**2. PR proteins and their classifications**

PR proteins are defined as "Proteins encoded by the host plant but induced only in pathogenic or related conditions" [10]. Plant PR proteins were discovered and published for the first time in tobacco plants infected with the tobacco mosaic virus [11] and initially, only PR-1, PR-2, PR-3, PR-4, and PR-5 classes of PR proteins were reported from tobacco plants, but later different PR proteins were found from numerous plants [12]. These low molecular weight proteins (6–43 kDa) are heat stable, protease-resistant and soluble at acidic pH (<3) [13]. PR proteins are currently classified into 19 major families based on their enzymatic activity, biological roles, and amino acid sequences, as indicated in **Table 1** [31, 32]. These include antifungal (PR1), hydrolytic β-1,3- Glucanase (PR2), chitinases (PR 3, 4, 8,11), thaumatin (PR5), proteinase inhibitors (PR6), endo-proteinase (PR7), peroxidase (PR9), ribonuclease-like (PR10), plant defensins (PR12), plant thionins (PR13), lipid transfer proteins (PR14), oxalate oxidase protein family (PR15 and PR16) secretory protein (PR17) and carbohydrate oxidases (PR 18) [7, 32]. A novel antimicrobial protein from *Pinus sylvestris* was isolated and classified as PR19 [31].

### **3. PR proteins: functional characterization and mode of action**

Plants are constantly being challenged by disease-causing organisms that have co-evolved with the evolution of plant hosts' defense mechanisms. Many PR proteins have been shown to possess antifungal, antibacterial, antiviral and antinematode properties [13]. Different PR proteins have a distinct mode of action against the pathogen depending upon the type of pathogen and the activities of the majority of these protein families are known or can be inferred. PR-1 protein, one of the dominant groups of PRs induced by the pathogen, inhibits pathogen growth by binding and sequestration of sterols from the pathogen. Moreover, the programmed cell death is also inhibited by PR1 upon pathogen infection by releasing a defense signal peptide CAPE1 (CAP-derived peptide 1) [33]. Some PR proteins function as hydrolytic enzymes, *viz.* the PR-2 (endo-β-1,3-glucanases) and PR-3, −4, −8 and − 11 (endochitinases) [34, 35]. They function as antifungal proteins by catalyzing hydrolytic cleavage of major components of fungal and oomycete cell wall, i.e. β-1,3-glucan (by the breakdown of β-1,3-glucosidic linkages) or chitin (by the breakdown of internal β-1,4-glycoside bonds) respectively, resulting in the breakdown of the fungal cell wall [36, 37]. Different isoforms of glucanases and chitinases are produced depending upon the plant-pathogen interaction.

Thaumatin-like proteins or Osmotin-like proteins such as PR5 inhibit hyphal growth and spore germination by producing transmembrane pores leading to fungal cell leakiness and blocking the function of plasma membrane receptors molecules involved in cAMP/RAS2 signaling pathways. Also, antifungal action has been demonstrated in some family members, predominantly against oomycetes. PR-5 was also demonstrated to exhibit potato cell's defense against *Phytophthora infestans* by forming a cytoplasmic aggregation through an actin-binding complex [38]. Proteinase inhibitors (PIs) such as trypsin inhibitors and serine inhibitors) belonging to PR6 family proteins, implicated in broad-spectrum defense activity, including suppressing pathogenic nematodes, insects and other herbivores, fungi and bacteria [39]. PIs can provide defense against pathogens, decreasing the lyase activity essential for fungal pathogenicity [40], inhibiting the viral replication cycle [41] and restricting the

digestive enzyme activity of nematodes and insects, limiting amino acid release [42]. In addition, HyPep (proteinase inhibitor peptide) also causes cell aggregation and pseudo-mycelia development by inhibiting amylase and serine proteinases [43]. Also, PIs can block chitin synthesis in fungal cell walls by inhibiting endogenous trypsin that is essential for chitin synthase, thus inhibiting fungal growth and development [44].

PR-7 is a major protein that has only been examined in tomatoes as an endoproteinase. It is an antifungal auxiliary protein that aids in destroying fungal cell wall proteins, chitinases, and glucanases [45]. The PR-9 family of peroxidases is believed to have a role in plant cell wall strengthening by facilitating lignin deposition in response to microbial invasion [46]. In susceptible wheat varieties, the transcription level of PR9 is considerably reduced after infestation with the aphid-transmitted fusarium virus and hessian flies [47]. This showed that PR9 catalyzes lignin deposition to protect susceptible cultivars from BPH.

The members of PR10 protein families exhibit ribonuclease activity required to inhibit the growth of pathogenic fungi. The antifungal activity of ribonucleases develops due to penetration of the pathogen and the destruction of cellular RNAs due to phosphorylation of PR10. It further leads to plant cell death at the inoculation site, causing apoptosis and the hypersensitivity reaction [48]. These intracellular PRs may be active against viruses due to their ribonuclease activity, although their ability to cleave viral RNA has yet to be shown.

The PR-12 type defensins, PR-13 type thionins, and PR-14 type lipid transfer proteins show antifungal and antibacterial activity, interacting with the target microorganism's biological membrane, leading to altered membrane permeability [49, 50]. Plant defensins are divided into two groups based on the structure of their precursor proteins: class I and class II. Class I defensins have endoplasmic reticulum (ER) signaling sequences along with defensin domains. In contrast, class II defensins contain an additional domain of 27–33 amino acid residues called C-terminal prepropeptide (CTPP) [51]. Due to a lack of signal sequences, class I defensins do not undergo post-translational modification or subcellular targeting. They accumulate in the cell wall and extracellular space directly upon synthesis through the secretory pathway [52]. However, class II defensins undergo proteolysis in the vesicles due to CTPP signal peptides targeting vesicles and releasing mature short peptides. Mature defensins consist of five segments of non-conserved loops, linking α-helices and β-strands to form high-level structures. Differences in the loop sequences confer different functions, including inhibition of protein synthesis, antimicrobial activity, heavy metal tolerance, plant development, and blocking of ion channels [53].

Oxalate oxidases (PR-15 family) and oxalate-oxidase-like proteins (PR-16 family) play an important role in plant defense [54]. These are essential enzymes to produce reactive oxygen species (ROS) during apoplastic oxidative burst [55]. ROS are produced in the apoplast by an enzyme that produces H2O2 and CO2 when it reacts with oxalic acid. Proteolytic enzymes of the PR17 family play an important role in defense against fungi and viruses. PR19 protein binds to fungal cell wall glucans altering cell wall structure, leading to morphological distortion of hyphae [31].

### **4. PR protein activation as a defense response**

Plant cells have evolved to activate and recruit the cellular machinery in response to various stresses to optimally utilize resources and sustain life. Accordingly, plants modulate genes' expression, activating a wide range of plant protectants and defense *Pathogenesis-Related Proteins and Their Transgenic Expression for Developing Disease-Resistant… DOI: http://dx.doi.org/10.5772/intechopen.106774*

#### **Figure 1.**

*Overview of the activation of defense response against the pathogen including induction of PR proteins locally as well as systematically.*

genes [56]. The pathogenesis-related (PR) protein activation and production are crucial in response to an invading pathogen [57]. While healthy plants may produce a trace amount of PR proteins, they are produced in higher concentrations in response to pathogen attacks, elicitor treatment, wounding, or other stress.

Plants defend themselves against pathogen attacks by employing a variety of defense mechanisms for their survival and fitness [58]. After the pathogen challenge, plants trigger basal defense mechanism, i.e., pattern triggered immunity (PTI), by recognizing the pathogen-associated molecular patterns (PAMPs) and induced defense mechanism, i.e., effector-triggered immunity (ETI) [59]. PTI and ETI are accompanied by a set of preformed defenses (structural and biochemical barriers) and/or induced defense responses (hypersensitive reactions) that usually combat pathogen attacks [60]. Depending upon the plant-pathogen interaction, these defense responses are associated with a coordinated and integrated set of metabolic alterations that lead to induction of systemic acquired resistance (SAR) or induced systemic resistance (ISR) through activation of defense signaling pathways viz., salicylic acid (SA) and jasmonic acid (JA)/ ethylene (ET) respectively. The activation of SA or JA signaling pathway leads to downstream activation and accumulation of PR gene products locally as well as systematically (**Figure 1**). As a result, PR proteins are related to the development of systemic acquired resistance (SAR) or a hypersensitive response (HR) to pathogenic fungi, bacteria, and viruses. Many plant species from many families have been shown to be induced by PRs, implying that PRs have a broad protective effect against biotic stress [57].

### **5. Role of signaling pathways in PR protein induction**

Depending upon the host-pathogen interaction, different signaling systems are activated, producing different sets of PR proteins that provide disease resistance in plants. Basically, pathogens can be categorized into two types depending on the mode of infection: biotrophic and necrotrophic. Based on the type of pathogen, the pathogenic elicitors induce the production of different secondary signals such as ROS, jasmonates, salicylic acid or ethylene, which further induce the expression of different PR genes. Within the plant species, these secondary signals' spatial and temporal

production vary depending on pathogen type [61]. Classically, the resistance against biotrophic pathogens is conferred through the salicylic acid (SA) pathway, whereas against necrotrophic pathogens is conferred through activation of jasmonic acid/ ethylene (JA/ET) pathways [62].

In plant-biotrophic pathogen interaction, the SA signaling system induces the expression of signature PR genes related to this pathway *viz* PR1, PR2, PR5, PR8, PR9 and PR10 [63]. The transcription studies (overexpression of PR genes), as well as mutational studies (SA mutants such as *nim1, npr1, sai1, nahG*), have provided evidence of the dependence of these PR genes on SA signaling pathway [64]. SA-mediated defense signaling regulates the expression of the PR genes through binding with and activating the NPR1 (due to conformational changes). Activated NPR1 interacts with transcription factors such as TGACG-binding factor (TGA), thus inducing defense gene expression [65]. However, in plant-necrotrophic pathogen interaction, it has been found through transgenic expression of PR genes as well as JA mutant analysis that the JA/ET signaling pathway induces the expression of PR3, PR4, PR10, PR11, PR12 and PR13 genes [64]. ET signaling pathway induces the expression of PR genes by activating the ETHYLENE RESPONSE FACTOR (ERF) transcription factor through activation of EIN2 and EIN3 proteins. However, in the JA signaling pathway, JAZ (jasmonate ZIM domain) protein is degraded by COL1 (coronatine insensitive 1) mediated 26S proteasome leading to activation of MYC2 transcription factor and hence transcription of JA responsive genes [66]. Furthermore, applying JA or SA hormones (defense hormones) increases the PR genes' transcription level, providing a broad spectrum of resistance [67]. During plant-pathogen interaction, hormonal crosstalks also occur, which can provide novel insights for disease resistance. PR-6 in tomato leaves generated by systemic and jasmonic acid was suppressed by exogenous application of SA. When a pathogen infects tobacco, ethylene may operate downstream of jasmonic acid to activate PR-2 and 3.

The ERF branch's ET/JA-regulated transcription factors are inhibited by the negative regulators of the SA signaling pathway. Also, SA biosynthesis is inhibited upon activation of the ET/JA signaling pathway, depicting these pathways' antagonistic role in defense response [68].
