to parasitize the mycelium and
chasmothecia in turn limiting PM spread. Its reduced abundance in MLO6/7 lines can be due to the lower colonization
of PM (its natural hosts). On the other hand, the unaffected
relative abundance in NPR3 lines suggests that, even
though NPR3-edited lines showed improved defense
responses, such adjustments are not influencing the
Ampelomyces activity. Finally, a much wider effect on
the immune system of NPR3-edited lines can be envisaged
by the improved resilience against DM and by an exclusive
reduction of Dactylonectria, Ilyonectria, and Pleosporales
incertae sedis genera, all hosting well-known grape pathogens (Probst et al., 2019), only in NPR3-edited plants.
Interestingly, NPR3-edited plants exhibited an
enhanced regeneration efficiency, suggesting an indirect
effect on the embryogenesis process. Indeed, if compared
to MLO-edited plants we were able to recover a higher
number of embryos even if the starting material was the
same (same embryogenic calli stock and same starting
quantity). Such a result was quite unexpected since, as
previously reported (Dal Santo et al., 2022), the elicitation
of stress responses and secondary metabolism was associated with a reduced embryogenic potential in grapevine.
This unforeseen finding is quite interesting, as recalcitrance to regenerate is one of the main bottlenecks impairing the wide application of gene editing in many plant
species (Atkins & Voytas, 2020). A more in-depth evaluation of the effects of NPR3-like editing during the regeneration phase is needed in order to uncover the molecular
and biochemical signals controlling the observed enhancement of embryo regeneration.
Finally, to improve the understanding of the complex
responses occurring in the edited lines, leaf samples were
subjected to molecular and biochemical analyses. In particular, both NPR3 and MLO-edited lines accumulated higher
levels of stilbenes and polyphenols. Specifically, MLO6/7
mutants displayed a significant overaccumulation of polymerized stilbenes (viniferins). This observation can be
explained by the fact that the overproduction of secondary
metabolites can lead to polymerization within specific cellular environments. Although the exact mechanism
remains unclear, the polymerization is a well-known process occurring for catechins (yielding proanthocyanidins
and tannins) (Mannino et al., 2021) and resveratrol (yielding viniferins) (Fuloria et al., 2022). These obtained molecules possess improved stability and bioavailability
compared to their monomeric equivalents, potentially
enhancing their efficacy in preventing and ameliorating
various pathological conditions (Langcake, 1981). In our
experimental conditions, we observed a substantial reduction in total peroxide content for both mutant types,
coupled with a concurrent increase in total polyphenol
content. The elevation in total polyphenol contents closely
correlates with the increased levels of phytoalexins. In
NPR3-edited lines the increase in polyphenols also correlates with the strong overexpression (FC >2) of three polyphenol oxidase-encoding genes (VIT_00s0480g00060,
VIT_00s0480g00070, VIT_10s0116g00560). Additionally,
phenolic compounds are renowned for their antioxidant
properties, and along with other secondary metabolites
could have contributed to maintain the cellular redox
Ó 2024 The Author(s).
The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.,
The Plant Journal, (2025), 122, e17204
12 of 20 Loredana Moffa et al.
balance in edited grapevine plants by efficiently mitigating
free radicals (Amarowicz et al., 2008). The derivatives of tresveratrol, such as piceid and viniferins, also hold robust
antioxidant capabilities. In fact, if the greater stability and
aqueous solubility of the piceid compared to t-resveratrol
allows a greater bioavailability, viniferins show an even
greater reductive capacity due to their polymeric architecture enhancing their ability to sequester oxidizing agents
(Delmas et al., 2011). Moreover, both resveratrol and its
derivatives augment the activity of endogenous antioxidant enzymes within cells, including superoxide dismutase
(SOD) and catalase (CAT), thereby further fortifying cells’
resilience against oxidative stressors. SOD and CAT work
synergistically neutralizing free radicals and peroxides,
thereby mitigating oxidative damage to cells and safe€ lcßin, 2010). In edited
guarding their structural integrity (Gu
lines, in particular in NPR3 ones, we observed an overexpression of catalase genes and several Glutathione STransferase (GST ) genes (Tables S4 and S5) encoding a
large protein family that catalyzes the conjugation of
reduced glutathione (GSH) to various substrates preventing the oxidative stress (Neuefeind et al., 1997). For example, GST expression was higher in asymptomatic leaves of
grapevine canes infected by esca disease (Valtaud
et al., 2009). Resveratrol and its derivatives possess the
ability to modulate the activity of SOD and CAT through
various mechanisms. Firstly, as previously discussed,
these compounds can act as direct antioxidants, effectively
neutralizing free radicals and reducing peroxide generation. Furthermore, resveratrol and its derivatives can
directly shield SOD and CAT from inactivation induced by
€ lcßin, 2010). The observed reduction in
oxidative agents (Gu
total peroxide content in our experimental conditions
could likely be ascribed experimentally to the combined
action of both enzymatic activity and polyphenol content
(Tang et al., 2010). Finally, the accumulation of stilbenes
and higher activity of the antioxidant enzymes, both having a cytoprotective outcome, could also explain the reason for more stable Fv/Fm values, potentially mitigating the
pleiotropic effects of MLOs knock-out mutants (as depicted
by SPAD and Fv/Fm values) as reported in other species
(Consonni et al., 2010).
CONCLUSIONS
The recent legislative proposal adopted by the European
Parliament
(https://www.europarl.europa.eu/doceo/
document/TA-9-2024-0067_EN.html) reflects a consensus
among scientific institutions, industry, and farmers on the
need to exploit biotechnological approaches to start a new
green revolution in agriculture (Sheridan, 2024). Indeed,
gene editing represents a novel chance to exploit the
potential of plant biotechnology in Europe, allowing the
amelioration of cultivated plants in terms of nutritional
profile, resistance to stress, and yield (Zhan et al., 2021).
The present work, exploiting a Cre-lox recombinase
approach, demonstrated the huge potential of biotechnology to improve viticulture sustainability producing a
transgene-free edited grape plant. Indeed, by targeting two
different sets of genes, we obtained immune plants as well
as plants showing an improved tolerance to both E. necator and P. viticola (Figure 6). Metabolic analyses revealed a
possible correlation with the accumulation of phenolic
compounds, including diverse stilbenes, in both MLO6/7
and NPR3-like mutants. Such an improvement not only
positively influenced resilience against PM and DM, but
also potentially mitigated the pleiotropic effects of MLO6-7
defective plants already reported in other species. These
results suggest that the application of NPBTs for the
obtainment of improved genotypes with high level of these
strong fungitoxic compounds could help to improve biotic
stress resilience. Finally, we uncovered an intriguing side
effect of NPR3-like defective calli, which displayed an
improved ability to regenerate embryos when compared to
MLO-edited calli. Additional studies are warranted to elucidate the underlying mechanisms contributing to such an
effect.
EXPERIMENTAL PROCEDURES
Embryogenic calli
From late April to the beginning of May depending on the phenological stage, flower clusters of V. vinifera cv. Chardonnay were
harvested from a vineyard located in Rauscedo (PN) and managed
by Vivai Cooperativi Rauscedo (VCR). Only the basal half of the
inflorescence was retained during collection. The right developmental stage of pollen was assessed following established methodologies (Gambino et al., 2007; Gribaudo et al., 2004).
Subsequently, the collected flowers underwent a sterilization process involving a 15-min treatment with a sodium hypochlorite
solution (1.5% available chlorine) containing 100 lL L 1 of Tween
20, followed by multiple rinses with sterile distilled water. Ovaries
and anthers, including their filaments, were excised and cultured
on PIV starter calli induction medium [Nitsch and Nitsch basal
salt, Murashige and Skooge vitamins, 6% sucrose, 0.3% Gelrite,
4.5 lM 2,4-Dichlorophenoxyacetic acid (2,4 D), and 8.9 lM 6Benzylaminopurine (BAP), pH of 5.8] (Dhekney et al., 2019; Franks
et al., 1998; Gambino et al., 2007, 2021). Three months after callus
induction, embryogenic calli were visually identified and transferred onto C1 proliferation medium (Nitsch and Nitsch basal salt,
Murashige and Skooge vitamins, 6% sucrose, 0.5% Gelrite, 5 lM
2,4 D, and 1 lM BAP, pH of 5.8) and transferred monthly onto
fresh medium (Gribaudo et al., 2004).
Vector construction
Guide RNAs (gRNAs) targeting the VvMLO7, VvMLO6, and
VvNPR3 genes (Vitvi13g00578, Vitvi13g04070, and Vitvi10g01335)
(Shi et al., 2023) were evaluated using the online tool CRISPR-P
(http://crispr.hzau.edu.cn/CRISPR2/) (Lei et al., 2014). The gRNAs
(Table S6) were selected based on their on-target efficiency with a
single gRNA designed for each gene. Additionally, an off-target
analysis was performed using CRISPOR (Concordet & Haeussler, 2018) and Cas-OFFinder to ensure specificity and minimize
Ó 2024 The Author(s).
The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.,
The Plant Journal, (2025), 122, e17204
Characterization of grapevine MLO6/7 and NPR3 genes 13 of 20
Figure 6. Overview of the main findings.
Summary of the susceptibility degree to powdery mildew (PM) and of the molecular, physiological and biochemical adjustments observed in MLO6/7 edited (left
side) and NPR3 edited (right side) plants. Metabarcoding analysis revealed that, while in MLO6/7 plants PM development was almost impaired, in NPR3 plants
PM development significantly lower than in wild-type plants (WT) but not that much as in MLO6/7. Additionally, while in MLO6/7 plants no influence was
observed on the relative abundance of other pathogens, in NPR3 ones a significant reduction was observed. From the physiological point of view, leaf thickness,
chlorophyll content index and photosystem II efficiency (Fv/Fm) were evaluated, highlighting a thicker leaf in NPR3 plants. From the molecular point of view, similarity between MLO6/7 and NPR3-edited plants were observed. From the biochemical point of view we observed an enhancement of stilbenes in both type of
plants but involving different type of molecules. In MLO6/7 plants increase in d- and e-viniferin was observed while piceid and resveratrol increased more in
NPR3 plants. Finally, the oxidative status was monitored revealing an increase of scavenging potential in both type of plants but with a larger extent in NPR3
plants.
potential off-target effects (Bae et al., 2014). Used guides are listed
in Table S6.
Phylogenetic placement of VvMLO6, VvMLO7, and NPR3 was
calculated as previously described (Brambilla et al., 2023; Nerva
et al., 2022). Briefly, sequences of MLO and NPR genes were
retrieved from repositories (TAIR for Arabidopsis, SolGenomics
for tomato, NCBI for others) and aligned using Muscle
(Edgar, 2004). For MLO, 143 sequences were used and for NPR 20
sequences were used (including the VvACT as outgroup for both
analyses). Alignments in fasta format are made available as
Ó 2024 The Author(s).
The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.,
The Plant Journal, (2025), 122, e17204
14 of 20 Loredana Moffa et al.
Data S1 (MLO) and Data S2 (NPR). Phylogenetic relationship was
then calculated using the IQ-TREE webserver (Trifinopoulos
et al., 2016) and then visualized in MEGA (Kumar et al., 2016). The
pDIRECT_22c vector (Cerm
ak et al., 2017) was customized, and a
recombinase system based on the Cre-lox system combined to
the heat-shock inducible promoter (Chong-Perez et al., 2012; Gilbertson, 2003) was integrated (Figure S8 and sequence reported in
Data S3). The vector assembly involved the insertion of a gRNA
targeting each gene. In detail, for MLO knock-out, one gRNA targeting VvMLO6 and one targeting VvMLO7 were co-integrated,
otherwise a gRNA was inserted to achieve VvNPR3 knock-out.
PCR products of gRNAs obtained following the procedure
described by Cerm
ak et al. (2017) were cloned into the previously
constructed plasmid following the protocols by Lampropoulos
et al. (2013). Competent cells of Agrobacterium tumefaciens strain
GV3101 were used to transform embryogenic calli as previously
described by Dhekney et al. (2008).
A. tumefaciens was initially cultured on LB agar and subsequently sub-cultured in MG/L medium to reach a final optical density (OD600) of 0.8, according to Amarowicz et al. (2008) and Li
et al. (2008), with only minor modifications: after MG/L culture,
the cell suspension was centrifuged, resuspended in LCM medium
supplemented with 100 lM acetosyringone and incubated at 28°C
and 210 rpm for 3 h.
Embryogenic calli, previously conditioned according to Gribaudo et al. (2004), were co-cultured with A. tumefaciens (Torregrosa et al., 2015) using 600 mg L 1 of cefotaxime as selective
agent in LCM medium to rinse embryogenic calli. Subsequently,
the embryogenic calli were dried on sterile filter paper, transferred
onto GS1CA medium [Nitsch and Nitsch basal salt, Murashige
and Skooge vitamins, 6% sucrose, 1% Agar, 4.5 lM 2,4 D, 1 lM
BAP, 10 lM 2-Napthoxyacetic acid (NOA); 20 lM Indole-3-acetic
acid (IAA) pH of 5.8] (Franks et al., 1998; Gambino et al., 2007) supplemented with 450 mg L 1 cefotaxime, and maintained at 26°C
for 9 days. Calli were sub-cultured monthly on GS1CA media supplemented with 450 mg L 1 cefotaxime and 100 mg L 1 kanamycin for 4 months. Thereafter, calli were transferred monthly onto
GS1CA medium supplied with 100 mg L 1 kanamycin.
Embryos at the torpedo stage were transferred onto MS-SH
medium (Murashige and Skooge basal salt, Murashige and
Skooge vitamins, 1.5% sucrose, 0.4% Gelrite, and 10 lM BAP, pH
of 5.6). After 2 months, embryos were transferred onto halfstrength MS basal salts at 26°C, 200 lmol m 2 sec 1 (PPFD), and
a 16/8-h day/night photoperiod. When 5 leaves were fully
expanded, each node was transferred onto half-strength MS basal
salts supplemented with 4 lM BAP for 1 month. Subsequently,
the nodal explants were moved to MS supplemented with 2.5 lM
NAA and 2.5 lM IBA for 20 days to induce root formation. The
resulting explants were transferred to half-strength MS basal salts
for 1 month and then to sterilized peat pellet. Among all regenerated plants, three lines for the double mutants MLO6/MLO7 and
three for the NPR3-like were retained for further experiments. In
total 25 plants for each selected line, including wild-type
embryogenic-derived plants (WT), were acclimatized in a greenhouse at the beginning of June 2023.
DNA polymerase (Thermo Fisher Scientific) using specific primers
for Cas9 (Table S7). The initial editing efficiency was assessed
through Sanger sequencing (BioFab Research srl, Italy) of the
genomic regions targeted by gRNAs (Table S7). The sequencing
data were analyzed using TIDE (https://tide.nki.nl/) software (Brinkman et al., 2014).
The VvMLO7, VvMLO6, and VvNPR3 target regions of the
greenhouse-acclimatized plants were screened by highthroughput sequencing. The target regions were amplified using
primers and overhang Illumina adapters to generate Illumina
library amplicons (Table S7). Subsequently, the library was
sequenced by the Illumina NovaSeq 6000 platform at the IGA
(Udine, Italy). Approximately 100 000 reads per sample were produced. Raw paired-end reads were analyzed using the CRISPResso
pipeline (https://crispresso2.pinellolab.org/) and removing reads
with low quality (Clement et al., 2019).
Editing efficiency analysis
Nucleic acid extraction, sequencing, and bioinformatics
Genomic DNA was extracted from a single leaf of each plant using
the Genomic DNA Isolation Kit NORGEN (Norgen Biotech Corp).
The DNA concentration was determined using a NanoDrop One
Spectrophotometer (Thermo Fisher Scientific) and subsequently
diluted to a final concentration of 10 ng lL 1. The diluted DNA
samples were used for PCR reactions using DreamTaq Green
For molecular assays, three independent biological replicates
were obtained for each condition by pooling five asymptomatic
leaves randomly selected every five vines of the 25 acclimatized
plants (1 leaf 9 5 plants 9 3 biological replicates: each set of five
plants therefore represented an independent biological replicate).
Attention was paid to randomly collect asymptomatic leaves also
Susceptibility assay and leaf ecophysiology
After 3 months in the greenhouse, plants were attacked by a naturally occurring PM infection. When all control plants had at least
three leaves with clear powdery mildew symptoms, the disease
indices (incidence and severity) were collected using the Grape
Assess Mobile App (University of Adelaide). For each line, four
independent biological groups of observation were considered.
Each biological group was made up by at least 50 observations.
Five non-infected leaves for each plant were collected, lyophilized,
and stored at 80°C for further analyses.
Besides PM evaluation, a controlled infection using downy
mildew was performed. Ten leaves from each regenerated line
were randomly collected during July. Leaves were surface washed
using water and paper towel and then laid on Petri dishes containing sterile water-agar (1%). The inoculum of P. viticola was prepared collecting sporangia from infected leaves and then diluting
to 5 9 105 sporangia/mL. After inoculation (1 mL per leaf) leaves
were incubated at 24°C with a 16–8 light–dark cycle. Development
of leaf symptoms (i.e., emergence of sporangia from the leaves)
were checked 7 days after inoculation. Descriptor 452-1 of the
International Organization of Vine and Wine (OIV) were used to
determine leaf resistance to downy mildew. Briefly, each leaf was
visually checked and assigned to a class among the following:
class 1—61 to 100% of surface covered with sporulation, class 2—
41 to 60% of surface covered with sporulation, class 3—21 to 40%
of surface covered with sporulation, class 4—1 to 20% of surface
covered with sporulation, class 5—0% of surface covered with
sporulation. Control leaves (n = 3) were kept without inoculation
to exclude previous infections naturally occurring in the greenhouse. The experiment was repeated twice with a 15-day delay
between the two trials, in order to confirm the results.
Measurements to determine chlorophyll fluorescence parameters (Fv/Fm), relative chlorophyll content (CCI), and leaf thickness
were carried out using a MultispeQ v2.0 device controlled by
PhotosynQ software (Kuhlgert et al., 2016). Records were taken on
ten fully formed non-senescing leaves for each line, selecting one
leaf per plant growing between the 4th and 6th shoot from
the top.
Ó 2024 The Author(s).
The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.,
The Plant Journal, (2025), 122, e17204
Characterization of grapevine MLO6/7 and NPR3 genes 15 of 20
from untreated plants. This approach was adopted with the aim of
investigating the specific defense responses established in the edited vines regardless of the occurrence of visible symptoms.
Genomic DNA and total RNA were isolated from lyophilized
leaves using Genomic DNA Isolation Kit NORGEN (Norgen Biotech Corp) and Spectrum plant total RNA kit (Sigma-Aldrich, St.
Louis, MO, USA), respectively, according to the manufacturer’s
instructions.
DNA and RNA concentrations were evaluated using a NanoDrop one Spectrophotometer (Thermo Fisher Scientific, Waltham,
MA, USA) and integrity was confirmed on agarose gel. To proceed
with RNA-seq analysis, at least 2 lg for each sample was sent to
Macrogen Inc. (Europe) for cDNA library construction and
sequencing using NovaSeq. Furthermore, Illumina tag screening
of the ITS2 region of the rDNA was performed by Macrogen Inc.
(South Korea) using primers ITS3-ITS4 on a MiSeq Illumina apparatus. Trimmed sequences were analyzed with DADA2 and
sequence variants were taxonomically classified using the UNITE
database, as previously described (Nerva et al., 2021). Analysis of
transcripts was performed using the Artificial Intelligence RNAseq Software AIR (accessible at https://transcriptomics.cloud).
Transcripts were annotated using the V1 annotation of the grapevine genome (Grimplet et al., 2012); functional classification, GO
enrichment analysis, and identification of differentially expressed
genes (DEGs) were performed as previously described (Nerva
et al., 2022).
Heat shock treatments and evaluation of copy number
variation through qPCR
Three-week-old grapevine plants acclimatized in the greenhouse
and displaying knock-out of target genes were incubated in a
temperature-controlled chamber at 45°C for 24 h for three cycles
with a 48-h interval between treatments. To assess the CNV resulting from heat treatments, a single leaf (developed after the heat
treatment) from each plant was collected, lyophilized, and the
genomic DNA was extracted and used for qPCR analysis using a
custom-made calibration curve (Dalla Costa et al., 2009; Lee
et al., 2006). Quantification of the NPTII copy number was
employed to quantify T-DNA insertions (Table 1).
Calibration curves were constructed by amplifying the VvCHI
fragment from Chardonnay leaf using primers listed in Table S7.
Briefly, PCR product was cloned into pGEM-T Easy vector, which
already contained a fragment of the NPTII gene cloned from pDIRECT_22c. The final plasmid pGEM:CHI:NPTII was purified and
quantified using NanoDrop One Spectrophotometer and its molarity was calculated using the NEBioCalculator online tool (version
Costa et al. (2009).
Each amplification was followed by melting curve analysis
(60–94°C) with a heating rate of 0.5°C every 15 sec. All reactions
were performed in duplicate with nuclease-free water as negative
control. Estimation of the NPTII insertion copies in transgenic
grapevines was calculated using the following formula: (NPTII
gene copy number/VvCHI copy number) 9 2.
Biochemical analyses
About 100 mg of lyophilized leaves (the same used for RNA-seq
analysis) were used for the biochemical analyses. Briefly, extraction was performed using 1 mL of a 1:1 (v/v) mixture of Methyl-tButyl Ether (MTBE) and Methanol (MetOH), as previously
described (Liu et al., 2013). Following vortex mixing, the samples
were sonicated for 15 min at 4°C. Subsequently, 0.5 mL of 0.1%
(v/v) HCl was added. After a 5-min incubation at 4°C, the samples
were centrifuged at 10 000g for 10 min at 4°C. The upper MTBE
phase was transferred, dried under nitrogen flow at constant flow,
and resuspended in 100 lL of 50% (v/v) propanol. Accordingly,
samples were injected into a HPLC-DAD-ESI-MS/MS apparatus,
and the analytical separation of phytoalexins was conducted
employing a binary solvent system consisting of water acidified
with 0.1% (v/v) formic acid (solvent A) and methanol acidified to
0.1% (v/v) with formic acid (solvent B). A Kinetecs C18 column
(130
A, 1.7 lm, 2.1 9 100 mm) was utilized for the separation. The
gradient profile was as follows: 0–8 min with 95% (v/v) A,
8–12 min decreasing A to 75% (v/v), 12–16 min decreasing A to
55% (v/v), 16–32 min decreasing A to 25% (v/v), and subsequently
reduced to 5% (v/v) at 45 min. Solvent A concentration was maintained for 10 min. Throughout the chromatographic run, the flow
rate was set at 0.2 mL min 1, with a 10 lL injection volume. Chromatographic conditions were restored to initial conditions and
maintained for 8 min before the next injection (Sun et al., 2006).
Phytoalexins were quantified using different concentrations of tresveratrol, d-viniferin, and e-viniferin by plotting peak areas
against concentrations, and linear regression was applied for
curve fitting. Original standards of t-resveratrol (purity ≥99%),
piceid (purity ≥99%), and viniferins (purity ≥95%) purchased from
Merck KGaA were used to prepare the calibration curves. Limits of
detection (LOD) and limits of quantification (LOQ) were calculated
for each standard following the method described by Cao
et al. (2020).
Total phenolic content (TPC) was quantified preparing the
ethanolic extracts of the lyophilized leaves and assessed using
the Folin–Ciocalteu method, which entails the reduction of
phosphotungstic-phosphomolybdic acid to blue pigments in an
alkaline medium. This method follows the procedure outlined by
Folin and Denis, as previously described by Singleton (1966). For
quantification, a calibration curve with gallic acid (GA) was used,
and the results were expressed as milligrams of GA equivalents
(GAE) per 100 g of fresh weight (FW). Each sample was measured
in triplicate to ensure accuracy and reliability of the data. This
approach allows for a precise evaluation of the phenolic content,
which is crucial for understanding the antioxidant properties of
the extracts.
The total peroxide content was measured using a Peroxide
Assay Kit from Abnova (St. Louis, MO, USA). To begin, 50 mg of
lyophilized leaves were ground in liquid nitrogen and extracted
with milli-Q water at a ratio of 1:10 (w/v). Following extraction, the
samples were centrifuged at 15 0009g for 10 min, and the resulting supernatant was used for the assay. For the assay, 200 lL of
detection reagent was added to each 40 lL sample in a flatbottom 96-well plate. After a 30-min incubation period, the absorbance was measured at 600 nm using a microplate reader. The
H2O2 content was determined by comparing the absorbance
values to a standard curve generated from serial dilutions of a 3%
H2O2 standard.
Lyophilized leaves were pulverized and homogenized with a
mortar and pestle to determine enzyme activities. The ground
material was then extracted in a solution containing 62.5 mM
Tris–HCl (pH 7), 10% (v/v) glycerol, 2% (w/v) Sodium Dodecyl Sulfate (SDS), 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride
(PMSF). The mixture was centrifuged at 5000g for 10 min at 4°C,
and the supernatant was collected for subsequent enzymatic
assays. The total protein concentration was measured using the
Lowry method to normalize the enzymatic activities (Assink Junior
et al., 2023). Enzymatic activities for Superoxide Dismutase (SOD;
ab65354) and Catalase (CAT; ab83464) were determined using
Ó 2024 The Author(s).
The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.,
The Plant Journal, (2025), 122, e17204
16 of 20 Loredana Moffa et al.
commercial kits from Abcam, following the manufacturer’s
instructions and according to Agliassa et al. (2021).
Statistical analysis
For the PM disease indices, biochemical data, and MultispeQ data,
analysis of variance (ANOVA) was conducted using the SPSS software package (v. 23, SPSS Inc). For DM disease resistance analysis chi-squared test was calculated. The Tukey’s HSD post hoc test
was used when ANOVA showed significant differences at a probability level of P ≤ 0.05. The standard deviation (SD) of all means
was calculated.
ACCESSION NUMBERS
The GenBank accession numbers of the sequences reported in
this paper are:
88785.
• NGS amplicon on-target genomic sequences: BioProject
All the other study data are included in the article and supporting information.
AUTHOR CONTRIBUTIONS
LN and WC conceived and supported this study; LN, WC
and LM designed the experiments; LM performed research
with assistance from GM, IB, GG, IP, CP, CMB, AS, EZ, RV,
WC and LN; LM, GM, GG, WC and LN analyzed the data;
LM, GM, IB, WC and LN wrote the manuscript; and all
authors reviewed and edited the manuscript.
ACKNOWLEDGMENTS
Part of the work was conducted within the Shield4Grape project
in the framework of the Horizon Europe program and within the
BIOTECH-VITECH project funded by the Italian Ministry of Agriculture, Food Sovereignty and Forestry. Views and opinions
expressed are, however, those of the authors only. Neither the
European Union nor the granting authority nor the European
Commission can be held responsible for them. The authors thank
Alison Garside for the manuscript English editing service. Figure 6
has been created in BioRender (https://biorender.com/z80s157).
CONFLICT OF INTEREST
The authors declare that they have no conflict of interests.
This article does not contain any studies with human or
animal participants.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are openly
available. The GenBank accession numbers of the
sequences reported in this paper are indicated in the
Accession numbers section. All the other study data are
included in the article and supporting information.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article.
Data S1. Alignments of MLO proteins. Sequences of 141 MLO proteins, including protein sequences from grapevine were aligned
using Muscle.
Data S2. Alignments of NPR proteins. Sequences of 19 NPR proteins, including protein sequences from grapevine were aligned
using Muscle.
Data S3. Vector sequence. The modified sequence of the pDIRECT_22C vector including Cre/loxP cassette and sites.
Figure S1. Phylogenetic placement of VvMLO proteins. The phylogenetic placement of Vitis vinifera (Vv) MLO proteins has been
inferred using the IQ-TREE webserver. Sequences from Malus
domestica (Md ), Glycine max (Gm), Prunus persica (Pp), Solanum
lycopersicum (Sl ), Fragaria vesca (Fv), and Arabidopsis thaliana
(At) have been used. The red arrow indicates the clade hosting
MLO6 and MLO7, the two targets of the present work.
Figure S2. Phylogenetic placement of VvNPR proteins. The phylogenetic placement of Vitis vinifera (Vv) NPR proteins has been
inferred using the IQ-TREE webserver. The red arrow indicates the
clade hosting the target (NPR3) of the present work.
Figure S3. Picture of representative plants. Plants acclimatized
from the in vitro condition did not show phenotypic differences
when compared to WT plants. From left to right, one representative plant for each line (NPR3#4, #9, and #15, MLO6-7#1, #3, #9,
and WT) has been chosen.
Figure S4. Gel electrophoresis of the Cre and Cas PCR products.
PCR products from the negative MLO6-7#3 plant were run in a 1%
agarose gel together with positive and negative controls. (A) is the
PCR product from the NCED3 gene, representing a positive control
for the plant DNA. (B)–(D) are the amplicon from PCR at 35, 40,
and 45 cycles, respectively. In each panel, from left to right: MM,
molecular marker (1 kb plus, the first thicker band represents the
500 bp, the second thicker band represents the 1000 bp); (1) plant
harboring T-DNA with a copy number of 1; (2) MLO6-7#3 T-DNAfree plant; (3) MLO6-7#3 plant with a copy number of 0.3; (4) WT
plant (negative control); (5) MLO6-7#3 plant with a copy number
of 0.1.
Figure S5. Upregulated metabolic pathways as identified through
RNA-seq analysis. Upregulated genes in MLO6-7 edited versus
WT lines (A-B-C), NPR3-like edited versus WT (D-E-F) and MLO6-7
edited versus NPR3-like edited were analyzed to identify genes
belonging to specific metabolic routes. (A, D, G) Pathways showing an enrichment of terms in the dataset. Darker colors (from yellow to orange) refer to a more represented pathway. (B, E, H)
percentages of gene ontology (GO) terms belonging to each specific group. (C, F, I) percentages of genes belonging to specific
terms or functions.
Figure S6. Redox-active balance measured through UV/Vis determination. (A) displays the total peroxide content expressed as
mmol H2O2 g 1 of dry (DW) material (n = 3), while (B) shows the
total phenolic compound content expressed as mg of gallic acid
equivalent (GAE) g 1 of dry weight material (n = 3). Enzymatic
activities of superoxide dismutase (SOD) and catalase (CAT) are
depicted in (C) and (D), respectively, measured as enzymatic activity (U) relative to the total protein content determined as
described in the materials and methods section (n = 3 each). Lowercase letters above bars indicate significant differences among
the samples, as determined by ANOVA followed by Tukey’s post-
Ó 2024 The Author(s).
The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.,
The Plant Journal, (2025), 122, e17204
Characterization of grapevine MLO6/7 and NPR3 genes 17 of 20
hoc test. All data in bar charts represent mean values standard
deviation bars.
Figure S7. Map of plasmid developed in the present study. The
main sequences of the vector have been annotated, including the
HSP promoter, the Cre encoding sequence, and the loxP sites.
Figure S8. Map of the modified pDIRECT_22C vector.
Table S1. Differential abundances of taxa across edited lines.
Average abundances (Mean), standard deviation (SD), and significance in comparison with WT plants (P-value) of each edited line
were calculated using the ANCOM-BC functions.
Table S2. Differential abundances of taxa across edited targets.
Average abundances (Mean), standard deviation (SD), and significance in comparison with WT plants (P-value) were calculated
using the ANCOM-BC functions.
Table S3. List of differentially expressed genes in MLO6-7 edited
lines. Differentially regulated genes with a fold-change (FC) higher
than 1 are reported considering the comparison between MLO6-7
edited lines and WT plants.
Table S4. List of differentially expressed genes in NPR3-like edited
lines. Differentially regulated genes with a fold-change (FC) higher
than 1 are reported considering the comparison between NPR3like edited lines and WT plants.
Table S5. List of differentially expressed genes in MLO6-7 versus
NPR3-like edited lines. Differentially regulated genes with a foldchange (FC) higher than 1 are reported considering the comparison between MLO6-7 versus NPR3-like edited lines.
Table S6. Selected gRNAs. List of the gRNAs used in this study.
For each gRNA, the PAM site is underlined.
Table S7. Oligonucleotides. List of the oligonucleotides used in
this study.
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