Grapevine Gene Systems for Resistance to Gray Mold Botrytis cinerea and Powdery Mildew Erysiphe necator

Grapevine is one of the world’s most economically important fruit crops. It is known that Vitis vinifera is a host for a large number of pathogenic agents, which significantly reduce the yield and berry quality. This forces the agronomists to use a huge amount of fungicides. Over the last few decades, alternative methods for solving this problem have been developed and continue to be developed. Such new technologies as marker-assisted selection, bioengineering of the rhizosphere, genetic engineering (transgenesis, cisgenesis and intragenesis) allow the production of pathogen-resistant cultivars. However, they are linked to a number of problems. One of the most promising methods is the creation of modified non-transgenic cultivars via CRISPR/Cas9-targeted mutagenesis. Therefore, researchers are actively looking for target genes associated with pathogen resistance and susceptibility. This review elucidates the main mechanisms of plant—pathogen interactions, the immune systems developed by plants, as well as the identified genes for resistance and susceptibility to the biotrophic pathogen Erysiphe necator and the necrotrophic pathogen Botrytis cinerea.


Introduction
The grape family (Vitaceae) includes about 16 genera and 900 species [1][2][3][4]. The genus Vitis L. includes over 70 woody climber species, spread mostly in the temperate regions of the northern hemisphere. These species are classified into 3 groups of origin: European/West Asian; East Asian; American. The European/West Asian group includes only one species-Vitis vinifera L., which includes almost all varieties cultivated for high quality fruits. However, these cultivars are characterized as being unstable to abiotic and biotic factors. The East Asian and American groups include all the rest species of grapes. They are mostly of no practical value due to the low quality of the fruit and still remain poorly studied [1,3,5]. Two subgenera of Vitis have been commonly recognized: (1) subg. Vitis (2n = 38) comprises the vast majority of species and is distributed in northern South America, Central and North America, Asia, and Europe. (2) subg. Muscadinia (2n = 40) includes only 2-3 species and its distribution includes only the southeastern United States, northeastern Mexico, Belize, Guatemala, and the Caribbean [1][2][3]5,6]. The recognition of the two distinct subgenera, defined on the basis of morphological, karyological and cytological differences, is still debated [3,5]. The grape production is widespread throughout the world. Grapevine is one of the economically most important crops yielding berries and is mainly used for wine production [7,8]. According to data from the Food and Agriculture Organization (FAO, 2019), its cultivation area covers over 7.2 million hectares and the total grapes in the Russian Federation have been increasing annually over the past 10 years (Figure 1).
One of the main global problems of viticulture is the decrease in quantity and quality of harvest due to biotic stresses, mainly caused by bacteria, fungi and oomycetes, which causes severe diseases of grapes, including gray mold (caused by Botrytis cinerea Pers: Fr.), downy mildew (caused by Plasmopara viticola de Bary), powdery mildew (caused by Erysiphe necator (Schw.) Burr), also formerly known as Uncinula necator (Schwein.) Burr.) and grape black rot (Guignardia bidwellii (Ellis)). The majority of European grapevine cultivars of V. vinifera are susceptible to pathogen attack [10][11][12].
Gray mold causes significant economic losses in viticulture all around the world, reaching 20-50% of yield losses in grapes, due to rotting of ripe bunches in the post-harvest period. The high relative humidity and moderate temperatures during the grapevine vegetative cycle favour the development of this fungal pathogen [13]. The causative agent of the disease is Botrytis cinerea, a necrotrophic fungus with a short biotrophic phase. B. cinerea infects more than 1400 different plant species [14]. In the vineyard, this pathogen is a part of the microflora and usually lives in the soil on dead plant parts [15]. The disease infects fruits during ripening, causing necrotic areas with extensive fungal growth, which gives the characteristic appearance of gray rot. As a consequence, the grapes become unsuitable for wine making. Infection of berries is usually initiated by airborne conidia from overwintered sources [16,17]. During contact with the plant, B. cinerea causes cell death by producing phytotoxins and cell wall degrading enzymes and controls the host metabolism to facilitate colonization [13,18,19].
Powdery mildew (PM) is one of the most serious fungal diseases of grapes (V. vinifera) caused by the ascomycete E. necator, an obligate biotrophic pathogen that spreads through the air by conidial sporulation. It was introduced from America to Europe in the middle of the nineteenth century. Powdery mildew easily infects all green tissues (leading to chlorosis and premature aging and shedding of leaves, and forming white or gray powdery bloom on green stems), inflorescences and berries. Fruit infection leads to uneven ripening, wrinkling or cracking of the berries, resulting in fruit rotting, reduced yields and deterioration of the wine quality [20,21]. Fruit quality deteriorates greatly, acidity rises, and anthocyanins and sugar levels decrease. Even a 5% of infection can lead to unpleasant odors of the wine [22]. Host defense, which conditions ontogenic resistance, is known to operate early in the infection process, in the absence of major anatomical barriers. Until recently, grape berries were thought to remain susceptible to powdery mildew (Erisyphe necator) until late in their development. However, the development of ontogenic resistance is actually quite rapid in berries, and fruits become nearly immune to after fruit One of the main global problems of viticulture is the decrease in quantity and quality of harvest due to biotic stresses, mainly caused by bacteria, fungi and oomycetes, which causes severe diseases of grapes, including gray mold (caused by Botrytis cinerea Pers: Fr.), downy mildew (caused by Plasmopara viticola de Bary), powdery mildew (caused by Erysiphe necator (Schw.) Burr), also formerly known as Uncinula necator (Schwein.) Burr.) and grape black rot (Guignardia bidwellii (Ellis)). The majority of European grapevine cultivars of V. vinifera are susceptible to pathogen attack [10][11][12].
Gray mold causes significant economic losses in viticulture all around the world, reaching 20-50% of yield losses in grapes, due to rotting of ripe bunches in the post-harvest period. The high relative humidity and moderate temperatures during the grapevine vegetative cycle favour the development of this fungal pathogen [13]. The causative agent of the disease is Botrytis cinerea, a necrotrophic fungus with a short biotrophic phase. B. cinerea infects more than 1400 different plant species [14]. In the vineyard, this pathogen is a part of the microflora and usually lives in the soil on dead plant parts [15]. The disease infects fruits during ripening, causing necrotic areas with extensive fungal growth, which gives the characteristic appearance of gray rot. As a consequence, the grapes become unsuitable for wine making. Infection of berries is usually initiated by airborne conidia from overwintered sources [16,17]. During contact with the plant, B. cinerea causes cell death by producing phytotoxins and cell wall degrading enzymes and controls the host metabolism to facilitate colonization [13,18,19].
Powdery mildew (PM) is one of the most serious fungal diseases of grapes (V. vinifera) caused by the ascomycete E. necator, an obligate biotrophic pathogen that spreads through the air by conidial sporulation. It was introduced from America to Europe in the middle of the nineteenth century. Powdery mildew easily infects all green tissues (leading to chlorosis and premature aging and shedding of leaves, and forming white or gray powdery bloom on green stems), inflorescences and berries. Fruit infection leads to uneven ripening, wrinkling or cracking of the berries, resulting in fruit rotting, reduced yields and deterioration of the wine quality [20,21]. Fruit quality deteriorates greatly, acidity rises, and anthocyanins and sugar levels decrease. Even a 5% of infection can lead to unpleasant odors of the wine [22]. Host defense, which conditions ontogenic resistance, is known to operate early in the infection process, in the absence of major anatomical barriers. Until recently, grape berries were thought to remain susceptible to powdery mildew (Erisyphe necator) until late in their development. However, the development of ontogenic resistance is actually quite rapid in berries, and fruits become nearly immune to after fruit set [23]. High relative humidity is conducive to production of conidia. Atmospheric moisture in the 40% to 100% relative humidity range is sufficient for germination of conidia and infection. Free moisture, especially rainfall, is detrimental to survival of conidia [24][25][26][27][28][29]. Pesticides and fungicides, inorganic substances, above all sulfur, are widely used against powdery mildew and gray mold. However, this has a number of disadvantages, such as their negative impact on the environment, the high cost of chemicals and of their use. Worldwide, on average 35% of all pesticides are used for viticulture, which accounts for only 0.005% of the world's arable land [30][31][32]. In addition, populations of pathogens rapidly develop resistance to fungicides [33]. All this makes fungicide application program development a challenging task for winegrowers. Thus, there is a growing interest of modern viticulture in searching for new alternative environmentally friendly methods of protection against pathogens [11,[33][34][35]. Since the 19th century, breeding programs based on generative hybridization have been developed [36,37]. High pathogen resistance is observed among grapevine species from East Asia (V. pseudoreticulata, V. romanetii, V. amurensis and V. piasezkii Maximowicz) and North America (Vitis riparia Michx., V. berlandieri Planch (syn. V. cinerea var. helleri (Bailey) M.O. Moore), V. rupestris Scheele, V. aestivalis Michx (Muscadinia rotundifolia (syn. Vitis rotundifolia Michx.), V. labrusca L.). Due to its ability to be easily crossed with V. vinifera, these wild species are actively and successfully used in breeding as a rootstock, providing resistance to abiotic and biotic stresses [5,12,[37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52]. However, grape breeding is a slow process, for example, only after six successive crosses between Vitis vinifera and Muscadinia rotundifolia the complete resistance to powdery mildew was developed, which was associated with the transfer of the Run1 resistance gene to V. vinifera [53].
One of the alternative strategies to reduce the use of pesticides is related to the bioengineering of the rhizosphere, which represents the development of methods to stimulate the spread of introduced or local populations of beneficial microbes, the creation of synthetic microbial communities to stimulate plant growth, disease and stress resistance [54]. The plant ecosystem functioning and for sustainable farming the quality of the rhizosphere microbiome is of great importance [55,56]. It is known that the use of beneficial bacteria inhabiting the rhizo and/or endosphere of plants as biocontrol agents provides protection of their host plants against pathogens either by direct interaction with the pathogen or by promoting the development of plants induced resistance (IR) [57][58][59]. Inoculation with biocontrol agents could lead to changes in the whole plant root transcriptome due to a complex plant response [60]. After inoculation of grapes with various potential biocontrol agents (P. oligandrum, Trichoderma spp., Streptomyces sp., Pseudomonas protegens), the development of some pathogens was suppressed and plant growth was stimulated [8,[60][61][62][63][64][65][66]. In addition, arbuscular mycorrhizas (AM) fungal inoculation enhanced the growth of grapevine, together with rhizobacterium, increasing plant height and total dry weight [67][68][69]. Putatively effective and good candidates are known for bacterial biocontrol of E. necator, B. pumilus B-30,087 [70], Bacillus strains ATCC 55,608 and 55,609, which produce antifungal substances including zwittermicinA, playing a vital role in the interaction [71], B. subtilis [8,72]. The following biocontrol beneficial strains demonstrated a protective activity against B. cinerea on dual culture plates, on in vitro plantlets and in field conditions: Pantoea agglomerans, Acinetobacter Iwoffii, Burkholderia phytofirmans, Micromonospora spp., Streptomyces spp., Cupriavidus sp., P. fluorescens, P. putida, Pseudomonas aeruginosa, Bacillus circulans, Bacillus subtilis [8,57,[73][74][75][76][77][78][79][80][81][82][83][84][85]. Pathogen resistance was correlated to a different extent with phytoalexins and oxidative burst production [77,78,85]. In addition, it was concluded that different strains could be more appropriate for treatment of specific organs [75]. The bacterial strain oxB (which is closely related to Cupriavidus campinensis) was detected, which actively decomposes oxalic acid, an important virulence factor of B. cinerea, thus limiting the symptoms of gray mold on the leaves and greatly reducing the symptoms of the disease caused by B. cinerea in inflorescences in laboratory conditions [84]. However, there are several problems linked to this strategy-the success of bioinoculants or biocontrol agents utilization in the field depends on the target culture, product availability and application options, environmental conditions, and costs [86]. Expansion of the knowledge of plant-microbial interactions and its application may be useful in future plant breeding and farming programs [87].
Enormous efforts to develop resistant varieties have prompted researchers to search for new breeding technologies based on the knowledge of genes and genomes. One approach is a combination of classical selection with marker-assisted selection (MAS) for introducing certain loci into new varieties [88]. Different countries have developed various new breeding programs aimed at obtaining grape varieties combining disease resistance with high quality berries [4,[89][90][91][92]. The use of molecular markers allows to speed up and to make the selection process cheaper, and also to ensure the creation of varieties carrying new combinations of genes, which are almost impossible by traditional breeding methods [93].
The second approach is associated with the interference into the genome with the use of genetic engineering. It includes several methods that can purposefully alter the genome of plants in order to obtain resistant grape varieties with the desired grape properties for growers and consumers [94][95][96][97][98][99][100]. One of the methods is the genetic modification of grapes to provide resistance to fungal diseases. For example, transgenic grape plants were created, carrying the rice chitinase gene, which increased resistance to powdery mildew and anthracnose (Elisinoe ampelina (de Bary) Shear). However, transgenesis raises concern among winemakers due to the introduction of alien genes into elite grape varieties and their potential impact on wine characteristics, and genetically modified organisms (GMOs) are prohibited from being grown in many countries [101].
As alternatives to transgenesis, two methods of crop transformation and plant breeding have been developed-cisgenesis and intragenesis. Both concepts imply the modification of plants only with genetic material obtained from the same or closely related species [102,103]. Cisgenesis is a closer method to traditional breeding, since plants are subjected to genetic modification by one or more genes that retain their natural genetic composition with all its regulatory elements. However, unlike traditional breeding, cisgenic crops contain exclusively the gene or genes of interest without any undesirable genetic element. Intragenesis allows the use of hybrid genes that can incorporate genetic elements from different genes and loci, thus promoting the creation of new genetic combinations, new expression models and new plant properties [104]. The application of cisgenesis and intragenesis is limited to several species, mainly due to the lack of knowledge about the required regulatory sequences. Cisgenic grape lines are currently being developed. Chardonnay grape was transformed with the use of the construct, containing PR protein VVTL-1 (Vitis vinifera thaumatin-like protein), which inhibits spore germination and hyphal growth. The resulting transformants were characterized by a decreased severity of powdery mildew and black rot [105,106].
CRISPR/Cas9 technology is one of the most promising genome-editing methods, which can be used for targeted gene modification, suppression/activation of gene expression, subtle modification of gene function, and epigenome editing. CRISPR/Cas9-targeted mutagenesis opens the possibility to obtain non-transgenic modified plants carrying specifically determined mutations that are stably inherited over generations. CRISPR/Cas9targeted mutagenesis. The legislation of some countries does not prohibit the cultivation of such modified non-transgenic organisms; therefore, CRISPR/Cas9 editing is considered a promising tool for improving the resistance of grape varieties. Researchers conduct search for new target genes of grapes for being modified with CRISPR/Cas9 technology that will increase resistance to pathogens [93,99,100].
To date, the genomes of cultivated grapes V. vinifera [107,108] and wild V. vinifera sylvestris [109], as well as the B. cinerea, have been sequenced [110,111]. Modern genetic studies of grapes have also allowed to identify gene systems of resistance/susceptibility to the most common pathogens-gray mold and powdery mildew. In the frameworks of this review, we have systematized the available to date scientific data about these gene systems.

Gene Systems for Resistance and Susceptibility to Botrytis cinerea 2.1. Mechanisms
Infection of grapevine inflorescences with B. cinerea occurs at the stage of flowering with air conidia, which settles in the surface cells of the host. At the stage of immature green berries, the pathogen spends a long time in the host tissues without symptoms [15][16][17][112][113][114][115]. The resistance of immature berries to B. cinerea is the result of several associated mechanical and chemical processes, but they have not yet been fully studied [13]. The transition of the pathogen from the resting stage to the phase of active infection occurs during the berry ripening, which is facilitated by physiological and biochemical changes that are activated in berries during ripening, as well as by signals associated with ripening [13,116,117]. Although B. cinerea has been well studied in various plant species, there is limited information regarding the mechanisms of resistance and susceptibility of the Vitis ssp. genotypes. to B. cinerea. causing gray mold. Transcriptome and metabolomic analyzes of grapes and B. cinerea at different stages of plant-pathogen interaction allowed identification of 3610 genes with differential expression [116][117][118][119]. The genes VIT_01s0011g05420 and VIT_11s0016g02070 were the only genes shared at three different stages of infection and they were always activated by B. cinerea. The function of the VIT_01s0011g05420 gene is not yet known [116]. The VIT_11s0016g02070 gene presumably belongs to the basic helix-loop-helix family; it is a transcription factor (TF) involved in hormonal interactions and host immunity [120].
For B. cinerea, the expression of 9927 genes was identified, both unique as well as common for different stages of infection. 222 common genes were identified for all stages of infection, most of them were predicted/hypothetical proteins, ribosomal proteins, and housekeeping genes [116]. It is known that the fungus B. cinerea actively promotes plant susceptibility using many virulence factors [121,122]. At the early stages B. cinerea utilises mRNA and effector proteins to suppress the early host cells death and immune responses, which allows the fungus to anchor inside the host and to accumulate its biomass before the necrotrophic phase. In response to plant infection, a whole cascade of interrelated defense mechanisms is activated, which come into action at certain stages of the pathological process [123,124]. The plant recognizes peptides and proteins such as extracellular pathogen-associated molecular patterns PAMP, or intracellular pathogens effectors delivered to host cells, which trigger signal transduction and activate rapid defense responses that involve massive reprogramming of transcription within the plant. This leads to activation of gene expression (for example VvGLP3), accumulation of reactive oxygen species (ROS, oxidative burst), activation of genes encoding antimicrobial proteins, and of genes of the polyphenol biosynthesis pathway for the production of phytoalexins and precursors for strengthening the cell wall (biosynthesis of monolignols (VvPAL, VvCOMT, VvCCoAMT), stilbenoids (VvSTSs) [119].
The plant cuticle is the first barrier against the pathogen conidia germination. It consists mainly of two types of lipids, cutin and cuticular waxes, which may be the main candidates for signaling early interactions between plants and pathogens. It is known that the absence of any of these components in mutants led to changes in genes expression at the early stages of infection and germination of B. cinerea decreased consequently [125,126]. Oleanolic acid (OA) and n-fatty alcohols (C22, C24 and C26) are the main components of the cuticular waxes of mature grapes [127]. OA is a naturally occurring triterpenoid that is associated with membrane fluidity and potentially affects signaling by many ligands and cofactors. The presence of OA can play a regulatory or a stabilizing role in the germination of B. cinerea. The second component, n-fatty alcohols, play an important role as inducers of germination at the early stages; however, they participate in the acceleration of germination only in a certain combination-the complete mixture of alcohols (C22, C24 and C26) was designated as "full-fatty-alcohols" (FFA) [126]. The involvement of pectin methylesterases (PMEs) and PME inhibitor families in maintaining cell wall integrity has also been detected [116].

Initiation of Infection
One of the earliest cellular responses to flower infection with B. cinerea is the production of ROS, and the genes encoding oxidative stress enzymes (GST, ascorbate oxidase, 2OG-FeII oxygenase, cytochrome P450 monooxygenases activation [12]). In the investigation of Wan et al. (2015) the grape leaves resistance to B. cinerea was analyzed in the genotypes of Chinese wild grape species and of cultivated V. vinifera. The low rates of infection and good resistance to fungi were characteristic to the Chinese wild species: V. amurensis; V. yenshanensis; V. qinlingensis P.C. He (Qinling grape from Qinling Mountain region)clone "Pingli-5", V. adstricta. Many Chinese wild species demonstrated simultaneous resistance to several fungi: for example, "Pingli-5" was resistant to gray mold, anthracnose, powdery mildew and downy mildew [12,41]. Oxidative stress disrupts the redox balance in the affected tissues, thereby contributing to the progression of the disease. Antioxidant enzymes such as Peroxidase (POD), catalase (CAT) and superoxide dismutase (SOD) protect plant cells from oxidative stress and maintain redox balance by removing ROS generated during pathogens attack. Pathogen-resistant "Pingli-5" was characterized by CAT and POD increased activities throughout the experiment, but almost no change in SOD activity was observed, with the exception of its increase at 4 hpi (hours post inoculation), which corresponded to the minimal induction of ROS [12,128]. Similarly, in the genotype of V. vinifera "Ju mei gui", a small amount of ROS was found after inoculation, which indicates the antioxidant enzymes role in maintenance of redox balance and cells protection from ROS destruction. The leaves of "Ju mei gui"were characterised by reduced germination of fungi and spread of infection. The elevated levels of POD activity and no significant changes in SOD activity were detected in "Ju mei gui" as well [129].
At the beginning of the initiation of infection, in 24 hpi, the genes encoding membrane receptor-like kinases (RLK), such as CLV1, WAK1, BAK1, associated with the immune system response to necrotrophic pathogens, are activated. WAK1-damage-related receptor recognizes oligogalacturonides derived from the plant cell wall due to cell wall degradation [117,119]. There is a rapid and strong induction of the expression of genes encoding about 100 transcriptional regulators (TFs) in infected flowers, which promotes the activation of specific defense pathways. The genes encoding WRKY, NAC, MYB, and ethylene-responsive element-binding proteins were among the most well-studied and most important identified TF genes. TF WRKY33 is involved in the defense reaction of the host plant, and its expression level was detected to increase strongly already after 12 hpi, but it decreased after 48 hpi [117,119,130]. TF Myb14 regulates the biosynthesis of stilbenes [131]. At the early stage of infection, grapevine genes encoding various classes of pathogenesis related proteins (PR), like osmotin, thaumatin, chitinases, Beta 1-3 glucanase, Bet v I allergen, PR1 and PR10, regulated by TF, are activated. As a result, oligosaccharides are formed, which in turn enhance the synthesis of phytoalexins, low molecular weight substances of a secondary origin, which are synthesized at the sites of pathogen penetration and suppress its development. Phytoalexins maintain species immunity to non-specialised pathogens, and varietal resistance to specialised pathogens [117,119]. Even during the green berry stage, when the pathogen was dormant, the genes encoding PR proteins were still activated. The involvement of these genes in response to B. cinerea was also observed in ripe berries, but their action was ineffective against bunch rot development [116]. In response to the infection of grapes with B. cinerea, the phenylpropanoid defense pathway is activated. At the stage of infection initiation, an increase in the concentration of compounds such as resveratrol, viniferin, miyabenol, isogopeaphenol, catechin and proanthocyanidins was observed. These substances mediate plant protection, suggesting that they contributed to the inhibition of the pathogen before maturation [117][118][119][132][133][134].
Genes involved in the synthesis and signaling of phytohormones in response to infection are differentially expressed. Their production starts relatively fast in plant tissues after its interaction with fungal elicitors [135,136]. Salicylic acid (SA) is associated with local resistance to biotrophs and with the onset of systemic acquired resistance (SAR). Biotrophic pathogens generally induce the SA-mediated defense response, which activates various downstream physiological immune responses such as programmed cell death and ROS accumulation [137]. Jasmonic acid (JA) and ethylene (ET) control the resistance to necrotrophs and induce systemic resistance. Semi-biotrophic pathogens induce both SAand JA-mediated signaling responses [135,136,138]. It has been suggested that SA-and JA-mediated signaling pathways are antagonistic, and if plants defend against a particular pathogen via the SA-dependent pathway first, then JA-signaling is inhibited [136,139]. The phytohormones ET, JA and SA have been found to play an important role in the interaction between the flower and B. cinerea. The involvement of gibberellic acid (GA) and abscisic acid (ABA) has also been identified [119]. High content of JA in resistant cultivar "Ju mei gui", blocked B. cinerea infection [129].

The Green Berry Stage, 4 Wpi (Weeks Post Inoculation)
At the green berry stage, the amount of expressed B. cinerea genes is relatively small. The pathogen is able to maintain its main metabolic activity during dormancy, high expression of fungal ribosomal genes, elongation factors, ATP synthesis, ATP-dependent molecular functions related genes, as well as 34 CAZyme genes, is registered, which implies the preservation of the pathogen ability to obtain energy from the host plant [117,119]. The expression level of the CWDE genes and genes encoding phytotoxins is either strongly reduced or not detected-the virulence factors of the fungus are disabled, probably due to the dormant state [116][117][118]140]. There is a molecular relationship between B. cinerea and immature berries during the dormant period-the virulence genes of the pathogen are inactive, and an increased immune response is observed in plants [117]. Preformed and induced defense mechanisms, including skin features of immature berries such as polyphenols in cell walls of berry skin and the thickness of the epidermal cell layer complex, have been proposed as part of ontogenic resistance to B. cinerea [119,133].
At the green berry stage, the VvPAL and VvSTS genes are induced in grape plants, which are associated with the immune pathway of phenylpropanoid biosynthesis, leading to the production of stilbenes (resveratrol and its derivatives) and lignin, which inhibit the growth of B. cinerea. The activation of the expression of 2 grape genes encoding putative enzymes involved in lignin biosynthesis, VvC3H (p-coumarate 3-hydroxylase) and VvCCR (cinnamoyl CoA reductase), was characterized, and the increase in the amount of the corresponding metabolites (affeic acid, ferulic acid, and chlorogenic acid) was confirmed at the green berry stage. At the ripe berry stage, their expression decreased. The expression of VvEXT (extensin-like protein) gene encoding proline-rich extensin-like protein was also induced at the green berry stage. Papillas (callose-enriched encasement of haustorium) were formed under the appressoria in the green berry, during early infection, but they were not detected in the mature berry [141]. The activation of the VvRbohD gene was identified, which encodes the putative superoxide-generating NADPH-oxidase, that leads to the formation of O2-, and its transformation into H 2 O 2 depends on the activation of the VvGLP3 gene (germin-like proteins). GLPs were among the most inducible genes during the green berry stage and the early ripening stage [142,143]. Since the expression of the SA marker gene VvPR1 was increased, it was suggested that the SA pathway may also be involved in basal resistance at this stage [118]. Similar to the stage of infection initiation in the flower, in the green berry there was a detected activation of the expression of the genes encoding membrane-localized RLKs: Clavata1 receptor kinase (CLV1), Brassinosteroid insensitive 1-associated kinase 1 (BAK1), and Wall-associated kinase 1 (WAK1) [117,119]. In response to infection, genes of various PR protein families, including PR10, were strongly induced due to dormant B. cinerea. At this stage B. cinerea induces the expression of key TFs that play an important role in the interactions between plants and microbes. Lignification at the site of entry is one of the main defense mechanisms used by plants to arrest the development of B. cinerea [118,119]. It was observed that there was an up-regulation of SA, ET signaling marker genes, and genes related to auxin metabolism, that contributed to the increase in the protective ability of the hard green berry [117,118].

The Ripe Berry Stage, 12 Wpi
During the process of ripening, grape berries undergo several modifications that reduce their natural resistance to the pathogen. The mechanical resistance in the cell wall decreases, contributing to the appearance of microcracks. Moreover, the sugar concentration increases and the concentrations of organic acids and of some compounds associated with biotic resistance decrease. The hormonal balance and pH also change [140]. VvJAZ1, a marker gene for JA, and transcripts of various enzymes of the JA biosynthesis pathway (phospholipase, lipase, allene oxide synthase, jasmonate O-methyltransferase) are the most activated at the ripe berry stage [118]. It is likely that the signals associated with ripening, as well as physical and chemical changes at this stage, play an important role in triggering the transition of a necrotrophic pathogen from prolonged dormancy to an active infection phase [13,116]. The sensitivity of ripe berries is significantly correlated with phenolic compounds in the cell walls of berry skin and is negatively correlated with the total content of tannins in the skin and with water activity (Aw) on the surface of the berry [144].

Gene Systems for Resistance and Susceptibility to Botrytis cinerea
Currently, a number of genes have been identified in grapevines that may confer resistance or tolerance to B. cinerea, including VvPLDs, VvPR10.1, VvPR10.3, VvNPR1.1, VaSTS19, WRKY57, VlWRKY3, VqWRKY52, VqSTS21, VqAP13, ERF, VvXYLP, etc. The introduction of genes VaSTS19 and VvNPR1.1 into A. thaliana enhanced its resistance to B. cinerea. On the contrary, overexpression of the VqTLP29, VlWRKY3, VqWRKY52, VqSTS21, VqAP13 genes in A. thaliana increased the susceptibility to B. cinerea [139,[145][146][147][148][149]. The phospholipase D gene family (PLD) has been found to play an important role in the regulation of cellular processes in plants, including ABA signaling, programmed cell death, growth regulation and stress responses. Structural analysis showed that the PLD gene family can be divided into 6 subgroups: α, β/γ, δ, ε, ζ and ϕ, which had descended from 4 original ancestors as a result of a series of gene duplications [150]. PLD α1 participates in various plant processes, such as ROS production and accumulation of jasmonic acid at the wound site, and promotes ABA signaling inhibiting the negative regulator ABI1. PLD α1 and its lipid product PA are intermediates between important cellular regulators in plant cells. PLD α1 interacts directly with Ga proteins [151]. PLDδ positively regulates plant resistance to stress, including oxidative stress. Its role lies in signaling of damage caused by ROS. It is activated by oleic acid and strongly binds to the plasma membrane and the cytoskeleton of microtubules. PLDδ is activated by hydrogen peroxide (H 2 O 2 ) and the resulting PA reduces the programmed cell death caused by H 2 O 2 . PLD δ and PA participate in the H 2 O 2induced activation of MAP kinase cascades [152]. Thus, PLDδ mediates plant responses to ROS [151].
In grapes, the VvPLD genes were also characterized and classified into 6 types (VvPLDαs, VvPLDβs, VvPLDδs, VvPLDε, VvPLDρ, VvPLDζ) and 3 groups (C2-PLD, PXPH-PLD and SP-PLD). The function of VvPLD genes upon infection V. vinifera cv. Cabernet Sauvignon with B. cinerea was studied. It was revealed that the genes VvPLDβ1, VvPLDβ2, VvPLDδ2, VvPLDρ and VvPLDζ were activated, and expression of the genes VvPLDα and VvPLDδ was suppressed. Identified genes can serve as candidates for the role of resistance genes [153].
The WRKY TF family has been detected to play a role in biotic stress responses. The VqWRKY52 gene found in the Chinese wild Vitis quinquangularis encodes the WRKY III gene family member and plays an important role in the SA-dependent signal transduction pathway, inducing cell death during hypersensitive response (HR). In transgenic A. thaliana lines, its overexpression increased the sensitivity to B. cinerea due to increased HR [146]. When the VvWRKY52 gene was switched off in grapes by CRISPR/Cas9-targeted mutagenesis, a phenotype with increased resistance to B. cinerea was observed in transgenic lines [154]. In Arabidopsis gene WRKY33, a functional homologue of VvWRKY33, plays a key role in plant defense, regulating redox homeostasis, SA signaling, ET-JA-mediated cross-communication and phytoalexin biosynthesis, providing resistance to B. cinerea [130]. WRKY57 belongs to Group IIc of WRKY TF family [155]. Loss of function of WRKY57 en-hanced resistance against B. cinerea, and this resistance was associated with the JA signaling pathway, especially COI1. JAZ1 and JAZ5, direct targets of WRKY33, were identified as direct target genes of WRKY57. WRKY57, the same as WRKY33, binds to the promoters of the JAZ1 and JAZ5 genes via a W-box sequence. WRKY57 and WRKY33 competitively regulate JAZ1 and JAZ5. WRKY57 compromises B. cinerea resistance via competing with WRKY33 to transcriptionally regulate JAZ1 and JAZ5. WRKY33 acts as a transcriptional repressor of JAZ1 and JAZ5 [156]. Transgenic VlWRKY3 lines of A. thaliana showed increased sensitivity to B. cinerea, probably due to the interaction of the SA and methyl jasmonate (MeJA) signaling pathways, or the influence of other genes. It has previously been reported that WRKY3 responds to salt and drought stress as well as MeJA and ET in V. labrusca × V. vinifera cv. 'Kyoho' [147,157].
Among the Pr genes, the highest activation of expression was observed for genes VvPR10.1 and VvPR10.3. This suggests that they are the main candidates for the role of genes for resistance to B. cinerea. In grapes, VvPR10.1 is associated with resistance to P. viticola and is regulated by VvWRKY33 [119]. In grapes (V. vinifera), the regulation of 35 ERF genes (Ethylene responsive factor) in response to B. cinerea was analyzed, and their important functions in plant protection, in addition to their known role in development, were identified. In the study, the genes VvERF071, VvERF072, VvERF066 and VvERF099 of the IX ERF subfamily showed the highest levels of expression at key time points of infection. After leaves infection with B. cinerea of two cultivars (the susceptibile "Red Globe" and the resistant "Shuangyou") the destruction and strengthening of the cell wall was significantly induced respectively. The analysis of cis-elements of the promoter showed that most of the ERF genes contain elements of the CGTCA-motif and TGACG-motif, which are known to be involved in the response to MeJA, especially VvERF071 and VvERF072, suggesting that they may be involved in the response to the pathogen infection. The identified TGAelement and AuxRR-core are involved in the response to auxin. ABRE, TCA-element and ERE-elements are involved in the reaction for ABA, SA and ET, respectively. VvERF099 has a gibberellin responsive motif P-box [158]. It was also detected that expression levels of VvERF016 from group VII increased after infection by B. cinerea. In A. thaliana, the VvERF016 gene homologue (AtERF72, also named AtEBP) interacts with ACBP4 (acyl-CoA binding protein 4) and probably regulates ethylene-related signaling and defense mechanisms in plants [158,159].
Thaumatin-like protein (TLP) is a large family in plants, and individual members play different roles in various responses to biotic and abiotic stresses. TLP family is involved in pathogen resistance. The expression of the TLP gene is widely influenced by E. necator and B. cinerea. VqTLP29 overexpressing transgenic grape lines of diseases resistant V. quinquangularis increased resistance to powdery mildew, but increased susceptibility to gray mold, by regulating signal transduction through the SA and JA/ET pathways. Treatment with MeJA at concentrations of 10 to 100 µmol/L can effectively induce resistance to B. cinerea. Induced disease resistance was closely associated with increased production of H 2 O 2 , enhanced expression of the VvNPR1.1 gene, and accumulation of stilbene phytoalexins such as transresveratrol and its oligomer (trans-) ε-viniferine [160].
Recently identified sugar transporters SWEETs have been characterized as bi-directional, low-affinity sugar carriers, probably operating by an uniport mechanism [161]. On the one hand, pathogens can alter the expression level of genes encoding various sugar carrier proteins, including the SWEET genes using sugars as a source of carbon and energy [162]. On the other hand, it has been found that high sugar levels in plant tissues can also increase plant resistance enhancing the oxidative burst in the early stages of infection, thereby increasing cell wall lignification, stimulating flavonoid synthesis and inducing certain PR proteins [163]. VvSWEETs transporters play an important role in sugar mobilization during grape berry development, and their expression is transcriptionally reprogrammed in response to B. cinerea infection. In the grapevine, the SWEET family is composed by 17 members, and only VvSWEET4 and VvSWEET10 were functionally characterized [164,165]. VvSWEET7 and VvSWEET15 are the key SWEET genes at the stages of grapes development and maturation, localized in the plasma membrane. VvSWEET7 transports both glucose and sucrose. Expression of VvSWEET7 in berries peaks at the green berry stage and of VvSWEET15 at maturity. They are characterized by increased expression in response to infection with B. cinerea at the green berry and ripe berry stages, respectively. In addition, B. cinerea infection suppresses the expression of VvSWEET17a and activates the expression of VvSWEET2a at the green berry stage, suppresses the expression of VvSWEET10 and VvSWEET17d at the veraison stage and the expression of VvSWEET11 at the ripe berry stage. Since the grape resistance is high at the green berry stage, it is likely that overexpression of VvSWEET2a and VvSWEET7 may be a defense mechanism [19].
Xylogen-like proteins (XYLPs), a glycoprotein family with high molecular weights, are widespread in plants [166]. XYLPs are involved in various plant growth, development and stress responses, such as cell xylem differentiation, programmed cell death, abiotic tolerance and hormone signal transduction, such as SA, JA, and ABA. Six XYLP genes were identified in V. vinifera (named VvXYLP01-VvXYLP06 according to their chromosome location). It was detected that upon infection with B. cinerea the level of VvXYLP02 expression significantly increases in the resistant genotype, but decreases or slightly increases in the sensitive genotype. Overexpression of VvXYLP02 in A. thaliana significantly increased resistance to B. cinerea. In addition, the JA treatment significantly increased VvXYLP02 expression in V. vinifera [149]. Chitosan can play a key role in plants resistant to B. cinerea infection. Chitosan induces protective genes and protects berries quality from infection through JA signaling. Suppression of expression of negative regulators in fruit disease-resistance expression-VvHDAC19 (histone deacetylase 19) and VvTPR3 (Topless-related protein 3)-with the application of chitosan led to an increase in grape resistance to B. cinerea. TPR3 and HDAC19 interact with each other and their activity is suppressed by JA and chitosan. The synthesis of phenolic compounds was activated and the strengthening of the cell wall was observed, oxidative stress was modulated and the production of JA in ripe fruits was induced, the growth of B. cinerea was suppressed [167].
The VvAOS gene, which plays an important role in the biosynthesis of JA, and the VvCOI1 gene, which encodes the receptor of jasmonate [168] and regulates the level of JA signaling, both affect the process of berries ripening through the regulation of fruit pigmentation and cell wall metabolism, and also affect the process of berries infection by B. cinerea.
The accumulation of JA occurs relatively quickly in plant tissues and cells after injury or exposure to fungal elicitors [169,170]. Suppression of the VvAOS gene leads to a decrease in the JA content. The samples with overexpression of the VvCOI1 gene (VvCOI1-OE) were characterized by a high level of JA and a delay in the B. cinerea infection progression. Even though the overexpression of VvCOI1 in strawberries did not affect directly the JA content, it induced plant defense genes such as PPO, SOD, POD, PAL, BG and chitinase, which protected fruits from infection by gray mold [171]. Cysteine-rich receptor-like kinases (CRKs) play an important role in disease resistance of plants, including A. thaliana, Oryza sativa L. (rice), Hordeum vulgare L. (barley), Triticum aestivum L. (wheat) and Medicago truncatula Gaertn. [172][173][174][175][176][177][178]. Four CRKs have been identified in V. amurensis (VaCRK1-VaCRK4). Overexpression of VaCRK2 increases resistance to B. cinerea in A. thaliana. A grape cultivar resistant to gray mold had a significant increase in the VaCRK2 gene expression after inoculation with B. cinerea and treatment with methyl jasmonate. VaCRK2 expression induced genes associated with the JA signaling pathway, genes associated with pathogenesis (PR), and accumulation of ROS [179].

Gene Systems for Resistance and Susceptibility to Erysiphe necator
To resist pathogens, plants have developed an innate immune system that exists in two main forms: (1) the primary species-specific immune response known as PTI (PAMPtriggered immunity) is activated when the host's extracellular plasma membrane receptors (PRR-pattern-recognition receptors) interact with pathogen-specific molecules. For example, callose synthase GSL5/PMR4 is responsible for the biosynthesis of most of the callose. Disabling this gene leads to a dysregulation of SA-mediated defense reactions and a loss of susceptibility to powdery mildew pathogen. On the other hand, overexpression of GSL5/PMR4 leads to the increase of callose accumulation in the papilla and complete resistance to the penetration of E. necator [180]. This cellular rearrangement is observed in mlo (MILDEW RESISTANCE LOCUS O) mutants resistant to pathogen penetration [180,181].
(2) The secondary, or race-cultivar-specific immune response, known as ETI (effectertriggered immunity), is initiated by pathogen virulence factors, which are recognized directly or indirectly by the intracellular receptors of the host plant, usually linked with resistance genes (R-genes). Then, subsequent signaling pathways are activated, that ultimately leads to numerous protective reactions, localized hypersensitivity reactions at the site of infection, and programmed cell death [181,182]. It has been suggested that wild Chinese and wild American grapes interact with E. necator via different cellular and molecular defense mechanisms [46]. Wild North American grape species (Muscadinia rotundifolia (syn. Vitis rotundifolia), V. rupestris, V. riparia, and V. Aestivalis; V. labrusca, V. Berlandieri) [183], due to long co-evolution with E necator, have acquired ETI resistance to powdery mildew. As a consequence, this resistance can be quickly overcome by new strains of E. necator. Thus, the resistance of grapes obtained by the introduction of dominant R-genes into elite cultivars through classical breeding is unlikely to be durable, and a broad spectrum genetic resistance is required for breeding [46,[184][185][186][187]. Wild Chinese species (V. pseudoreticulata, V. romanetii and V. piasezkii) exhibit persistent immunity to E. necator after its penetration into cells via programmed cell death mediated by NBS-LRR, as well as the formation of papilla. Thus, wild Chinese grapes represent an important genetic source for enhancing the sustainability of cultivated grapes [46,[188][189][190][191].

R-Genes, NBS-LRR
The most characterized R-genes belong to the NBS-LRR (leucine-rich nucleotide-binding site repeat) gene superfamily, one of the largest gene families in plant genomes. The majority of plant disease resistance genes encode NBS-LRR proteins and contain conserved nucleotide binding motifs (NB) and leucine rich repeat (LRR) motifs. NBS-LRR proteins are divided into two subfamilies based on their N-termini: TIR-NBS-LRR (TNL) and non-TNL [192].
NBS-LRR genes have been extensively studied in various plant species such as A. thaliana, O. sativa, Populus trichocarpa (poplar), Zea mays (maiz), etc. [193]. These genes were also identified in grapes by various research groups [108,192,193]. It was revealed that they are widely represented in the grape genome, and localized in many chromosomes. Among all detected NBS-LRR genes, 63 are associated with response to powdery mildew infection. Some of them were found to localize at loci REN1 (chromosome 13), REN3 (chromosome 15), REN5 (chromosome 14), REN6 (chromosome 9), REN9 (chromosome 15) and RUN1 (chromosome 12) [193]. Based on the studies of the NBS-LRR genes in different plants, a correlation between the number of NBS-LRR genes and the size of plant genome was established (with the increase in size of plant genome the number of NBS-LRR genes increases) [194][195][196][197][198]. The E. necator susceptible NBS-LRR proteins were characterized as being about 1000 amino acids in size [193]. Various types and amounts of regulatory elements associated with stress-induced gene expression were identified in the sequence of promoter regions of NBS-LRR proteins: hormone responsive regulatory elements: Salicylic acid responsive element (TCA element), Ethylene responsive element (ERE), Methyl jasmonate responsive element (CGTCA and TGACG-motifs) and Abscisic acid responsive element (ABRE); defense and stress responsive element (TC-rich repeats); wound and pathogen responsive element (WUN-motif and W-box); stress responsive element (GT1 Box), cis-acting regulatory elements (CARE). TFs bind to these regulatory elements and activate the defense mechanisms of V. vinifera in response to stress conditions caused by powdery mildew, for example NAC TFs bind to GT1 box and WRKY binds to W-box [193]. It was observed that the levels and timing of expression of the NBS-LRR genes differ greatly in susceptible and resistant V. vinifera lines in response to E. necator. Some of the NBS-LRR genes are induced at later stages of powdery mildew infection, and its expression is higher in resistant lines.
Several NBS-LRR genes were detected to be expressed in resistant cultivars only, as well as several NBS-LRR genes of resistant cultivars that were involved in the early response to powdery mildew infection [193].

S-Genes, MLO
An alternative approach is based on obtaining recessive mutations of susceptibility genes (S-genes). Expression of S-genes is induced by pathogens, and encoded proteins suppress plant immunity system. Inactivation of S-genes required for the successful penetration of a particular pathogen does not cause a strong activation of plant defense. Their knock out leads to recessively inherited non-race-specific resistance, and is one of the main models of PTI non-host resistance [188,199]. S-gene-mediated resistance is usually more durable as the pathogen must overcome its dependence on S-factor of the host. The disadvantage of such resistance is a possible formation of pleiotropic phenotypes, for example, the formation of papilla in the absence of any pathogen and the premature onset of leaf senescence [200][201][202]. Some genes of the Mildew Locus O (MLO) family are a typical example of powdery mildew S-genes. For dicotyledons, these are all S-genes from group V of the MLO family [203][204][205]. In grapes, 17 representatives of the MLO gene family were identified, and suppression of a certain group of these genes led to resistance to E. necator [203][204][205][206][207]. For the first time resistance to powdery mildew mediated by inactivation of the MLO gene was revealed in barley in 1992 and for a long time has been considered as a unique form of resistance. Disruption of the MLO gene in barley provides persistent resistance to powdery mildew, which has been confirmed by the fact that during many decades no new E. necator strains capable to overcome this mlo-mediated resistance have been found in the fields [199]. Later, it was revealed that MLO genes are conserved within the plant kingdom, and their non-functional forms reduce the susceptibility to powdery mildew in some species, such as barley [208], Arabidopsis [209], pea [202], tomato [210], wheat [211] and pepper [212]. In Arabidopsis, 3 genes (AtMLO2, AtMLO6 and AtMLO12) were identified, which are induced in response to powdery mildew infection, and their joint inactivation led to complete resistance to powdery mildew [209].
In the study carried out by Feechan and colleagues (2008) and Winterhagen and colleagues (2008), three VvMLO genes were identified in grapes, in accordance with the Feechan nomenclature named: VvMLO3 (VvMLO11 (W) (W-Winterhagen's nomenclature)), VvMLO4 (VvMLO13 (W)) and VvMLO17, which showed similarity in homology of translation products sequences and transcriptional response to infection with other MLO genes of Arabidopsis and tomato, associated with susceptibility to powdery mildew. In response to E. necator rapid induction of genes VvMLO3, VvMLO4 and VvMLO17 and accumulation of large amount of their transcripts was detected [203,204,213]. MLOs are calmodulin-binding proteins. Pharmacological studies suggest that the influx of Ca2+ ions is important for MLO functioning [213,214]. Therefore, Ca2+ may be a candidate signal for the induction of the E. necator responsive genes VvMLO3, VvMLO4, VvMLO9 and VvMLO17, since plant cells generate a transient Ca2+ signal in response to pathogen attack [203,204,215,216]. Further research has revealed grape MLO genes in clade V, which are associated with susceptibility to powdery mildew: VvMLO6 (W), VvMLO7 (W) and VvMLO11 (W). It is likely that VvMLO6/VvMLO7 (W) can be a functional homologue of AtMLO2.
In the experiment, the incomplete knock out of these MLO genes via RNAi significantly reduced the susceptibility of V. vinifera to powdery mildew. VvMLO7 (W) is the main candidate gene for susceptibility to E. necator; it was inactive in resistant cultivars, and a decrease in its expression in transgenic mlo lines correlated with the level of susceptibility to powdery mildew. Inactivation of VvMLO6 (W) and VvMLO11 (W) was not directly related to susceptibility, the expression of these two genes was significantly reduced in both resistant and susceptible lines; however, both genes additionally contributed to increasing resistance [205,206]. Pleiotropic phenotypes were not found in mlo-lines under greenhouse conditions [205].

PM-Responsive REN and RUN Loci
In the genotypes of various grape species quantitative trait loci (QTLs) associated with powdery mildew resistance were identified: Run (Resistance to Uncinula necator) and Ren (Resistance to Erysiphe necator) [45]. These QTLs were mapped to chromosomes 9, 12, 13, 14, 15, 18, and 19, where various resistance gene analogs (RGAs), encoding resistance proteins of the TIR-NBS-LRR and CC-NBS-LRR types, are also localized. Loci Run, Ren provide qualitative resistance [193]. The main QTLs of the North American Muscat grape V. rotundifolia, associated with powdery mildew resistance, were located on chromosome 12-Run1 [217], and on chromosome 18-Run2.1 (V. rotundifolia "Magnolia") and Run2.2 (V. rotundifolia "Trayshed") [218]. The Run1 locus was found to comprise a family of seven putative Toll/ interleukin-1 receptor (TIR)-NB-LRR-type R genes [186]. Run1 confers ETI hypersensitivity phenotype, was introgressed from V. rotundifolia to V. vinifera. The locus Ren1 was identified on chromosome 13 in the cultivar «Kishmish vatkana» of V. vinifera [219]. Ren1 was located in the region of the genome containing NB-LRR sequences, which corresponds to its resistance phenotype (ETI). Hence, both Ren1 and Run1 may be non-durable considering their probable type of functioning. V. rupestris B38 showed an enhanced penetration resistance to a powdery mildew isolate with the ability to overcome the Run1 gene, making it an interesting resistance source to prolong the durability of this gene. V. rupestris B38 showed an enhanced penetration resistance to E. necator isolate with the ability to overcome the Run1 gene, making it an interesting resistance source to prolong the durability of this gene [44]. M.A. Dalbó and colleagues (2001) investigated a new locus of resistance on chromosome 14, later named Ren2, in a population of grape cultivars crossing: "Horizon" and resistant "Illinois 547-1" [45,220]. Run2, Ren1 and Ren2 have a lower incidence of programmed cell death after infection than Run1, which leads to further formation of secondary hyphae [221]. In the case of Ren1, the fungus can receive sufficient nutrition to maintain its life cycle; however, the level of sporulation is about 10 times lower than that observed in susceptible genotypes [219].
Locus Ren3 was identified on chromosome 15 in the cultivars of V. vinifera «Regent» and «Villard Blanc» [45,222]. Later, two regions were identified in this locus-more distinct boundaries of Ren3 and the new locus Ren9 [223]. Moreover, a new qualitative resistance locus, named Ren11, was found on chromosome 15. It was found to be effective in nearly all vineyard environments on leaves, rachis, berries, and stems. Locus on chromosome 8 and 9 had additive effects with Ren11 on the stems [224].
Locus named Ren4 was identified on chromosome 18 in the wild Chinese species V. romanetii [218,225]. Chromosome 18 contains large clusters of the TIR-NBS-LRR category of genes and surpasses all other chromosomes for the number of resistance genes [218]. Locus Ren8 was also identified on chromosome 18 during and after a dry growing season characterized by high temperatures. This locus is likely to mainly affect the leaf resistance [226].
The Ren5 locus was identified on chromosome 14 in V. rotundifolia, which affects both the development of mycelium and the intensity of sporulation. Ren5 manifests its effect after the formation of the first appressorium, delaying and then interrupting the development of mycelium. In the region of Ren5 locus, the presence of 7 NBS-LRR genes, as well as the VvEDR1 defense reaction regulator, was revealed. Thus, Ren5 is likely to belong to the class of NBS-LRR genes responsible for disease resistance [227].
The dominant powdery mildew resistance loci, designated as Ren6 and Ren7, were found in the wild Chinese V. piasezkii grapevine on chromosomes 9 and 19, respectively. Ren6 is associated with complete resistance, whereas Ren7 is responsible for partial resistant with reduced colony size. Genotypes containing Ren7 or Ren6 exhibited HR to powdery mildew infection. In the case of Ren7, HR was much more slower, it occurred in epidermal cells after the penetration of appressoria and the beginning of the secondary hyphae development, whereas in the Ren6 genotypes HR was more severe and was caused by appressoria of germinated spores [190].
The locus Ren10 was detected on chromosome 2, where the Myb-like genes controlling berries color are located [95,107,108,228,229]. Grapevine plants with the Ren10 haplotype showed less colonization by E. necator and decreased sporulation. Ren10 was probably introgressed from a North American species [95].
The major QTL associated with powdery mildew susceptibility in V. vinifera cultivar "Chardonnay" was located on chromosome 9 and named Sen1 (Susceptibility to Erysiphe necator 1) [230].
The majority of loci (Run1, Run2, Ren4, Ren5, Ren6 and Ren7) confer resistance to powdery mildew after the pathogens penetration in cells by inducing programmed cell death (ETI) [188,190,217,227]. The Ren6 locus contributes to a strong restriction of hyphal growth [187,190]. In the case of Ren4, resistance is associated with papilla formation [187,188].

Other Gene Systems for Resistance and Susceptibility to Erysiphe necator
The VvDOF3 TF was identified in V. vinifera, the overexpression of which enhanced resistance to powdery mildew through the SA signaling pathway. DOF proteins are plant-specific TFs that play an important role in plant growth, development, and defense responses. The highest level of VvDOF3 expression was observed in leaves and roots. Rapid induction of VvDOF3 expression occurred under the attack of E. necator and under the influence of SA and JA phytohormones. Transgenic A. thaliana overexpressing VvDOF3 was characterised by increased expression of the SA-sensitive PR1 gene and high concentration of SA [231]. Overexpression of VqTLP29 in V. quinquangularis caused activation of the SA signaling pathway, and could promote JA biosynthesis after infection with powdery mildew. Studies have shown that the level of PR5 expression increases after infection with powdery mildew, with an increase in the expression of PR1 and NPR1. Overexpression of PR5 can increase resistance to biotic and abiotic factors by activating many protective genes in the SA or JA/ET signaling pathway [160].
The WRKY gene family is one of the largest TF families in plants, playing an important role in diverse plant developmental and physiological processes, including plant defense signaling pathways. In grapes 59 identified WRKY genes were classified into 3 groups (I-III) [157]. Upon infection of grapes with E. necator, TFs VvWRKY19, VvWRKY48 and VvWRKY52 are activated [205]. The genes VvWRKY48 and VqWRKY52 belong to Group III, and gene VvWRKY19 belongs to Group II. VqWRKY52 was strongly induced by SA, but not by JA. In transgenic A. thaliana lines its expression was strongly induced 24 h after inoculation with powdery mildew, which contributed to an increase in resistance to E. necator, but at the same time it increased sensitivity to B. cinerea [146,157].
During investigations carried out on grapes, defense-related genes were identified in basal resistance, belonging to the innate immune system of plants, which does not depend on resistance genes: EDS1 (ENHANCED DISEASE SUSCEPTIBILITY 1), EDL2 (EDS1-LIKE 2), EDL5 (EDS1-LIKE 5), and PAD4 (PHYTOALEXIN DEFICIENT 4). The EDS1-PAD4 complex takes part in the SA-mediated defense pathway, and was detected to be more complicated in grapes than in Arabidopsis. EDS1 is a key regulator of basal resistance in various plants, including grapes. Pathogen effectors target EDS1. EDS1 expression is induced by SA and grapes resistance increases with increasing SA levels. Grapevines gene VvEDS1 is a functional ortholog of Arabidopsis gene AtEDS1, VvEDS1-LIKE (EDL)-paralog of AtEDS1. VvEDS1 is expressed higher in veins than in the rest of the leaf infected by E. necator [232]. In Arabidopsis, EDS1 interacts with PAD4 and SAG101, which in turn compete for EDS1 to form nuclear heterodimers. In the grapevine there was reported up to five SAG101-like genes. VvEDS1, VvEDL2 and VvPAD4 may form a complex to regulate the defense system. VvEDL2 was proposed to be a non-functional version of VvEDS1 and either competes with VvEDS1 when interacting with VvPAD4 or regulates the amount of functional VvEDS1 dimers. These protein interactions provide resistance to E. necator [232].
It is well known that an increase in the concentration of stilbene phytoalexins has an important role in global defense mechanisms and is observed in pathogen-resistant grape cultivars, when the growth of pathogen appressorium is suppressed [233]. In the Chinese wild grape species V. pseudoreticulata, resistant to powdery mildew, the VpSTS29/STS2 encoding stilbene synthase was identified. Stilbene synthase catalyzes the synthesis of various stilbenoids, including an antimicrobial low molecular weight natural phytoalexin-resveratrol and piceid, derivative glucosides of resveratrol. Overexpression of VpSTS29/STS2 in powdery mildew susceptible V. vinifera cultivars increases the amount of STS, which induces reprogramming of gene expression. The SA signaling pathway is activated, which induces transcriptional reprogramming of the downstream WRKY-MYB complex, which in turn regulates the synthesis of stilbenes and PR to enhance plant defense at the loci of infection. Expression of STS then promotes the production of the more toxic pterostilbene and ε-viniferin, which enhance basic immunity by inducing programmed cell death. Expression of the grape STS gene improves resistance to biotrophic and semi-biotrophic pathogens, but not necrotrophic organisms. The VqSTS5 gene from V. quinquangularis contributed to the resistance to powdery mildew infection [234]. Among the studied 31 VqSTS genes from V. quinquangularis, it has been revealed that VqSTS21 was up-regulated in response to E. necator. VqSTS21 overexpressing lines of Arabidopsis exhibited enhanced resistance to E. necator, via up-regulation of genes involved in SA-mediated signaling pathway, but displayed increased susceptibility to necrotrophic B. cinerea, with suppressed JA-mediated signaling pathway [139]. Another STS gene VaSTS19, isolated from a Chinese wild grape, V. amurensis. cv. "Tonghua-3", enhanced resistance to powdery mildew and gray mold in Arabidopsis transgenic lines, and contributed to the accumulation of more callose amount in comparison to nontransgenic control plants. Analysis of the expression of several diseaserelated genes suggested that VaSTS19 expression enhanced defense responses though SA and/or JA signaling pathways [235].
The Early-Response to Dehydration six-like (ERD6l) is one of the largest families of sugar transporters in plants. For grapevines, 18 genes of the ERD6l family were identified, one of which (VvERD6l13) was characterized in detail. When berries were infected either by necrotrophic pathogen B. cinerea or by the biotrophic E. necator, the VvERD6l13 gene was activated. VvERD6l13 localizes in the plasma membrane and mediates H+-dependent sucrose transport, thereby suggesting that VvERD6l13 is a sugar transporter involved in the sugar mobilization process and is induced in response to biotic stress. VvERD6l13 expression was detected at all developmental stages, with the highest level at the ripe berry stage. Two sucrose-sensitive elements were detected in the promoter region of this gene, as well as regulatory cis-acting elements, associated with both biotic and abiotic stress and responding to various hormones. It can likely facilitate the extraction of apoplasmic sucrose, thereby limiting the availability of nutrients for the pathogen and reducing the progression of infection [18].
The VvCSN5 gene (subunit 5 of the COP9 signaling complex), negatively associated with grapevine powdery mildew resistance, has been identified. Inactivation of VvCSN5 led to higher levels of expression in leaves of several marker genes linked to plant protection processes (VvPR1, VvPR3, VvPAD4 and VvRBOHD). As a result, an increased resistance to E. necator was noted to be manifested by the deposition of callose on the cell walls at the sites of pathogen penetration attempts and the death of infected epidermal cells. Gene VvCSN5 may be positively correlated with the JA-signaling pathway [191].
Aspartic proteases (AP), one of the superfamilies of proteolytic enzymes, are the extracellular enzymes that may play a role in the degradation of pathogenesis-related proteins in leaves. APs are involved in the regulation of many biological processes, such as the recognition of pathogens, pests and the induction of protective reactions [236,237]. In grapes, 50 AP genes have been identified. It was revealed that the AP13 gene is susceptible to infection caused by E. necator, as well as to salicylic acid exposure. As a consequence of infection caused by B. cinerea and exposure to MeJA, the VqAP13 transcript levels in V. quinquangularis cv. 'Shang-24 decreased, but increased when exposed to ET and SA. Transgenic A. thaliana lines overexpressing VqAP13 showed increased resistance to E. necator and accumulated more callose than wild-type plants, while its resistance to B.
cinerea inoculation was reduced. The results suggest that functioning of VqAP13 promotes the SA-dependent signaling pathway but suppresses the JA signaling pathway [148].

Genetic Editing Capabilities
CRISPR/Cas9-targeted mutagenesis technology allows editing of plant genomes, potentially disabling one or more genes in order to study their function, and is a valuable tool for creating disease-resistant varieties [99,100]. This two-component system is based on the Cas9 nuclease, which is able to cut a double-stranded DNA molecule in a selected target in the presence of a guide RNA (gRNA). To date, there have been several reports on the use of CRISPR-Cas9 for genome editing in grapevine [154,207,[238][239][240][241]. The application of CRISPR/Cas9-targeted mutagenesis allowed to reveal that VvMLO11 (W) belongs to S-genes promoting infection of grapes with powdery mildew, and the targeted mutation of VvMLO11 (W) leads to enhanced resistance to powdery mildew in edited lines, which was associated with host cell death, cell wall apposition (CWA) and H 2 O 2 accumulation. A homozygous Vvmlo3 mutant seedling with knocked out VvMLO3 (VvMLO11 (W)) died from massive leaf necrosis in tissue culture medium. Significantly increased resistance to powdery mildew was observed in several heterozygous VvMLO11 (W) lines in comparison with WT plants, which suggested that VvMLO11 (W) is one of the functional homologues of AtMLO2 in grapevines. Simultaneous directed mutagenesis of all three MLO genes (VvMLO6 (W), VvMLO7 (W) and VvMLO11 (W)) may be necessary for the development of complete resistance of grapevines to powdery mildew in the future [207]. The VvWRKY52 gene knock out in grapes by means of CRISPR/Cas9-targeted mutagenesis led to a transgenic line phenotype with increased resistance to B. cinerea [154].

Conclusions
Over the past decade, significant progress has been made in the study of the grapes system of genes for resistance and susceptibility to Erysiphe necator and Botrytis cinerea. The most important direction of the future viticulture is based on infections suppression with the least amount of chemicals, development and introduction of new pathogen-resistant varieties, preservation and enhancement of nutrient content, obtaining higher yields, and restoration of crop properties unintentionally lost during domestication. Currently, breeding and interspecific hybridization have been replaced by new technologies of cisgenesis / intragenesis and editing of genes (or genome) as more relevant new biotechnologies. This has improved the breeding efficiency and has dramatically increased the range of plant properties that can be obtained.
Author Contributions: Writing-original draft preparation and editing, J.F.; writing-review and editing, Y.U., N.T., E.K. and R.I.; conceptualization and supervision, R.I. and E.K. All authors have read and agreed to the published version of the manuscript.

Funding:
The work is supported by the Sirius University of Science and Technology project: GNZH-RD-2008.