Summary
The clustered regularly interspaced short palindromic repeats‐associated protein 9 (CRISPR/Cas9) system is a powerful tool for editing plant genomes. Efficient genome editing of grape (Vitis ...vinifera) suspension cells using the type II CRISPR/Cas9 system has been demonstrated; however, it has not been established whether this system can be applied to get biallelic mutations in the first generation of grape. In this current study, we designed four guide RNAs for the VvWRKY52 transcription factor gene for using with the CRISPR/Cas9 system, and obtained transgenic plants via Agrobacterium‐mediated transformation, using somatic embryos of the Thompson Seedless cultivar. Analysis of the first‐generation transgenic plants verified 22 mutant plants of the 72 T‐DNA‐inserted plants. Of these, 15 lines carried biallelic mutations and seven were heterozygous. A range of RNA‐guided editing events, including large deletions, were found in the mutant plants, while smaller deletions comprised the majority of the detected mutations. Sequencing of potential off‐target sites for all four targets revealed no off‐target events. In addition, knockout of VvWRKY52 in grape increased the resistance to Botrytis cinerea. We conclude that the CRISPR/Cas9 system allows precise genome editing in the first generation of grape and represents a useful tool for gene functional analysis and grape molecular breeding.
Abstract
Drought stress severely affects grapevine quality and yield, and recent reports have revealed that lignin plays an important role in protection from drought stress. Since little is known ...about lignin-mediated drought resistance in grapevine, we investigated its significance. Herein, we show that
VlbZIP30
mediates drought resistance by activating the expression of lignin biosynthetic genes and increasing lignin deposition. Transgenic grapevine plants overexpressing
VlbZIP30
exhibited lignin deposition (mainly G and S monomers) in the stem secondary xylem under control conditions, which resulted from the upregulated expression of
VvPRX4
and
VvPRX72
. Overexpression of
VlbZIP30
improves drought tolerance, characterized by a reduction in the water loss rate, maintenance of an effective photosynthesis rate, and increased lignin content (mainly G monomer) in leaves under drought conditions. Electrophoretic mobility shift assay, luciferase reporter assays, and chromatin immunoprecipitation-qPCR assays indicated that
VlbZIP30
directly binds to the G-box
cis
-element in the promoters of lignin biosynthetic (
VvPRX N1
) and drought-responsive (
VvNAC17
) genes to regulate their expression. In summary, we report a novel
VlbZIP30
-mediated mechanism linking lignification and drought tolerance in grapevine. The results of this study may be of value for the development of molecular breeding strategies to produce drought-resistant fruit crops.
Abstract
The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) system is a powerful tool for targeted genome editing, with applications that include plant ...biotechnology and functional genomics research. However, the specificity of Cas9 targeting is poorly investigated in many plant species, including fruit trees. To assess the off-target mutation rate in grapevine (
Vitis vinifera)
, we performed whole-genome sequencing (WGS) of seven Cas9-edited grapevine plants in which one of two genes was targeted by CRISPR/Cas9 and three wild-type (WT) plants. In total, we identified between 202,008 and 272,397 single nucleotide polymorphisms (SNPs) and between 26,391 and 55,414 insertions/deletions (indels) in the seven Cas9-edited grapevine plants compared with the three WT plants. Subsequently, 3272 potential off-target sites were selected for further analysis. Only one off-target indel mutation was identified from the WGS data and validated by Sanger sequencing. In addition, we found 243 newly generated off-target sites caused by genetic variants between the Thompson Seedless cultivar and the grape reference genome (PN40024) but no true off-target mutations. In conclusion, we observed high specificity of CRISPR/Cas9 for genome editing of grapevine.
WRKY transcription factors are known to play important roles in plant responses to various abiotic and biotic stresses. The grape WRKY gene,
was previously reported to respond to salt and drought ...stress, as well as methyl jasmonate and ethylene treatments in
×
cv. 'Kyoho.' In the current study, WRKY3 from the 'Kyoho' grape cultivar was constitutively expressed in
under control of the cauliflower mosaic virus 35S promoter. The
transgenic
plants showed improved salt and drought stress tolerance during the germination, seedling and the mature plant stages. Various physiological traits related to abiotic stress responses were evaluated to gain further insight into the role of
, and it was found that abiotic stress caused less damage to the transgenic seedlings than to the wild-type (WT) plants.
over-expression also resulted in altered expression levels of abiotic stress-responsive genes. Moreover, the
transgenic
lines showed improved resistance to
, but increased susceptibility to
, compared with the WT plants. Collectively, these results indicate that
plays important roles in responses to both abiotic and biotic stress, and modification of its expression may represent a strategy to enhance stress tolerance in crops.
Drought stress limits the growth and development of grapevines, thereby reducing productivity, but the mechanisms by which grapevines respond to drought stress remain largely uncharacterized. Here, ...we characterized a group A bZIP gene from "Kyoho" grapevine,
, which was shown to be induced by abscisic acid (ABA) and dehydration stress. Overexpression of
in transgenic
enhanced dehydration tolerance. Transcriptome analysis revealed that a major proportion of ABA-responsive and/or drought-responsive genes are transcriptionally regulated by
during ABA or mannitol treatment at the cotyledon greening stage. We identified an
G-box motif (CACGTG) and a potential grapevine G-box motif (MCACGTGK) in the promoters of the 39 selected
genes upregulated in the transgenic plants and in the 35 grapevine homologs, respectively. Subsequently, using two grapevine-related databases, we found that 74% (23/31) and 84% (21/25) of the detected grapevine genes were significantly upregulated by ABA and drought stress, respectively, suggesting that these genes are involved in ABA or dehydration stress and may be regulated by
in grapevine. We propose that
functions as a positive regulator of dehydration-responsive signaling in the ABA core signaling pathway.
Summary
Powdery mildew (PM) is a severe fungal disease of cultivated grapevine world‐wide. Proanthocyanidins (PAs) play an important role in resistance to fungal pathogens; however, little is known ...about PA‐mediated PM resistance in grapevine.
We identified a WRKY transcription factor, VqWRKY56, from Vitis quinquangularis, the expression of which was significantly induced by PM.
Overexpression (OE) of VqWRKY56 in Vitis vinifera increased PA content and reduced susceptibility to PM. Furthermore, the transgenic plants showed more cell death and increased accumulation of salicylic acid and reactive oxygen species. Transient silencing of VqWRKY56 in V. quinquangularis and V. vinifera reduced PA accumulation and increased the susceptibility to PM. VqWRKY56 interacted with VqbZIPC22 in vitro and in planta. The protein VqWRKY56 can bind to VvCHS3, VvLAR1, and VvANR promoters, and VqbZIPC22 can bind to VvANR promoter. Co‐expression of VqWRKY56 and VqbZIPC22 significantly increased the transcript level of VvCHS3, VvLAR1, and VvANR genes. Finally, transient OE of VqbZIPC22 in V. vinifera promoted PA accumulation and improved resistance to PM, while transient silencing in V. quinquangularis had the opposite effect.
Our study provides new insights into the mechanism of PA regulation by VqWRKY56 in grapevine and provides a basis for further metabolic engineering of PA biosynthesis to improve PM resistance.
•We transformed Arabidopsis with VqAP13 under the control of constitutive promoter.•We reported the function of VqAP13 through characterization of transgenic plants.•The transgenic plants showed ...enhance resistance to powdery mildew.•The resistance of transgenic plants to B. cinerea inoculation was reduced.•VqAP13 promotes the SA dependent signal transduction pathway.
The grape aspartic protease gene, AP13 was previously reported to be responsive, in Chinese wild Vitis quinquangularis cv. ‘Shang-24’, to infection by Erysiphe necator, the causal agent of powdery mildew disease, as well as to treatment with salicylic acid in V. labrusca×V. vinifera cv. ‘Kyoho’. In the current study, we evaluated the expression levels of AP13 in ‘Shang-24’ in response to salicylic acid (SA), methyl jasmonate (MeJA) and ethylene (ET) treatments, as well as to infection by the necrotrophic fungus, Botrytis cinerea, and the transcript levels of VqAP13 decreased after B. cinerea infection and MeJA treatment, but increased following ET and SA treatments. Transgenic Arabidopsis thaliana lines over-expressing VqAP13 under the control of a constitutive promoter showed enhanced resistance to powdery mildew and to the bacterium Pseudomonas syringae pv. tomato DC3000, and accumulated more callose than wild type plants, while the resistance of transgenic A. thaliana lines to B. cinerea inoculation was reduced. In addition, the expression profiles of various disease resistance- related genes in the transgenic A. thaliana lines following infection by different pathogens were compared to the equivalent profiles in the wild type plants. The results suggest that VqAP13 action promotes the SA dependent signal transduction pathway, but suppresses the JA signal transduction pathway.
Difficulties in generating woody perennial fruit trees and a lack of molecular markers have impeded functional analysis of agronomically important genes and genetic engineering for improved fruit ...cultivars. The clustered regularly interspaced short palindromic repeats (CRISPR) technology has been applied in editing fruit tree genomes, providing timely opportunities for studying the functions of the genes in fruit development and stress tolerance. It also leads to the new era of genetic engineering of fruit trees. In this review, we intend to present recent progress in the application of CRISPR to genome editing of fruit trees. We reviewed the most widely used CRISPR systems developed in other plant species that may be adopted for fruit trees and describe three genetic transformation methods used for fruit trees that have potential application value for different CRISPR systems. We also discuss the potential of the CRISPR system to study basic fruit tree biology and for precision fruit tree breeding.
WRKY transcription factors are known to play important roles in plant responses to biotic stresses. We previously showed that the expression of the WRKY gene,
, from Chinese wild
was strongly induced ...24 h post inoculation with powdery mildew. In this study, we analyzed the expression levels of
following treatment with the defense related hormones salicylic acid (SA) and methyl jasmonate, revealing that
was strongly induced by SA but not JA. We characterized the
gene, which encodes a WRKY III gene family member, and found that ectopic expression in
enhanced resistance to powdery mildew and
pv.
DC3000, but increased susceptibility to
, compared with wild type (WT) plants. The transgenic
lines displayed strong cell death induced by the biotrophic powdery mildew pathogen, the hemibiotrophic
pathogen and the necrotrophic pathogen
. In addition, the relative expression levels of various defense-related genes were compared between the transgenic
lines and WT plants following the infection by different pathogens. Collectively, the results indicated that
plays essential roles in the SA dependent signal transduction pathway and that it can enhance the hypersensitive response cell death triggered by microbial pathogens.
Abstract
Anthocyanins are plant secondary metabolites that have a variety of biological functions, including pigmentation. The accumulation of anthocyanins is regulated by both transcriptional ...activators and repressors. Studies have shown that bZIP family members act primarily as positive regulators of anthocyanin biosynthesis, but there are few reports of negative regulation. Here, we report that a grapevine (Vitis vinifera) bZIP gene from group K, VvbZIP36, acts as a negative regulator of anthocyanin biosynthesis. Knocking out one allele of VvbZIP36 in grapevine with CRISPR/Cas9 promoted anthocyanin accumulation. Correlation analysis of transcriptome and metabolome data showed that a range of anthocyanin biosynthesis genes were activated in VvbZIP36 mutant plants relative to the wild type, resulting in the accumulation of related metabolites, including naringenin chalcone, naringenin, dihydroflavonols, and cyanidin-3-O-glucoside. The synthesis of stilbenes (α-viniferin), lignans, and some flavonols (including quercetin-3-O-rhamnoside, kaempferol-3-O-rhamnoside, and kaempferol-7-O-rhamnoside) was significantly inhibited, and several genes linked to their metabolism were downregulated in the mutant plants. In summary, our results demonstrate that VvbZIP36 is a negative regulator of anthocyanin biosynthesis that plays a role in balancing the synthesis of stilbenes (α-viniferin), lignans, flavonols, and anthocyanins.
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