Pathogenesis-Related Proteins in Grape

An overview of major pathogens and their control, plant defense mechanisms, and pathogenesis-related (PR) proteins and their roles in pathogen control is presented herein. Vitis vinifera, including wine grape and table grape, is one of the most valuable horticultural crops in the world because of its commercial use. However, V. vinifera cultivars are extremely susceptible to pathogens, particularly fungi and oomycetes, such as Botrytis cinerea and Plasmopara viticola, respectively. Plants have various defense mechanisms to counter these pathogens. One example is induced resistance, which involves the induction of the immune system in the event of a pathogen attack, including the generation of PR proteins. Some PR proteins possess antimicrobial activity. PR proteins are classified into 17 families, some of which are found in grape. Thus, their roles in grape have been actively studied. A new strategy to increase plant resistance to pathogens has been developed. A good understanding of grape defense mechanism through PR proteins is expected to open new doors to improve grape quality and yield by efficiently controlling pathogens in the future.


Introduction
Vitis vinifera is one of the most important grape species for wine making. However, it is extremely susceptible to such pathogens as fungi and oomycetes. An understanding of grape defense mechanism is important because infection by pathogens leads to marked loss of fruit quality and yield.
classified into 17 families according to molecular structure and enzyme activity. The functions of all the families have not been reported in grape.
In this chapter, we review PR proteins in V. vinifera by presenting an overview of the latest knowledge of PR proteins in grape.

Major pathogens and their control in grapevine
Grapevine pathogens are roughly divided into fungi, bacteria, virus, and others. V. vinifera cultivars in particular are extremely susceptible to diseases caused by fungi and oomycetes, which result in huge economic losses worldwide. Of those diseases, the most prevalent are powdery mildew, gray mould, and downy mildew. Their characteristics, the resistance of grapevine to these diseases, and pathogen control in grapevine are described below.

Powdery mildew
Powdery mildew caused by ascomycete Uncinula necator (syn. Erysiphe necator) is one of the most well-known fungal diseases in viticulture. In recent years, the decline of the effects of chemical fungicides and the emergence of climate suitable for fungal growth have led to the development of powdery mildew epidemic in European vineyards [1]. The symptoms are white powdery spots on the entire grapevine. Infected berries become brown and crack, resulting in loss of yield and seriously altering wine quality. Most V. vinifera cultivars are susceptible to U. necator, although some Vitis species show various levels of resistance [1][2][3].

Gray mould
Gray mould caused by ascomycete Botrytis cinerea is the major fungal disease in humid and temperate regions of the world [4]. B. cinerea invades a variety of agricultural crops and has a broad host range. In grape, the symptoms appear as 'gray mould' on the lesioned part of infected leaves, flower clusters, and ripening grape berries. B. cinerea infection likely occurs at all stages of grape growth. The development of gray mould in ripening berries results in loss of yield and berry quality.

Downy mildew
Downy mildew is caused by oomycete Plasmopara viticola. It is one of the most harmful diseases in grape grown in Europe and in the US [5]. Downy mildew appears as oily yellow spots on the surface of infected leaves. Loss of yield and lowered berry quality occur due to weakening of young shoot and death of leaf tissue. Boso et al. [6] investigated the susceptibility of V. vinifera cultivars and other Vitis species to downy mildew. V. vinifera 'Cabernet Sauvignon' was the least susceptible, whereas non-vinifera cultivars did not show symptoms of downy mildew in the field.

Pathogen control by chemical fungicide
The above diseases caused by ascomycete and oomycete are generally controlled by spraying chemical fungicides, such as quinone outside inhibiting (QoI) fungicides, in the vineyard. QoI fungicides, which act by inhibiting fungal mitochondrial respiration, is one of the most widely used agents against pathogens in viticulture. However, in addition to the adverse effects of these fungicides, the emergence of fungi resistant to these fungicides has been reported [7,8]. Therefore, restrictions and laws for use have been set by individual countries. The resistance of P. viticola to QoI fungicide is acquired by G143A mutation in cytochrome b that constitutes mitochondrial electron transport chain complex III [8,9].

Plant defense mechanism
Plant defense mechanism is roughly classified into two categories: constitutive (static) resistance and induced (active) resistance ( Table 1).

Category
Feature Reference

Constitutive (static) resistance [resistance inherent in plants]
Physical resistance Thickness and hardness of cell wall [10] Hydrophobic environment created by cuticle layer Chemical resistance Antimicrobial substances, such as phenol and saponin [11] Induced (active) resistance [resistance newly induced by pathogen attack] 1 Formation of papilla Physical and chemical barrier against penetration [12] 2 Hardening of cell wall Lignification [13] Crosslinked polymers with glycoprotein 3 Hypersensitive reaction Containment of pathogen by autocide activity of cells [14] Generation of ROS 4 Production of phytoalexins Low molecular weight antimicrobial substance [15] 5 Production of PR proteins With antimicrobial activity [16]  Constitutive resistance is a prophylactic resistance mechanism inherent in plants and is divided into physical resistance and chemical resistance. The former is the first barrier against pathogens created by the cell wall. The latter is realized by antimicrobial substances present in plants, such as polyphenols.
On the other hand, induced resistance involves the induction of the immune system by pathogen attack and is roughly divided into five types: (1) formation of papilla, (2) hardening of cell wall, (3) hypersensitive response (HR), (4) production of phytoalexins, and (5) produc-tion of PR proteins. (1) and (2) are resistance acquisition through the formation of a physical barrier. The generation of reactive oxygen species (ROS) by HR induces another type of defense mechanisms. Phytoalexins and some PR proteins, on the other hand, have antimicrobial activity. All the above-mentioned types of induced resistance are accompanied by changes in the metabolic system and take place in not only infected cells localized acquired resistance (LAR), but also the whole plant systemic acquired resistance (SAR).
These types of defense mechanisms in induced resistance operate by sensing a substance called elicitor on the receptor. The elicitors include abiotic substances, such as heavy metals and synthetic compounds in the form of fungicides, and biotic substances, such as proteins, lipids, oligosaccharides, and antibiotics of biological origin. In fact, elicitors are found in the cell wall of pathogens, and PR proteins, such as chitinase and β-1,3-glucanase, function indirectly by releasing oligosaccharide elicitors from the cell wall of pathogens [17,18].

Definition and classification of PR proteins
In this section, we define and classify in detail the PR proteins described above. PR proteins were discovered for the first time in tobacco leaves, indicating the plant's hypersensitive reaction to tobacco mosaic virus (TMV) [14,19]. These proteins are found in many plant species [20], including grape.

Definition
PR proteins are proteins encoded but not expressed in host plant in the absence of interaction with a pathogen. They are also defined as proteins generally induced in an infection [

Classification
PR proteins share many biochemical properties that render them easily distinguishable. They have relatively low molecular weights, are stably extractable at low pH [25,26], are highly resistant to proteases [27], and have extreme isoelectric points. Most of them are located in the apoplast [28,29]. In general, acidic PR proteins are located in the apoplast and basic ones, in the vacuole. PR proteins are classified on the basis of amino acid sequences, serological reaction, enzymatic activity, and others. Five groups of PR proteins (PR-1 to PR-5) were initially characterized in tobacco. Currently, PR proteins are categorized into 17 families [30], but not all are found in grape ( Table 2). In the next section, the roles and functions of PR proteins in grape ( Table 2) are described in detail.

PR-1 (unknown)
Although PR-1 proteins exhibit antifungal activity, their functions remain unclear. V. vinifera PR-1 was identified and cloned [31]. However, in grapes, signaling pathway related to PR-1 is remains to be determined. The signaling network of LAR and SAR has been well studied in recent years in Arabidopsis thaliana (Figure 1). LAR is induced by pathogen attack. SA and JA act as a second messenger [32]. In response to SA, positive regulator protein non-expresser of pathogenesis-related genes 1 (NPR1) is transported to the nucleus and activate the expression of PR protein genes, including the PR-1 [33]. On the other hand, JA dependent pathway is upregulated by MYC2, transcription factor, and induces plant defense-related genes, such as the plant defensin 1.2 (PDF1.2) [34]. As a result, LAR is induced in pathogen-infected cells. Further, when SA and JA signaling are transported to other cells, SA and JA similarly induce the expression of plant defense-related genes through the NPR1 and MYC2. Finally, SAR is expressed in other noninfected cells. Some classes of PR proteins, including PR-1, are known to be expressed in association with SAR.

PR-2 (β-1,3-glucanases) and PR-3 and -4 (chitinases)
PR-2 proteins are β-1,3-glucanases and PR-3 and -4 proteins are chitinases. Because β-1,3glucanases and chitinases were discovered as PR proteins early on, they had been widely studied for their roles in plant defense against pathogens in many species, including grape. They exert antimicrobial activity as a result of their ability to hydrolyze fungal cell wall components. The former hydrolyze β-1,3-glucan and the latter hydrolyze chitin. The synergistic effects of β-1,3-glucanases and chitinases inhibit the growth of fungal pathogens [35,36]. These proteins function indirectly by releasing the elicitor of oligosaccharides from the cell wall of pathogens, thereby inducing various plant defense mechanisms. As shown in Figure 1, the expression of PR-2 gene along with the PR-1, PR-5 gene is dependent on SA [33], expression of PR-3, PR-4 gene depends on the JA in Arabidopsis [32].
Jacobs et al. [37] showed that the hydrolytic activity in grape directly affects the extent of infection by powdery mildew at the pathogen infection site. β-1,3-Glucanase and chitinase activities were strongly induced in leaves and pre-véraison berries by ethephon treatment. Moreover, PR protein expression was decreased during grape maturation, which explains the increased susceptibility of grape to pathogen attack at the final stage of maturation [38]. Apoplasmic β-1,3-glucanase gene (VvGHF17) is expressed constitutively in grape leaves, berry pulp, and skin, and VvGHF17-overexpressing Arabidopsis plants exhibited disease resistance to B. cinerea and Colletotrichum higginsianum [39].

PR-5 (thaumatin-like proteins)
PR-5 proteins include thaumatin-like proteins and osmotin. The amino acid compositions and the NH 2 terminal sequences of thaumatin-like proteins showed that thaumatin-like proteins are actually osmotins, which are known to accumulate in tobacco cells in response to osmotic stress [40]. PR-5 proteins are believed to be involved in enhancing fungal membrane permeability and causing osmotic rupture of fungal plasma membrane [41]. Jayasankar et al. [42] demonstrated in vitro and in vivo that the constitutive expression of V. vinifera thaumatin-like protein 1 (VVTL-1) in V. vinifera protected grape from anthrax. Treatment with ethephon, which effects ethylene generation, induced grape PR-5 gene families in leaves and berries [37].

PR-10 (ribonuclease (like))
PR-10 proteins exhibit ribonuclease (RNase) activity. RNase contributes to plant defense in programmed cell death during HR or acts directly against pathogens [43]. PR-10 gene (VpPR10.2) isolated from Vitis pseudoreticulata exhibited resistance to fungi and had high homology with VvPR10.2 from susceptible V. vinifera cultivar. In contrast to VvPR10.2, VpPR10.2 was induced at high levels in response to P. viticola infection [44]. On the other hand, PR-10 proteins with cytokinin-binding activity were identified in mung bean [45] and moss [46]. Questions persist regarding the roles of PR-10.

PR-14 (lipid transfer proteins)
PR-14 proteins are lipid transfer proteins (LTPs). Some LTP-like polypeptides show antifungal or antibacterial activity [47,48]. Several isoforms are involved in the plant defense signaling pathway [49][50][51]. Type I LTP of tobacco binds jasmonic acid (JA), a signaling molecule, and the complex interacts with receptors on the cell membrane [52]. Some grape LTPs bind JA. The external application of the VvLTP4-JA complex to grape plantlets enhanced resistance to B. cinerea infection compared to the application of either VvLTP4 or JA alone [53]. Such elicitors as ergosterol triggered VvLTP1 upregulation by WRKY transcription factor and stilbene synthase gene expression in grape plantlets, and enhanced the production of resveratrol (grape phytoalexin) and the resistance to B. cinerea [54].

Physiological function
Some PR protein families have physiological functions. For example, PR-2 proteins (β-1,3glucanases) hydrolyze β-1,3-glucan in fungal cell wall, but because β-1,3-glucan (called 'callose' in plants), the substrate of β-1,3-glucanase, is widespread in plants, PR-2 proteins must perform various physiological functions, such as flower formation [56]. Many examples of PR-10 and PR-14 proteins have been reported in grape. The overexpression of grapevine PR-10 gene (Vvpr10) in Saccharomyces cerevisiae conferred salt tolerance in the yeast [57]. Some LTP isoforms are involved in somatic embryo development and epidermal layer formation. LTP gene is expressed during zygotic and somatic embryogenesis [58][59][60]. The overexpression of VvLTP1 gene interferes with somatic embryo development and invalidates the bilateral symmetry of the embryo in grape [61].

Application of PR proteins to pathogen control in V. vinifera grapevine
Fungicides are used to control fungal diseases. However, their adverse effects on the environment and the appearance of fungi resistant to the fungicides have been reported. Therefore, the development of new plant disease control methods is desired. In recent years, new strategies to increase plant resistance have been examined. Among them, PR-protein-related pathogen control methods are described here.

Molecular breeding
For a long time, researchers and breeders have used conventional breeding methods for the development of disease-resistant cultivars from available resources in genus Vitis. However, conventional breeding methods are hampered by a major problem, namely, hybrid produced by crossing often exhibits undesirable traits from hybrid parent resistant to fungi. In order to remove these traits, backcrossing is performed many times. On the other hand, molecular breeding by a transformation method can use breeding resources other than those from genus Vitis and offers the possibility of improving only the objective trait, such as disease resistance, without modifying other viticulturally desirable traits in the target cultivars.

Target cultivar Introduced gene Gene source Acquired resistance to Reference
'Merlot' Chitinase Trichoderma B. cinerea and U. necator [63] 'Chardonnay' 'Chardonnay' Chitinase Trichoderma B. cinerea and U. necator [65] 'Thompson Seedless' Chitinase Trichoderma B. cinerea [64] 'Neo Muscat' Chitinase Rice U. necator and Elsinoe ampelina [66] 'Pusa Seedless' Chitinase Rice U. necator [67] 'Crimson Seedless' Chitinase and β-1,3-glucanase Wheat P. viticola [68] 'Thompson Seedless' TLP (cisgenic) Chardonnay U. necator [70] 'Seyval blanc' Chitinase and RIP Barley No effect [69] β-1,3-Glucanase (PR-2); chitinase (PR-3 and -4); TLP, thaumatin-like protein (PR-5); RIP, ribosome-inactivating protein. Most commercially valuable V. vinifera cultivars are susceptible to pathogens, particularly fungi, as described above. Many studies that have attempted to confer fungal resistance to V. vinifera cultivars, including wine grape and table grape, via the transformation technique have been reported. The major findings appear in Table 3. The first report of a transformation technique for V. vinifera was by Kikkert et al. [62,63]. The introduction of chitinase from Trichoderma conferred disease resistance to V. vinifera cultivars and established a transformation system with embryogenic cultures of V. vinifera 'Merlot' and 'Chardonnay' by a biolistic method. Thereafter, examples of the Agrobacterium-mediated transformation method using transgenic chitinase were reported [64][65][66][67][68]. Nookaraju and Agrawal [68] also reported that the introduction of both β-1,3-glucanase (PR-2) and chitinase in transgenic plants increased resistance to downy mildew. Transgenic plants that displayed no resistance in field tests have also been reported [69]. The increase of disease resistance by chitinase introduction has been reported in other plants as well. Chitin is the main component of the cell wall in major pathogens, U. necator, B. cinerea, and P. viticola. As higher plants do not contain chitin, chitinase does not affect growth and development in plants. Therefore, this control strategy using chitinase is considered to be very effective.
Recently, PR proteins aside from the above have been used and strategies other than transgenic approaches have been attempted. V. vinifera 'Crimson Seedless' expressing cisgenic thaumatinlike proteins (TLP, PR-5) from V. vinifera 'Chardonnay' displayed resistance to powdery mildew [70]. Although the transformation method is adopted, cisgenic plants are produced by using a gene derived from a closely related species originally present in nature as genetic resources. Although cisgenic plants fall under regulations for transgenic plants, discussions are underway as to whether cisgenic plants should be excluded from the regulations in the EU and the US [71].

New chemical control method using elicitors
Transgenic grapevines are forbidden in French vineyards. Aziz et al. [72] proposed an alternative strategy for controlling pathogens, which is the activation of plant defense mechanism by elicitors. Defense reactions elicited by laminarin increase PR protein (chitinase and β-1,3-glucanase) activities and confer resistance to B. cinerea and P. viticola growth [72]. It is considered that the induction of natural plant defense mechanisms by elicitors is an attractive and powerful method compared with chemical fungicides [73]. Indeed, a number of positive results against downy and powdery mildew and gray mold have been reported in laboratories and greenhouses [74]. However, little research has been carried out in vineyards. In addition, results comparable to those obtained in laboratories have not been generated and its effect seems to be weaker than that of the conventional fungicide method. This variability is likely due to environmental, plant, pathogen, and external conditions. To quote Delaunois et al. [74], 'Additional research needs to be pursued to fully understand defense stimulation under vineyard conditions.'

Conclusions
Knowledge of the roles and functions of genes encoding PR proteins in grape has been accumulated from previous studies of other plants, such as tobacco, and used in the develop-ment of new methods for controlling pathogens in grape. However, questions remain, such as the presence of PR proteins of other classes in grape and the regulation PR protein genes involved in the plant defense mechanism. Elucidating the answers to these questions at the molecular level will help further our understanding of PR proteins in V. vinifera and create possibilities to improve grape quality and yield by efficiently preventing pathogen growth in the future. [20] Van Loon, L. C. (1985). Pathogenesis-related proteins. Plant Molecular Biology, 4(2), 111-116.