QTL detection and candidate gene analysis of grape white rot resistance by interspecific grape (Vitis vinifera L. × Vitis davidii Foex.) crossing

Grape white rot,a devastating disease of grapevines caused by Coniella diplodiella(Speg.)Sacc.,leads to significant yield losses in grape.Breeding grape cultivars resistant to white rot is essential to reduce the regular use of chemical treatments.In recent years,Chinese grape species have gained more attention for grape breeding due to their high tolerance to various biotic and abiotic factors along with changing climatic conditions.In this study,we employed whole-genome resequencing(WGR)to genotype the parents of‘Manicure Finger’(Vitis vinifera,female)and‘0940’(Vitis davidii,male),along with 101 F1 mapping population individuals,thereby constructing a linkage genetic map.The linkage map contained 9337 single-nucleotide polymorphism(SNP)markers with an average marker distance of 0.3 cM.After 3 years of phenotypic evaluation of the progeny for white rot resistance,we confirmed one stable quantitative trait locus(QTL)for white rot resistance on chromosome 3,explaining up to 17.9%of the phenotypic variation.For this locus,we used RNA-seq to detect candidate gene expression and identified PR1 as a candidate gene involved in white rot resistance.Finally,we demonstrated that recombinant PR1 protein could inhibit the growth of C.diplodiella and that overexpression of PR1 in susceptible V.vinifera increased grape resistance to the pathogen.

Control of ovule development in Vitis vinifera by VvMADS28 and interacting genes

Seedless grapes are increasingly popular throughout the world,and the development of seedless varieties is a major breeding goal.In this study,we demonstrate an essential role for the grapevine MADS-box gene VvMADS28 in morphogenesis of the ovule.We found that VvMADS28 mRNA accumulated in the ovules of a seeded cultivar,‘Red Globe’,throughout the course of ovule and seed development,especially within the integument/seed coat.In contrast,in the seedless cultivar‘Thompson Seedless’,VvMADS28 was expressed only weakly in ovules,and this was associated with increased levels of histone H3 lysine 27 trimethylation(H3K27me3)within the VvMADS28 promoter region.RNAi-mediated transient suppression of VvMADS28 expression in‘Red Globe’led to reduced seed size associated with inhibition of episperm and endosperm cell development.Heterologous overexpression of VvMADS28 in transgenic tomatoes interfered with sepal development and resulted in smaller fruit but did not obviously affect seed size.Assays in yeast cells showed that VvMADS28 is subject to regulation by the transcription factor VvERF98,and that VvMADS28 could interact with the Type I/MβMADS-domain protein VvMADS5.Moreover,through DNA-affinity purification-sequencing(DAP-seq),we found that VvMADS28 protein specifically binds to the promoter of the grapevine WUSCHEL(VvWUS)gene,suggesting that maintenance of the VvMADS28–VvMADS5 dimer and VvWUS expression homeostasis influences seed development.Taken together,our results provide insight into regulatory mechanisms of ovule and seed development associated with VvMADS28.

欧亚种葡萄(Vitis vinifera L.)的驯化及分类研究进展

欧亚种葡萄是世界上广泛栽培的具有重要经济价值的果树。一般认为欧亚种葡萄栽培亚种是由其野生亚种驯化而来,这个驯化过程包括对色泽引人的大果穗、含糖量高、两性花类型的选择和无性繁殖技术的发现,欧亚种葡萄的驯化问题主要涉及驯化事件发生的时间、地点和数量。本文综述了欧亚种葡萄的驯化和生态地理起源与分布分类研究进展。

葡萄(Vitis vinifera L.)试管繁殖中芽增殖能力衰减的原因及其防治方法的研究

以20个葡萄品种试管苗作为实验材料,经四年、四十多代的继代培养反复对比实验发现:在葡萄试管繁殖中,连续恒温(25±1℃)条件下培养或只采用固体培养方法,不适期进行继代培养均会导致芽增殖能力的衰减,根据生物统计分析,当继代培养超过16～17代后,芽增殖能力即随着继代培养次数的增加而逐渐下降,采取变温培养、变换培养方法、适期继代培养或在增殖培养基中附加适量的椰子乳汁等方法均可防治芽增殖能力的哀减,保持高的芽增殖能力。

葡萄(Vitis Vinifera L.)X-射线能谱元素微区污染分析研究

对葡萄果实不同处理的细胞组织多位点进行X -射线能谱微区元素分析。结果 :葡萄不同处理的细胞组织中共发现Mg、Al、Si、P、S、Cl、K、Ca、Fe、Cu十种元素。且不同处理的组织细胞中各元素含量分布有差异 ,未清洗果实表皮处理的Al、Si、S、Ca、Cu含量均显著高于对照 。

VitisMod:一个葡萄基因共表达数据库

葡萄转录组已在各种组织、发育阶段、生物胁迫、非生物胁迫和其他条件下被测定。目前,仍没有简单实用的网络工具来探索这些宝贵的数据。本文从美国国立生物技术信息中心的基因表达数据库(NCBI GEO)下载1019个基因表达芯片数据,进行权重基因共表达网络分析(WGCNA)。鉴定到41个基因共表达模块。功能富集分析发现这些模块具有不同的功能,并与实验/表型相关。通过模块内连接度筛选枢纽基因,这些基因可能具有重要功能。通过关联推定(Guilt-by-association)原理对模块内功能未知的基因进行功能预测。最后,构建了免费的网络工具VitisMod,为葡萄的基因功能研究提供新资源,网址为:http://bioinformatics.fafu.edu.cn/grape。

Anthocyanin profi les and color properties of red wines made from Vitis davidii and Vitis vinifera grapes

Spine grape(Vitis davidii Foex.)is an important wild grape species native to China.Fifteen red spine grape clones and three red Vitis vinifera grape varieties were used to evaluate the differences in the anthocyanin profiles and color properties of wines made from V.davidii and V.vinifera grapes.Among spine wines,‘Junzi#2’wine had the highest total phenolic and total anthocyanin,‘Xiangzhenzhu’wine had the highest total flavonoids,and‘Junzi#1’wine had the highest total tannin.The anthocyanin compositions of all of the spine wines were dominated by Mv-3,5-diglucoside.The total individual anthocyanin contents in spine wines,except‘Gaoshan#5’,‘Junzi#5055’,‘Junzi#5061’,and‘Junzi#5044’,were signifi cantly higher than in V.vinifera wines.Most of the spine wines had a stronger red intensity and a brighter chroma with a bluer hue than V.vinifera wines.Correlation analysis revealed that the color properties were closely related to the anthocyanin composition.These results suggest that wines made from spine grapes may be useful for the wine industry for their color properties and high individual anthocyanin contents.

The Establishment of an Agrobacterium-Mediated Transformation Platform for the Non-Embryogenic Calli of Vitis vinifera L.

Non-embryogenic calli （NEC） was inevitably and heavily produced when grape embryogenic calli （EC） was induced from explants or during the subculture of EC.A stable and highly efficient NEC transformation platform is required to further sort out and verify key genes which determine/switch the identity of NEC and EC.In this research,a vector pA5 containing a chitinase signal sequence fused to gfp （green fluorescent protein） and an HDEL motive was used to target and immobilize into Agrobacterium strain EHA105 to establish a transformation platform for Vitis vinifera L.cv.Chardonnay NEC.It was determined that NEC 10 d after subculture was the best target tissue;30 min for inoculation followed by 3 d co-cultivation with the addition of 200 μmol L-1 acetosyringone （AS） was optimized as protocol.The use of bacterial densities as 1.0 at OD600 did not result in serious tissue hypersensitive reaction and it had higher efficiency.Kanamycin at 200 mg L-1 was picked for positive expression selection.The stable transformation of NEC was proved by reverse transcription-polymerase chain reaction techniques （RT-PCR） and fluorescent microscopy after three sub-cultures of the selected cell line.Highly efficient genetic transformation protocol of grape NEC was achieved and some of the optimized parameters were different from that reported for EC.This transformation platform could facilitate the verification of candidate somatic embryogenesis （SE） decisive genes,and the successfully transformed NEC with certain genes can also be used as bioreactors for the production of functional products,as NEC not only proliferates fast,but also keeps in a rather stable condition.

Genome and transcriptome analysis of the grapevine(Vitis vinifera L.) WRKY gene family

The plant WRKY gene family represents an ancient and complex class of zinc-finger transcription factors(TFs)that are involved in the regulation of various physiological processes,such as development and senescence,and in plant response to many biotic and abiotic stresses.Despite the growing number of studies on the genomic organisation of WRKY gene family in different species,little information is available about this family in grapevine(Vitis vinifera L.).In the present study,a total number of 59 putative grapevine WRKY transcription factors(VvWRKYs)were identified based on the analysis of various genomic and proteomic grapevine databases.According to their structural and phylogentic features,the identified grapevine WRKY transcription factors were classified into three main groups.In order to shed light into their regulatory roles in growth and development as well as in response to biotic and abiotic stress in grapevine,the VvWRKYs expression profiles were examined in publicly available microarray data.Bioinformatics analysis of these data revealed distinct temporal and spatial expression patterns of VvWRKYs in various tissues,organs and developmental stages,as well as in response to biotic and abiotic stresses.To also extend our analysis to situations not covered by the arrays and to validate our results,the expression profiles of selected VvWRKYs in response to drought stress,Erysiphe necator(powdery mildew)infection,and hormone treatments(salicilic acid and ethylene),were investigated by quantitative real-time reverse transcription PCR(qRT-PCR).The present study provides a foundation for further comparative genomics and functional studies of this important class of transcriptional regulators in grapevine.

Somatic embryogenesis and histological analysis from zygotic embryos in Vitis vinifera L.‘Moldova’

We examined the somatic embryogenesis from and histological studies of zygotic embryos of seeds in European Grape ＇Moldova＇ （Vitis vinifera U ＇Moldova＇）. Primary calli were initiated on Nitsch and Nitsch （NN） medium supplemented with 1.0 mg·L^-1 2,4-D and 0.5 mg·L^-1 6-BA. Embryogenic calli were produced upon transfer to a NN medium with 0.5 mg·L^-1 6-BA and 2 mg·L^-1 NAA and somatic embryos were obtained on a half strength MS medium without plant growth regulators. During the somatic embryo germination, an addition of 1.0 mg·L^-1 6-BA in the medium could accelerate somatic embryos to develop into normal plants and increase the conversion rate from 0 to 43.3%. Histological studies of embryogenic calli and somatic embryos demonstrated dynamic changes of proteins and starch grains. The developmental processes of somatic embryos were similar to those of zygotic embryos, including typical epiderma, cotyledon primordium and vascular tissue.

Identification and Characterization of MicroRNAs and Their Targets in Grapevine(Vitis vinifera)

MicroRNAs （miRNAs） are a class of newly identified, small, non-coding RNAs that play vital roles in regulation. Based on miRNAs unique features of expression pattern, evolutionary conservation, secondary structure and genetic requirements for biogenesis, computational predication strategy is adopted to predicate the novel miRNAs. In this research, potential miRNAs and their targets in grapevine （Vitis vinifera） were predicted. We used previously known plant miRNAs against grapevine genome sequence databases to search for potential miRNAs. A total of 81 potential miRNAs were detected following a range of strict filtering criteria. Using these potential miRNA sequences, we could further blast the mRNA database to find the potential targets in this species. Comparative analysis of miRNAs in grapevine and other species reveals that miRNAs exhibit an evolutional conservation, the number and function of miRNAs must have significantly expanded during the evolution of land plants. Furthermore divergence made versatile functions of miRNAs feasible. Cluster of miRNAs likely represents an ancient expression mechanism. Predicted target genes include not only transcription factors but also genes implicated in floral development, signal transduction, diseases and stress response. Till now, little is known about experimental or computational identification of miRNA in grapevine species. Increased knowledge of the biological mechanisms of the grapevine will allow targeted approaches to increase the quality of fruit and reduce the impact of parasites together with stress, which could enable a sustainable, environmentally-sound, farming policv.

Effects of Partial Rootzone Drying on the Growth of Vitis vinifera cv.Malvasia Grafted on Different Rootstocks

To lay a biological foundation for rootstocks and alternate irrigation （AI） popularization, the effects of partial rootzone drying （PRD） on the growth of the grapevine Malvasia grafted on different rootstocks were investigated. Biological effects of 1/2 divided root irrigation on three combinations, i.e., Malvasia/420A, Malvasia/3309C, and Malvasia/110R, were studied by wood-boxed plants. All the plants were separated into three groups for different irrigation strategies. Mass growth of new root in alternate-irrigated plants was remarkably promoted by about 7.8-22.2% higher than the well- watered ones. However, new shoot growth, especially the internode was reduced by alternate irrigation. The average root-shoot ratio of all the three combinations was increased from 1.1 to 1.46. New root growth and internode length were decreased by fixed partial rootzone irrigation （FI） at different amount, M/3309C at 37.9 and 36.9%, M/110R at 18.4 and 22.5%, respectively. Total biomass of all the three combinations under FI decreased at the rate of 19.2-34.3% compared with well-watered ones. Water stress adaptation of grapevine mainly depends on rootstock. 110R is more efficient than 3309C and 420A in water stress adaptation. PRD-AI benefited root growth, thus improved the drought-resistant ability of grapevine.

Comparative metabolic profiling of Vitis amurensis and Vitis vinifera during cold acclimation

Vitis amurensis is a wild Vitis plant that can withstand extreme cold temperatures.However,the accumulation of metabolites during cold acclimation(CA)in V.amurensis remains largely unknown.In this study,plantlets of V.amurensis and V.vinifera cv.Muscat of Hamburg were treated at 4℃ for 24 and 72 h,and changes of metabolites in leaves were detected by gas chromatography coupled with time-of-flight mass spectrometry.Most of the identified metabolites,including carbohydrates,amino acids,and organic acids,accumulated in the two types of grape after CA.Galactinol,raffinose,fructose,mannose,glycine,and ascorbate were continuously induced by cold in V.amurensis,but not in Muscat of Hamburg.Twelve metabolites,including isoleucine,valine,proline,2-oxoglutarate,and putrescine,increased in V.amurensis during CA.More galactinol,ascorbate,2-oxoglutarate,and putrescine,accumulated in V.amurensis,but not in Muscat of Hamburg,during CA,which may be responsible for the excellent cold tolerance in V.amurensis.The expression levels of the genes encodingβ-amylase(BAMY),galactinol synthase(GolS),and raffinose synthase(RafS)were evaluated by quantitative reverse transcription-PCR.The expression BAMY(VIT_02s0012 g00170)and RafS(VIT_05s0077 g00840)were primarily responsible for the accumulation of maltose and raffinose,respectively.The accumulation of galactinol was attributed to different members of GolS in the two grapes.In conclusion,these results show the inherent differences in metabolites between V.amurensis and V.vinifera under CA.

The potential role of grape (Vitis vinifera L.) in prevention of threatened abortion via immunomodulatory and anti-inflammatory abilities: a hypothesis

Threatened abortion is a common problem in early pregnancy. This early vaginal bleeding happens in about 25% of pregnant women. The medications including progesterone, uterine muscle relaxant or human chorionic gonadotropin have essential effects in developing pregnancy, but the clinical data are insufficient to prescribe them. In recent decades, medicinal herbs can help us to present new treatments. Grape (Vitis vinifera L.) can protect the fetus from the perspective of Persian medicine. So, we hypothesize about the salutary effects of grape in miscarriage prevention. We found five standard expected mechanisms of grape to prevent threatened abortion: immunomodulatory and anti-inflammatory, anti-oxidant, anti-contraction, hormonal and anti-stress activities. Grape reduces nitric oxide, prostaglandin E2, expression of nuclear factor κB and other pro-inflammatory cytokines like IL (Interleukin)-1β, IL-6, IL-8, and tumor necrosis factor-α. It also elevates anti-inflammatory mediators and expression of peroxisome proliferator-activated receptor-γ. Grape polyphenols have a crucial role in fetus protection with high antioxidant power and other functions such as prevention of stress-triggered abortion with proanthocyanidins, or hormonal effects and inhibition of uterine contractions with resveratrol. So according to these studies, grape probably has effects on the immune and endocrine factors involved in threatened miscarriage.

Cloning and Characterization of WOX4 Gene from Vitis vinifera L. Involved in Stem Cell Regulation

Wuschel-related homeobox （WOX） genes play essential, specific, and sometimes redundant roles in plant embryo development, shoot and root meristem maintenance, and plant development. Though much information was quickly gained with members of the WOX gene family of Arabidopsis, monocotyledonous crops, and gymnospermous conifers, little is known about perennial woody plants. In this study, we isolated the first WOXgene family member from grape （Vitis vinifera L. cv. Cabernet Sauvignon）, and named it VvWOX4 based on its characteristic domains and phylogenetic analysis. The identity of VvWOX4 was validated by MALDI-TOF MS and Western blot with polyclonal antibody against Arabidopsis thaliana Wuschel. Functional analysis showed that VvWOX4 markedly increased shoot primordia structures when overexpressed under CaMV 35S promoter in tobacco. A different expression pattern was found for VvVOX4 compared with AtWUCHEL and its expression was detected in unique organs of grapevines. Besides the expression in the vegetative shoot apical meristem （SAM） of grape shoot tips, VvWOX4 is expressed in dormant winter buds, inflorescence, young leaves, and tendril tips, but not in root tips. In young leaves, the expression of VvWOX4 is strongly upregulated by wounding, and also by plant growth regulators such as 2 mg L-1 2,4-D, 1 mg L-1 NAA and 1 mg L-1 BAP treatments, while downregulation was monitored by 1 mg L^-1 IBA treatment, and there was no response to 0.5 mg L-1 GA3 treatment. Together, our results revealed the first member of grape WOX gene family and indicated different roles and regulation of VvWOX4 in the perennial woody crop grapevine.

Divergent molecular and growth responses of young“Cabernet Sauvignon”(Vitis vinifera)plants to simple and mixed infections with Grapevine rupestris stem pitting-associated virus

Grapevine rupestris stem pitting associated virus(GRSPaV)is one of the most widely distributed viruses;even so,little is known about its effect on Vitis vinifera.To provide new insights,the effects of single and mixed GRSPaV infections on the V.vinifera cultivar“Cabernet Sauvignon”were studied by evaluating growth parameters,such as measurements of the total plant length,the number and distance of internodes and the number of leaves per shoot.In addition,parameters relating to gas exchange,i.e.,the stomatal conductance,net photosynthetic rate,internal CO2 concentration and leaf transpiration,were also assessed.All the measurements were performed in one-and two-yearold plants with a single GRSPaV infection or mixed infections of GRSPaV and Grapevine fanleaf virus(GFLV).The results show that the plant phytosanitary status did not significantly alter the growth and gas exchange parameters in oneyear-old plants.However,in two-year-old plants,single GRSPaV infections increased shoot elongation,which was accompanied by the overexpression of genes associated with the gibberellic acid response pathway.The gas exchange parameters of these plants were negatively affected,despite exhibiting higher LHCII gene expression.Plants with mixed infections did not have modified growth parameters,although they presented a greater reduction in the primary photosynthetic parameters evaluated with no change in LHCII expression.The results presented here confirm the co-evolution hypothesis for V.vinifera and GRSPaV during the early stages of plant development,and they provide new evidence about the effects of GRSPaV and GFLV co-infections on the“Cabernet Sauvignon”cultivar.

CRISPR/Cas9-mediated mutagenesis of VvMLO3 results in enhanced resistance to powdery mildew in grapevine(Vitis vinifera)

Grapevine(Vitis vinifera),one of the most economically important fruit crops in the world,suffers significant yield losses from powdery mildew,a major fungal disease caused by Erysiphe necator.In addition to suppressing host immunity,phytopathogens modulate host proteins termed susceptibility(S)factors to promote their proliferation in plants.In this study,CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR-associated 9)technology was used to enable the targeted mutagenesis of MLO(mildew resistance Locus O)family genes that are thought to serve as S factors for powdery mildew fungi.Small deletions or insertions were induced in one or both alleles of two grapevine MLO genes,VvMLO3 and VvMLO4,in the transgenic plantlets of the powdery mildew-susceptible cultivar Thompson Seedless.The editing efficiency achieved with different CRISPR/Cas9 constructs varied from 0 to 38.5%.Among the 20 VvMLO3/4-edited lines obtained,one was homozygous for a single mutation,three harbored biallelic mutations,seven were heterozygous for the mutations,and nine were chimeric,as indicated by the presence of more than two mutated alleles in each line.Six of the 20 VvMLO3/4-edited grapevine lines showed normal growth,while the remaining lines exhibited senescence-like chlorosis and necrosis.Importantly,four VvMLO3-edited lines showed enhanced resistance to powdery mildew,which was associated with host cell death,cell wall apposition(CWA)and H2O2 accumulation.Taken together,our results demonstrate that CRISPR/Cas9 genome-editing technology can be successfully used to induce targeted mutations in genes of interest to improve traits of economic importance,such as disease resistance in grapevines.

Not All Shrivels Are Created Equal—Morpho-Anatomical and Compositional Characteristics Differ among Different Shrivel Types That Develop during Ripening of Grape (<i>Vitis vinifera</i>L.) Berries

An understanding of physiological disorders associated with ripening of fruits triggered by abiotic stress relies on anatomical and physico-chemical analyses, as it provide insights into their origin and probable causes. The objective of this study was to analyze different ripening disorders of grape (Vitis vinifera L.) berries by dissecting their morpho-anatomy, shriveling nature, and composition. Four well-defined disorders—sunburn, prolonged dehydration (PD), late-season bunch stem necrosis (LBSN), and berry shrivel (BS) were analyzed in field-grown grapevines of the cultivar Cabernet Sauvignon. Early bunch stem necrosis (EBSN) that occurred before ripening was also included in the study. Unlike healthy spherical berries, the pericarp of disordered berries except for sunburn shriveled causing concomitant reductions in fresh weight and volume. The exocarp of PD berries developed well-ordered indentations as distinct from the wrinkles in LBSN berries, whereas BS berries were flaccid with numerous skin folds. The epicuticular wax occurred as upright platelets in all shrivel forms excluding the sun-exposed hemisphere of sunburned berries. A chlorophyllous inflorescence framework persisted in all shrivel forms but in LBSN, wherein the necrotic regions developed tylosis. Unlike the translucent mesocarp of healthy, sunburned, and PD berries, the mesocarp was collapsed in BS and LBSN berries, nevertheless all had well-developed seeds. The composition of healthy berries was optimal, whereas the disordered berries were compositionally distinct from each other, which as a whole differed from the healthy berries. The BS berries had the lowest sugar content, and although sugar concentration was higher in LBSN, sunburned and PD berries, sugar amount per berry was highest in the healthy berries, the same was true for hexoses. Healthy and BS berries exhibited highest amounts of tartaric acid followed by sunburn and PD berries, whereas the LBSN berries had the lowest values. Conversely, healthy and PD berries had the highest amounts of malic acid followed by LBSN, sunburn and BS berries, which collectively displayed similar amounts. The PD berries exhibited the highest calcium content followed by LBSN, healthy, and finally BS and sunburned berries. A linear relationship existed between potassium (K) and pH of the berries. The PD berries had the highest amounts of K followed by healthy, sunburn, LBSN, and BS berries. Overall, the results reported here provided combined morpho-anatomical and compositional analyses of different shrivel types that occurred during a single growing season. Such analysis is needed to make a progress on understanding these ripening disorders culminating in the development of remedial measures.

Understanding Differential Responses of Grapevine (Vitis vinifera L.) Leaf and Fruit to Water Stress and Recovery Following Re-Watering

Among all fruit crops of horticultural importance, grapevines (Vitis vinifera L.) stand out as the most drought tolerant crop species whose tolerance is credited to their proficiency to recover from water stress in both the natural and vineyard growing conditions. However, information on the recovery responses is relatively scant. Studies were conducted to address this issue using potted vines of the grapevine cultivar, Cabernet Sauvignon, which was subjected to water stress and along with anatomical and ultrastructural characterizations, physiological status was assessed in healthy and water stressed vines, and following recovery via rewatering from the water stressed vines. Water stress induced wilting of leaves, drooping of tendrils, and desiccation followed by abscission of shoot tip leaving behind a brown scar at the shoot apex. The wilted leaves accumulated ABA, which correspondingly reduced stomatal conductance and leaf water potential. Upon re-watering, both these parameters made a recovery with values similar to healthy leaves. Likewise, leaf anatomical features following rewatering resembled to that of healthy leaves. In clusters, water stress caused shriveling of preveraison (unripened) berries, which regained full turgor following water resupply, whereas the postveraison (ripening) berries in the same cluster remained unaffected as evidenced by the presence of viable mesocarp cells and epicuticular wax in the form of platelets. The study revealed that shoot tip with leaf primordia was most sensitive to water stress followed by fully expanded leaves and preveraison berries, whereas the postveraison berries remained unaffected. This information could be valuable to implementing irrigation strategies towards sustaining grape production in existing vineyards experiencing episodic droughts and targeted areas prone to drought.

CRISPR/Cas9-mediated VvPR4b editing decreases downy mildew resistance in grapevine(Vitis vinifera L.)

Downy mildew of grapevine(Vitis vinifera L.),caused by the oomycete pathogen Plasmopara viticola,is one of the most serious concerns for grape production worldwide.It has been widely reported that the pathogenesis-related 4(PR4)protein plays important roles in plant resistance to diseases.However,little is known about the role of PR4 in the defense of grapevine against P.viticola.In this study,we engineered loss-of-function mutations in the VvPR4b gene from the cultivar“Thompson Seedless”using the CRISPR/Cas9 system and evaluated the consequences for downy mildew resistance.Sequencing results showed that deletions were the main type of mutation introduced and that no off-target events occurred.Infection assays using leaf discs showed that,compared to wild-type plants,the VvPR4b knockout lines had increased susceptibility to P.viticola.This was accompanied by reduced accumulation of reactive oxygen species around stomata.Measurement of the relative genomic abundance of P.viticola in VvPR4b knockout lines also demonstrated that the mutants had increased susceptibility to the pathogen.Our results confirm that VvPR4b plays an active role in the defense of grapevine against downy mildew.