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Proanthocyanidins (PAs) are an important group of flavonoids that contribute to astringency, color, and flavor in grapes (Vitis vinifera) and wines. They also play a crucial role in enhancing plant resistance to various stresses. However, the underlying regulatory mechanism governing PAs biosynthesis, particularly in relation to conferring resistance to powdery mildew, has not been extensively explored. This study focused on identifying a key player in PAs biosynthesis, namely the plant U-box (PUB) E3 ubiquitin ligase VvPUB26. We discovered that overexpression of VvPUB26 in grapes leads to a significant increase in PAs content, whereas interfering with VvPUB26 has the opposite effect. Additionally, our findings demonstrated that overexpression of VvPUB26 in transgenic grapevines enhances defense against powdery mildew while interfering with VvPUB26 results in increased susceptibility to the pathogen. Interestingly, we observed that VvPUB26 interacts with the WRKY transcription factor VvWRKY24, thereby facilitating ubiquitination and degradation processes. Through RNA-Seq analysis, we found that VvWRKY24 primarily participates in secondary metabolites biosynthesis, metabolic pathways, and plant-pathogen interaction. Notably, VvWRKY24 directly interacts with the promoters of dihydroflavonol-4-reductase (DFR) and leucoanthocyanidin reductase (LAR) to inhibit PAs biosynthesis. Meanwhile, VvWRKY24 also influences the expression of MYB transcription factor genes related to PAs synthesis. In conclusion, our results unveil a regulatory module involving VvPUB26-VvWRKY24-VvDFR/VvLAR that plays a fundamental role in governing PAs biosynthesis in grapevines. These findings enhance our understanding of the relationship between PAs biosynthesis and defense mechanisms against powdery mildew., Competing Interests: Conflict of interest statement. The authors have no conflict of interest to declare., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Chitinases (Chi), an important resistance-related protein, act against fungal pathogens by catalyzing the fungal cell wall, whereas are involved in different biological pathways in grape. In this study, we found 42 Chi family genes in Vitis vinifera L. (VvChis) and evaluated their expression levels after Botrytis infection, stress hormones like ethylene (ETH) and methyl-jasmonate (MeJA), and abiotic stresses like salinity and temperature changes in ripened fruits. VvChis were categorized into five groups including A, B, C, D, and E belonged to glycoside hydrolase family 18 and 19 (GH18 and GH19) according to genes structure, which expression analysis showed distinct temporal and spatial expression patterns changed in different tissues and various development stages. Different responsive elements to biotic and abiotic stresses were determined in the promoter regions of VvChis, specially elicitor-responsive element that was conserved among all VvChis genes. The expression levels of VvChis in groups A, B, and E increased after Botrytis cinerea infection in leaves and berries. Meanwhile, VvChis in glycoside hydrolase family 18 (GH18) were up-regulated under MeJA and ETH treatment, although the induction of VvChis by low temperature was more significant than high temperature. The expression of VvChis was also positively correlated with the concentration of NaCl treatment. Furthermore, differential gene-overexpression of VvChi5, VvChi17, VvChi22, VvChi26, and VvChi31 in strawberry and tomato fruits demonstrated the involvement of various isoforms in resistance to Botrytis infection through antioxidant system and lignin accumulation, which led to a reduction of damage. Among different isoforms of VvChis, we confirmed the interaction of Chi17 with Metallothionein (MTL) as oxidative stress protection, which suggests VvChis can modulate oxidative stress during postharvest storage in ripened fruits.

Main Conclusion: In grape (Vitis), stilbene phytoalexins can either be in situ synthesized or transported to the site of response during powdery mildew infection, enhancing disease resistance. Resveratrol is a phytoprotective stilbenoid compound that is synthesized by stilbene synthase (STS) in response to biotic and abiotic stresses, and is also known to have health benefits in the human diet. We have previously shown that transgenic Vitis vinifera cv. Thompson Seedless plants overexpressing a stilbene synthase gene, VqSTS6, from wild Chinese Vitis quinquangularis had a higher stilbenoid content, leading to an enhanced resistance to powdery mildew (Uncinula necator (Schw.) Burr). However, the biosynthesis and transportation in the plant tissue under powdery mildew infection are still unclear. Here, inhibitor and micro-grafting technologies were used to study the accumulation of resveratrol following powdery mildew infection. We observed that the levels of STS expression and stilbenoids increased in response to powdery mildew infection. Powdery mildew and inhibitor treatment on detached grape branches showed that resveratrol was in situ synthesized. Experiments with grafted plantlets showed that the abundance of stilbenoid compounds increased in the shoot during VqSTS6 overexpression in the root, while VqSTS6-Flag fusion was not tranported to the scions and only expressed in the transgenic rootstocks. Compared with wild-type Thompson Seedless plants, the non-transgenic/VqSTS6 transgenic (scion/rootstock) grafted Thompson Seedless plantlets exhibited increased resistance to powdery mildew. In addition, overexpression of VqSTS6 in roots led to increased levels of stilbenoid compounds in five other European grape varieties (V. vinifera cvs. Chardonnay, Perlette, Cabernet Sauvignon, Riesling and Muscat Hamburg). In conclusion, stilbenoid compounds can be either in situ synthesized or transported to the site of powdery mildew infection, and overexpression of VqSTS6 in the root promotes stilbenoids accumulation and disease resistance in European grapevine varieties.

Background: Melatonin is a ubiquitous molecule and exists across kingdoms. Studies on melatonin in plants have mainly focused on its physiological influence on growth and development, and on its biosynthesis. A number of studies have been conducted on the melatonin content and exogenous melatonin treatment of grapevine (Vitis vinifera L.). However, key genes or enzymes of the melatonin biosynthetic pathway remain unclear., Results: In this study, we cloned and identified the gene encoding serotonin N-acetyltransferase (SNAT) in grapevine (VvSNAT2). The VvSNAT2 protein was identified from a collection of 30 members of the grapevine GCN5-related N-acetyltransferase (GNAT) superfamily. Phylogenetic and protein sublocalization analyses showed that the candidate gene VvGNAT16 is VvSNAT2. Characterization of VvSNAT2 showed that its enzymatic activity is highest at a pH of 8.8 and a temperature of 45 °C. Analysis of enzyme kinetics showed the values of K m and V max of VvSNAT2 using serotonin were 392.5 μM and 836 pmol/min/mg protein, respectively. The expression of VvSNAT2 was induced by melatonin treatment and pathogen inoculation. Overexpression of VvSNAT2 in Arabidopsis resulted in greater accumulation of melatonin and chlorophyll and enhanced resistance to powdery mildew in the transgenic plants compared with the wild type (WT). Additionally, our data showed that the marker genes in the salicylic acid (SA) signaling pathway were expressed to higher levels in the transgenic plants compared with the WT., Conclusions: The VvSNAT2 gene was cloned and identified in grapevine for the first time. Our results indicate that VvSNAT2 overexpression activates the SA and JA signaling pathways; however, the SA pathway plays a central role in VvSNAT2-mediated plant defense.

Sour rot, caused by Geotrichum citriaurantii (G. citri-aurantii.), is one of the most serious grapevine secondary infection diseases in China. We have determined that pterostilbene, the most important phytoalexin, effectively inhibits the activity of G. citri-aurantii. To study the synthesis mechanism of pterostilbene against G. citri-aurantii in grape, we initially detected the content of pterostilbene present in grapes infected by G. citri-aurantii with the use of UHPLC-QQQ-MS 2 . Pterostilbene levels are controlled by the resveratrol O-methyltransferase (ROMT) gene, and within grape samples is positively related to the accumulation of resveratrol. The pPIC9k-ROMT vector and plasmid pCAMBIA2300-GFP-ROMT were constructed for expression purposes. The pPIC9k-ROMT vector was expressed in Pichia pastoris GS115 and plasmid pCAMBIA2300-GFP-ROMT was expressed in onion. Analysis of qRT-PCR amplification samples revealed that gene expression was induced rapidly in grape as a defense against G. citri-aurantii. Western blot analysis verified that the recombinant protein was successfully expressed. PRACTICAL APPLICATIONS: Pterostilbene is a trace and efficient phytoalexins produced by plant' secondary metabolism, which exhibits good pharmacological activity. As an important protective agent in plants, it can improve the antioxidant capacity and resistance to adversity of plants. However, the method which could be used for mass production of pterostilbene has not been reported currently. The key gene of pterostilbene biosynthesis was investigated and verified in this paper, which provides the theoretical basis for the industrial production of pterostilbene. The study of pterostilbene is significant for the prevention and treatment of G. citri-aurantii disease, and has important practical applications for the development and production of pesticides., (© 2019 Wiley Periodicals, Inc.)

Background: To limit the impact of the downy mildew disease of grapevine and reduce the need to recur to chemical treatments, an effective strategy might be recovering adaptive resistance traits in both cultivated and wild V. vinifera germplasm. Considering that stilbenes represent the most important class of phytoalexins in the Vitaceae, the constitutive expression and transcriptional activation of all the functional members of the stilbene synthase gene family were analysed in a group of nine grapevine genotypes following artificial infection with the oomycete Plasmopara viticola, the causal agent of the disease. In addition, in the same genotypes we analyzed the expression of genes encoding for two transcription factors involved in the transcriptional regulation of the stilbene synthase genes, namely VvMYB14 and VvMYB15, and of genes encoding for chalcone synthases., Results: Downy mildew incidence and severity ranged from nihil to high in the grapevine genotypes considered, being low to moderate in a subgroup of V. vinifera genotypes. The constitutive expression of the stilbene synthase genes as well as the extent of their transcriptional activation following P. viticola inoculation appeared to be inversely related to the proneness to develop disease symptoms upon infection. In a specular manner, following P. viticola inoculation all the chalcone synthase genes were up-regulated in the susceptible grapevine genotypes and down-regulated in the resistant ones. The infection brought by P. viticola appeared to elicit a co-ordinated and sequential transcriptional activation of distinct stilbene synthase genes subsets, each of which may be regulated by a distinct and specific MYB transcription factor., Conclusions: The present results suggest that the induction of stilbene biosynthesis may contribute to the basal immunity against the downy mildew of grapevine, thus representing an adaptive resistance trait to recover, in both cultivated and wild V. vinifera germplasm. During the early stages of P. viticola infection, an antagonistic interaction between flavonol and stilbene biosynthesis might occur, whose outcome might determine the subsequent extent of disease symptoms. Further studies are needed to decipher the possible regulatory mechanisms involved in the antagonistic crosstalk between these two metabolic pathways in resistant and susceptible genotypes in response to P. viticola.

Chitin, a major component of fungal cell walls, is a well-known pathogen-associated molecular pattern (PAMP) that triggers defense responses in several mammal and plant species. Here, we show that two chitooligosaccharides, chitin and chitosan, act as PAMPs in grapevine (Vitis vinifera) as they elicit immune signalling events, defense gene expression and resistance against fungal diseases. To identify their cognate receptors, the grapevine family of LysM receptor kinases (LysM-RKs) was annotated and their gene expression profiles were characterized. Phylogenetic analysis clearly distinguished three V. vinifera LysM-RKs (VvLYKs) located in the same clade as the Arabidopsis CHITIN ELICITOR RECEPTOR KINASE1 (AtCERK1), which mediates chitin-induced immune responses. The Arabidopsis mutant Atcerk1, impaired in chitin perception, was transformed with these three putative orthologous genes encoding VvLYK1-1, -2, or -3 to determine if they would complement the loss of AtCERK1 function. Our results provide evidence that VvLYK1-1 and VvLYK1-2, but not VvLYK1-3, functionally complement the Atcerk1 mutant by restoring chitooligosaccharide-induced MAPK activation and immune gene expression. Moreover, expression of VvLYK1-1 in Atcerk1 restored penetration resistance to the non-adapted grapevine powdery mildew (Erysiphe necator). On the whole, our results indicate that the grapevine VvLYK1-1 and VvLYK1-2 participate in chitin- and chitosan-triggered immunity and that VvLYK1-1 plays an important role in basal resistance against E. necator., (© 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

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., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The deposition of callose is a common plant defense response to intruding pathogens and part of the plant's innate immunity. In this study, eight grapevine callose synthase (CalS) genes were identified and characterized. To investigate biological function of CalS in grapevine against the infection of Plasmopara viticola, expression patterns of grapevine CalS family genes were analyzed among resistant/susceptible cultivars. After P. viticola infection, expression of CalS1, 3, 7, 8, 9, 10, and 11 were significantly modified among the grapevine cultivars. For example, the expression of CalS1 and CalS10 were greatly increased in downy mildew (DM)-immune Muscadinia rotundifolia 'Carlos' and 'Noble'. Transient expression assay with promoters of the CalS1 and CalS10 genes confirmed that they were regulated by the oomycete pathogen P. viticola. CalS1 promoter activity was also significantly up-regulated by ABA in DM-immune M. rotundifolia 'Noble', but down-regulated in DM-susceptible Vitis vinifera 'Chardonnay'. The CalS1 promoter, however, was also down-regulated by GA in 'Chardonnay', but not affected in 'Noble'. The promoter activity of CalS10 was significantly up-regulated by GA in 'Chardonnay', but not regulated by ABA at all. It is proposed that CalS1 and CalS10 were involved in grapevine defense against DM disease.

Abiotic factors inducing osmotic stress can influence the plant immune response and resistance to pathogen infections. In this study, the effect of polyethylene glycol (PEG)- and sucrose-induced osmotic stress on polyamine (PA) homeostasis and the basal immune response in grapevine plantlets before and after Botrytis cinerea infection was determined. Pharmacological approaches were also addressed to assess the contribution of osmotic stress-induced PA oxidation to the regulation of defence responses and the susceptibility of grapevine to B. cinerea. Following osmotic stress or pathogen infection, PA homeostasis was linked to enhanced activity of diamine oxidases (CuAO) and PA oxidases (PAO) and the production of 1,3-diaminopropane. These responses paralleled the accumulation of the main stilbenic phytoalexins, resveratrol and ε-viniferin and upregulation of gene transcripts including STS (a stilbene synthase), PR-2 (a β-1,3-glucanase), PR3-4c (acidic chitinase IV), and PR-5 (a thaumatin-like protein), as well as NCED2 involved in abscisic acid biosynthesis. It was also demonstrated that leaves pre-exposed to osmotic stress and later inoculated with B. cinerea showed enhanced PA accumulation and attenuation of CuAO and PAO activities. This was consistent with the impaired production of phytoalexins and transcript levels of defence- and stress-related genes following infection, and the enhanced susceptibility to B. cinerea. Pharmacological experiments revealed that, under osmotic stress conditions, CuAO and PAO were involved in PA homeostasis and in the regulation of defence responses. Specific inhibition of CuAO and PAO in osmotically stressed leaves strongly attenuated the induction of defence responses triggered by B. cinerea infection and enhanced susceptibility to the pathogen. Taken together, this study reveals a contribution of PA catabolism to the resistance state through modulation of immune response in grapevine following osmotic stress and/or after B. cinerea infection.

Aldehyde dehydrogenases (ALDHs) catalyze the irreversible oxidation of a broad spectrum of reactive aldehydes to their corresponding carboxylic acids. Although the proteins have been studied from various organisms and at different growth stages in plants, their potential roles in pathogen infection have not been examined. Here we isolated and functionally characterized a pathogen-inducible ALDH gene (VpALDH2B4) from Chinese wild grapevine Vitis pseudoreticulata accession Baihe-35-1. When transiently expressed in Arabidopsis leaves, VpALDH2B4 was found to be localized in mitochondria. Escherichia coli expressed GST-VpALDH2B4 exhibited ALDH activity in vitro and was capable of utilizing malondialdehyde (MDA), acetaldehyde and glyceraldehydes as its substrate. Over-expression of VpALDH2B4 in Arabidopsis resulted in hypersensitive response-like cell death, enhanced resistance to downy mildew and powdery mildew presumably via the SA-signaling pathway. The same Arabidopsis transgenic plants also showed enhanced tolerance to salt stress, which is accompanied by less MDA accumulation and upregulation of the stress-responsive superoxide dismutase activity. Taken together, our results suggest that VpALDH2B4 and perhaps its orthologous genes may be involved in responses of plants to stresses imposed by both biotrophic pathogens and high salinity conditions.

The oomycete Plasmopara viticola is responsible for downy mildew, a severe grapevine disease. In infected grapevine leaves, we have observed an abnormal starch accumulation at the end of the dark period, suggesting modifications in starch metabolism. Therefore, several complementary approaches, including transcriptomic analyses, measurements of enzyme activities, and sugar quantification, were performed in order to investigate and to understand the effects of P. viticola infection on leaf starch and-to a larger extent-carbohydrate metabolism. Our results indicate that starch accumulation is associated with an increase in ADP-glucose pyrophosphorylase (AGPase) activity and modifications in the starch degradation pathway, especially an increased α-amylase activity. Together with these alterations in starch metabolism, we have observed an accumulation of hexoses, an increase in invertase activity, and a reduction of photosynthesis, indicating a source-to-sink transition in infected leaf tissue. Additionally, we have measured an accumulation of the disaccharide trehalose correlated to an increased trehalase gene expression and enzyme activity. Altogether, these results highlight a dramatic alteration of carbohydrate metabolism correlated with later stages of P. viticola development in leaves.

Vitis pseudoreticulata glyoxal oxidase (VpGLOX) was previously isolated from the Chinese wild vine V. pseudoreticulata accession "Baihe-35-1" during a screen for genes that are upregulated in response to infection with grapevine powdery mildew (Erysiphe necator, PM). In the present study, a possible function of VpGLOX for defense against PM was investigated using Agrobacterium-mediated transient expression. After optimizing agro-infiltration, VpGLOX was transiently overexpressed in leaves of either PM-susceptible (accession "6-12-2") or PM-resistant (accession "6-12-6") plants. The efficiency of transfection was verified using a β-glucuronidase (GUS) reporter and was found to comprise most leaf areas regardless of the initial leaf position. Upon infection with E. necator, clear differences were observed with respect to hyphal development between agro-infiltrated leaves and control groups of both, the susceptible and the resistant, genotypes. The expression of VpGLOX was followed by real-time polymerase chain reaction in both genotypes. Whereas in the susceptible host ("6-12-2") expression was found to increase only in transfected leaves and remained transient, in the resistant host ("6-12-6"), a second peak appeared later in transfected leaves, probably representing the response of the endogenous VpGLOX. The data support the interpretation that VpGLOX is sufficient to confer resistance to E. necator.

The most devastating disease currently threatening to destroy the banana industry worldwide is undoubtedly Sigatoka Leaf spot disease caused by Mycosphaerella fijiensis. In this study, we developed a transformation system for banana and expressed the endochitinase gene ThEn-42 from Trichoderma harzianum together with the grape stilbene synthase (StSy) gene in transgenic banana plants under the control of the 35S promoter and the inducible PR-10 promoter, respectively. The superoxide dismutase gene Cu,Zn-SOD from tomato, under control of the ubiquitin promoter, was added to this cassette to improve scavenging of free radicals generated during fungal attack. A 4-year field trial demonstrated several transgenic banana lines with improved tolerance to Sigatoka. As the genes conferring Sigatoka tolerance may have a wide range of anti-fungal activities we also inoculated the regenerated banana plants with Botrytis cinerea. The best transgenic lines exhibiting Sigatoka tolerance were also found to have tolerance to B. cinerea in laboratory assays.

Grapevine species (Vitis sp.) are prone to several diseases, fungi being the major pathogens compromising its cultivation and economic profit around the world. Knowledge of the complexity of mechanisms responsible for resistance to fungus infection of cultivars, such as Regent, is necessary for strategies to be defined which will improve resistance in highly susceptible crop species. Transcript and metabolic profiles of the Vitis vinifera cultivars Regent and Trincadeira (resistant and susceptible to fungi, respectively) were analysed by cDNA microarray, quantitative real-time PCR, and nuclear magnetic resonance spectroscopy. The integration of datasets obtained through transcriptome and metabolome analysis revealed differences in transcripts and metabolites between both cultivars. These differences are probably associated with the innate resistance of Regent towards the mildews. Several transcripts related to stress and defence, namely a subtilisin-like protease, phenylalanine ammonia lyase, S-adenosylmethionine synthase, WD-repeat protein like, and J2P, were up-regulated in Regent suggesting an intrinsic resistance capability of this cultivar. A metabolic profile revealed an accumulation of compounds such as inositol and caffeic acid, which are known to confer resistance to fungi. The differences in transcripts and metabolites detected are discussed in terms of the metabolic pathways and their possible role in plant defence against pathogen attack, as well as their potential interest to discriminate among resistant and susceptible grapevine cultivars.