Categories of plant defense mechanisms against pathogens.
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.
- plant defense mechanism
- induced resistance
- pathogenesis-related (PR) protein
- pathogen control
Higher plants possess a variety of defense mechanisms against pathogens. Pathogenesis-related (PR) proteins are induced in response to infection by pathogens. PR proteins are 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
2. Major pathogens and their control in grapevine
Grapevine pathogens are roughly divided into fungi, bacteria, virus, and others.
2.1. Major pathogens
2.1.1. Powdery mildew
Powdery mildew caused by ascomycete
2.1.2. Gray mould
Gray mould caused by ascomycete
2.1.3. Downy mildew
Downy mildew is caused by oomycete
2.2. 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
3. Plant defense mechanism
Plant defense mechanism is roughly classified into two categories: constitutive (static) resistance and induced (active) resistance (Table 1).
|Constitutive (static) resistance [resistance inherent in plants]|
|Physical resistance||Thickness and hardness of cell wall|||
|Hydrophobic environment created by cuticle layer|
|Chemical resistance||Antimicrobial substances, such as phenol and saponin|||
|Induced (active) resistance [resistance newly induced by pathogen attack]|
|1||Formation of papilla||Physical and chemical barrier against penetration|||
|2||Hardening of cell wall||Lignification|||
|Crosslinked polymers with glycoprotein|
|3||Hypersensitive reaction||Containment of pathogen by autocide activity of cells|||
|Generation of ROS|
|4||Production of phytoalexins||Low molecular weight antimicrobial substance|||
|5||Production of PR proteins||With antimicrobial activity|||
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) production 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].
4. 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 , including grape.
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 [21, 22]. PR proteins are also induced under conditions of nonpathogenic origin, such as stress. Examples include cytoplasm separation  and high concentrations of plant hormones .
|PR-2||β-1,3-Glucanase||Cell wall (β-1,3-glucan)||[35–39, 56]|
|PR-3||Chitinase (types I, II, IV, V, VI, and VII)||Cell wall (chitin)|
|PR-4||Chitinase (types I and II)||Cell wall (chitin)|
|PR-5||Thaumatin-like||Plasma membrane||[37, 40–42]|
|PR-10||Ribonuclease (like)||RNA||[43–46, 57]|
|PR-14||Lipid-transfer protein||Involvement in defense signaling pathway||[47–54, 58–61]|
|PR-15||Oxalate oxidase||Production of H2O2 with|||
|PR-16||Oxalate oxidase-like protein||Antimicrobial activity|
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 , 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 , 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.
5. PR protein gene in
5.1. Pathological function
5.1.1. PR-1 (unknown)
Although PR-1 proteins exhibit antifungal activity, their functions remain unclear.
LAR is induced by pathogen attack. SA and JA act as a second messenger . 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
5.1.2. 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-1,3-glucanases 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 , expression of PR-3, PR-4 gene depends on the JA in
Jacobs et al.  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 . Apoplasmic β-1,3-glucanase gene (
5.1.3. PR-5 (thaumatin-like proteins)
PR-5 proteins include thaumatin-like proteins and osmotin. The amino acid compositions and the NH2 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 . PR-5 proteins are believed to be involved in enhancing fungal membrane permeability and causing osmotic rupture of fungal plasma membrane . Jayasankar et al.  demonstrated
5.1.4. 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 . PR-10 gene (VpPR10.2) isolated from
5.1.5. 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 [49–51]. Type I LTP of tobacco binds jasmonic acid (JA), a signaling molecule, and the complex interacts with receptors on the cell membrane . Some grape LTPs bind JA. The external application of the VvLTP4-JA complex to grape plantlets enhanced resistance to
5.1.6. PR-15 (oxalate oxidases) and PR-16 (oxalate-oxidase-like proteins)
PR-15 and PR-16 include germins (oxalate oxidases) and germin-like proteins (oxalate-oxidase-like proteins), respectively. Many germin-like proteins exhibit oxalic acid ester oxidase (OXO) or superoxide dismutase (SOD) activity. They are involved in the production of H2O2 a ROS, which has antimicrobial activity. Seven germin-like protein (GLP) cDNA clones were isolated from
PR-6, PR-7, PR-8, PR-9, PR-11, PR-12, and PR-13 are proteinase inhibitors, endoproteases, chitinases (type III), peroxidases, chitinases (type I), defensins, and thionins, respectively. To the best of our knowledge, these proteins have not yet been detected in grape.
5.2. Physiological function
Some PR protein families have physiological functions. For example, PR-2 proteins (β-1,3-glucanases) 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 . Many examples of PR-10 and PR-14 proteins have been reported in grape. The overexpression of grapevine PR-10 gene (
6. Application of PR proteins to pathogen control in
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.
6.1. 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
|Target cultivar||Introduced gene||Gene source||Acquired resistance to||Reference|
|‘Crimson Seedless’||Chitinase and β-1,3-glucanase||Wheat|||
|‘Thompson Seedless’||TLP (cisgenic)||Chardonnay|||
|‘Seyval blanc’||Chitinase and RIP||Barley||No effect|||
Most commercially valuable
Recently, PR proteins aside from the above have been used and strategies other than transgenic approaches have been attempted.
6.2. New chemical control method using elicitors
Transgenic grapevines are forbidden in French vineyards. Aziz et al.  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
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 development 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
Special thanks go to Ms. Kayo Arita, Mr. Masachika Mikami, and Mr. Yoshinao Aoki of the University of Yamanashi for their assistance.