Pathogenesis-Related Proteins and Their Transgenic Expression for Developing Disease-Resistant Crops: Strategies Progress and Challenges

Abstract

Various pathogenic microorganisms (such as fungi, bacteria, viruses and nematodes) affect plant viability and productivity. However, plants combat these pathogens by inducing their defense mechanism to sustain their fitness. The aggregation of pathogenesis-related (PR) proteins in response to invading pathogens is a crucial component of a plant’s self-defense mechanism. PR proteins induce innate resistance in plants through fungal cell wall disintegration, membrane permeabilization, transcriptional suppression, and ribosome inactivation. Earlier studies have demonstrated their crucial role in determining resistance against phytopathogens, making them a promising candidate for developing disease-resistant crop varieties. Plant genetic engineering is a potential approach for developing disease-resistant transgenic crops by employing several PR genes (thaumatin, osmotin-like proteins, chitinases, glucanases, defensins, thionins, oxalate oxidase, oxalate oxidases like proteins/germin-like proteins and LTPs). Furthermore, the overexpression of PR proteins enhances the resistance against phytopathogens. As a result, this chapter gives an overview of PR proteins, including their classification, functional characterization, signaling pathways, mode of action and role in defense against various phytopathogens. It also highlights genetic engineering advances in utilizing these genes singly or synergistically against various phytopathogens to impart disease resistance. Various challenges faced with the products of transgenic technology and synergistic expression of different groups of PR proteins were also discussed.

1. Introduction

With the rapid expansion in the world population, the area under cultivation has decreased [1]. Moreover, biotic stress has been a significant challenge for farmers since the dawn of agriculture. Global yield loss due to plant pathogens is estimated at 16% [2]. To overcome the economic loss in agricultural production, most research in this field focuses on protecting crops against pathogens, insect pests and nematodes. Crop production and productivity can be enhanced by significant breakthroughs in agricultural practices such as cultural controls, pesticide application, crop rotation, and plant breeding.

On the other hand, Pathogens frequently escape chemicals through strong selection and evolution, resulting in crop loss due to infection. Altering the genetic architecture of crops through breeding programmes is another option for crop protection, but it is a labor-intensive and time-consuming operation. In theory, genetic engineering, which refers to the use of biotechnology to alter an organism’s genetic material directly [3], is a potential tool for improving disease resistance. Furthermore, genetic engineering can overcome the limitations of traditional breeding technology, including the introduction/alteration of specific genes with minimum undesirable changes to the rest of the genome; cross-species exchange of genetic material; and introduction of variations/genes into asexually propagated crops like bananas [4]. As a result, research studies have been directed toward the genes that impart long-term resistance to many pests or pathogens and are safe for consumption.

In plants, tolerance and susceptibility to a particular pathogen are determined by a complex interaction of signals and responses corresponding to specific environmental conditions. So, the major difference between resistant and susceptible varieties is the ability to recognize an invading pathogen and further activate host defense mechanisms. Plants have evolved various defense mechanisms, including activating both constitutive and inducible defense responses to combat the diseases. When pathogens are detected, immune receptors in plants recognize specific molecules that signal the activation of effective defense responses. Despite extensive research, details of host defense mechanisms that limit pathogenic infections have yet to be elucidated. The majority of defense responses are characterized by the transcriptional activation of a large number of genes (>1% of the genome), many of which have unknown functions [5, 6]. Pathogen identification activates signaling pathways that result in the formation of reactive oxygen species (ROS), protein kinases, phytohormones, phytoalexins, phenolic compounds and pathogenesis-related (PR) proteins, and eventually a hypersensitive response (HR). Production and accumulation of PR proteins, which are low molecular weight proteins, in plants during pathogen attack is vital [7]. In most plant species, nineteen families of PR proteins (PR-1 to PR-19) have been identified to date. The role of PR proteins in plant defense includes altering the integrity of pathogen and activating other defense pathways through the generation of elicitors.

Due to improvements in transformation techniques and isolation of numerous pathogenesis-related genes, plants can now be engineered to have effective and broad-spectrum resistance against pathogens. The transgenic approaches using PR genes have been proven to be efficient for obtaining pathogen resistance in plants [8, 9]. Several transgenic plants have been developed that offer varying degrees of protection against certain fungal and oomycete diseases.

This chapter overviews the PR proteins, including their classification, activation as defense signaling indicators, and mode of action against the pathogens. It also highlights the success and challenges of the transgenic approach using PR genes for disease resistance.

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].

Families	Type member	Plant source	Gene accession no.	Classes/source	Size (kDa)	Properties	References
PR-1	Tobacco PR-1a	Nicotiana tabacum	YOO707		15–17	Antifungal	[14]
PR-2	Tobacco PR-2	N. tabacum	M59443.1	Classes III		β-1,3-Glucanase	[14]
				I plant vacuole	~33		[15]
				II, III extracellular proteins	~36		[15]
PR-3	Tobacco P, Q	N. tabacum	X77111.1	Classes V	25–30	Chitinase type I, II, IV, V, VI, VII	[16]
				I	~32		[15]
				II	27–28		[15]
				III	28–30		[15]
				IV	28–30		[15]
				V	41–43		[15]
PR-4	Tobacco “R”	N. tabacum	NW_015888419.1	Classes II	15–20	Chitinase type I, II	[16]
				I			[15]
				II			[15]
PR-5	Tobacco S	N. tabacum	NW_015793016		22–25	Thaumatin, antifungal, osmotin, zeamatin	[16, 17]
PR-6	Tomato inhibitor I	Solanum lycopersicum	NW_004196001.1		8	Proteinase inhibitor	[18]
PR-7	Tomato P69	S. lycopersicum	NC_015445.2		75	Endoproteinase	[19]
PR-8	Cucumber chitinase	Cucumis sativus	NC_026660.1		28	Chitinase type III	[20]
PR-9	Tobacco “lignin-forming peroxidase”	Solanum tuberosum	AJ401150		35	Peroxidase	[21]
PR-10	Parsley “PR1”	Petroselinum crispum	NC_026940.1	Classes III	17	Ribonuclease-like protein	[22]
				I	11 to 30		[15]
				II	~60		[15]
				III	~60		[15]
PR-11	Tobacco “class V” chitinase	N. tabacum	gi|899,342	—	40	Chitinase, type I	[23]
PR-12	Radish Rs-AFP3	Raphanus raphanistrum	NC_025209.1	Class IV	3–5	Defensin	[15, 24]
PR-13	Arabidopsis THI2.1	Arabidopsis thaliana	gi|1,181,531	—	5	Thionin	[25]
PR-14	Barley LTP4	Hordeum vulgare	gi|1,045,201	—	8.7–9	Lipid-transfer protein	[15, 26]
PR-15	Barley OxOa (germin)	H. vulgare	gi|2,266,668	—	20	Oxalate oxidase	[27]
PR-16	Barley OxOLP	H. vulgare	gi|1,070,358	—	20	Oxalate oxidase-like	[28]
PR-17	Tobacco PRp27	N. tabacum	—	—	27	Antifungal and antiviral	[29]
PR-18	Carbohydrate oxidases	Helianthus annuns	AF472608	—	60.9	Carbohydrate oxidases	[30]
PR-19	antimicrobial protein	Pinus Sylvestris	AF410954	—	—	antimicrobial protein	[31]

Table 1.

Classification and properties of PR proteins.

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 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].

Figure 1.

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

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].

6. Pathogenesis-related proteins (PR-proteins) with their transgenic expression

With the development of modern DNA technology, it is possible to engineer transgenic plants transformed with genes to provide resistance against specific diseases. Recently the transgenic expression of various groups of PR proteins has enhanced the resistance of the transformed plant against several plant pathogens (Table 2). PR proteins are found in all organisms and are part of their innate immune systems. They have a wide range of activities, including disrupting fungal cell walls, permeabilizing membranes, inhibiting transcription, and inactivating ribosomes [117]. Genes coding for various PR proteins have been identified, cloned, and expressed in plants, preventing the development of specific diseases and conferring resistance to affected plants. Using modern biotechnology tools, various crops have been engineered to express, or over-express the PR proteins from different sources, such as (i) that are produced during the plant’s defense response, (ii) derived from microorganisms or animal cells, (iii) synthetic peptides designed based on sequences of existing antimicrobial compounds [32, 118].

Enzyme	Genes	Source	Target pathogen	Transgenic plant	Transgenic system	Reference
Glucanase	β-1,3-glucanase	Linum usitatissimum	Fusarium culmorum	Potato	Agrobacterium; -mediated transformation	[69]
	HbGLU	Hevea brasiliensis	Rhizoctonia solani	Potato	Agrobacterium-mediated transformation	[70]
	β-1,3-glucanase II cDNA	Hordeum vulgare	Fusarium graminearum	Wheat	Particle gun bombardment	[71]
	chi-2, ltp	Hordeum vulgare, Triticum aestivum	Alternaria radicicola and Botrytis cinerea	Carrot	Agrobacterium-mediated transformation	[72]
	McCHIT1	Momordica charantia	Magnaporthe grisea and Rhizoctonia solani	Rice	Electroporation	[73]
	OsPR4a−e	Oryza sativa	Magnaporthe grisea	Rice	Agrobacterium-mediated transformation	[74]
	RC7	Oryza sativa	Rhizoctonia solani	Rice	Biolistic and PEG-mediated transformation system	[75]
	BjCHI1	Brassica juncea	Rhizoctonia solani	Potato	Agrobacterium-mediated transformation	[70]
	chit cDNA	Hordeum vulgare	Fusarium graminearum	Wheat	biolistic bombardment	[76]
	Chitinase-I	Oryza sativa	Verticillium dahliae and Fusarium oxysporum	Eggplant	Agrobacterium-mediated transformation	[77]
	RC24	Oryza sativa	Puccinia striiformis f.sp. tritici	Wheat	Particle bombardment	[78]
	rcc2 and rcg3	Oryza sativa	Puccinia striiformis f.sp. tritici	Wheat	Agrobacterium-mediated transformation	[79]
	LcCHI2	Leymus chinensis	Pseudomonas tabaci, A. alternata, Exserohilum turcicum, Curvularia lunata	Maize	Agrobacterium-mediated transformation	[80]
Thaumatin	Thaumatin-likeTaLr19TLP1	Triticum aestivum	Puccinia; triticina	Wheat	virus-induced gene silencing	[81]
	Tlp	Triticum aestivum	Fusarium graminearum	Wheat	biolistic transformation	[82]
	Tlp	Oryza sativa	Alternaria solani	Tomato	Agrobacterium-mediated transformation	[83]
	Tlp	Oryza sativa	Rhizoctonia solani	Rice	Particle bombardment	[84]
	tlp-1	Hordeum vulgare	Fusarium graminearum	Wheat	Agrobacterium-mediated transformation	[71]
	CsTLP	Camellia sinensis	Phytopthora infestans and Macrophomina phaseolina	Potato	Agrobacterium transformation	[85]
	AdTLP	Arachis diogoi	Rhizoctonia solani	Tobacco	Agrobacterium-mediated transformation	[86]
Osmotin-like proteins	OsOSM1	Oryza sativa	Rhizoctonia solani	Rice	Agrobacterium-mediated transformation	[87]
	OsmWS	Withania somnifera	A. solani	Potato	Agrobacterium-mediated transformation	[88]
	JIOsPR10	Oryza sativa	Magnaporthe oryzae	Rice	Agrobacterium-mediated transformation	[89]
Ribonuclease like protein	GmPRP	Glycine max	Phytophthora sojae	Soybean	Agrobacterium-mediated transformation	[90]
Ribonuclease inactivating protein	PAP	Phytolacca ameriacana	Cucumber mosaic virus, Potato virus X, Potato virus Y	Tobacco and Potato	Agrobacterium-mediated transformation	[91]
	PAP	Phytolacca ameriacana	Sclerotinia homoeocarpa	Beet grass	Particle bombardment	[92]
Proteinase inhibitor	mpi	Zea mays	Chilo suppressalis	Rice	Particle bombardment or Agrobacterium-mediated transformation	[93]
	cry1B	Zea mays	Chilo suppressalis	Rice	particle bombardment	[94]
Defensins	Wasabi	Wasabia japonica L.	Magnaporthe grisea	Rice	Agrobacterium-mediated transformation	[95]
	Wasabi	Wasabia japonica L.	Botrytis cinerea	Potato	Agrobacterium-mediated transformation	[96]
	MsDef1	Medicago sativa	Fusarium oxysporum f. sp. lycopersici	Tomato	-	[97]
	MtDef4.2	Medicago truncatula	Puccinia triticina	Wheat	Agrobacterium-mediated transformation	[67]
	RsAFP2	Raphanus sativus	Rhizoctonia solani and Magnaporthe grisea	Rice	Agrobacterium-mediated transformation	[98]
	RsAFP2	Raphanus sativus	Rhizoctonia cerealis, Fusarium graminearum	Wheat	Biolistic bombardment	[99]
	Wasabi	Wasabia japonica L.	Alternaria solani and Fusarium oxysporum	Melon	Agrobacterium-mediated transformation	[100]
	BoDFN	Brassica oleracea	Downy Mildew	Wild cabbage	Agrobacterium-mediated transformation	[101]
	VrPDF1	Vigna radiata	Weevils	mungbean	Agrobacterium-mediated transformation	[102]
	TAD1	Triticum aestivum	Typhula ishikariensis, Fusarium graminearum	Wheat	particle bombardment	[103]
Thionins	AT1G12660 and AT1G12663	A. thaliana	R. solani and F. oxysporum	Potato	Agrobacterium-mediated transformation	[104]
	Thionin	Brassica oleracea var. acephala, Nasturtium officinale and Barbarea vulgaris	B. cinerea	Potato	Agrobacterium-mediated transformation	[105]
	α-hordothionin (αHT)	Hordeum vulgare	Ceratocystis fimbriata	Sweet potato	Agrobacterium-mediated transformation	[106]
	Thi2.1	A. thaliana	Fusarium oxysporum	Tomato	Agrobacterium-mediated transformation	[107]
Oxalate Oxidase	OXO	Triticum aestivum	Sclerotinia sclerotiorum	Soybean	Agrobacterium-mediated transformation	[108]
	Osoxo4	Oryza sativa	Phytophthora infestans	Potato	Agrobacterium-mediated transformation	[109]
	OXO	Hordeum vulgare	Botrytis cinerea and Sclerotinia sclerotiorum	Tomato	Agrobacterium-mediated transformation	[110]
	OXO	Triticum aestivum	Phytophthora infestans	Potato	Agrobacterium-mediated transformation	[111]
Lipid Transfer Proteins	AtLTP4.4	A. thaliana	F. graminearum	Wheat	particle bombardment	[112]
	Ace-AMP1	Allium cepa	Sphaerotheca pannosa var. rosae, Blumeria graminis f. sp. tritici and Neovossia indica , Magnaporthe grisea and Rhizoctonia solani	Wheat and rice	Agrobacterium-mediated transformation, microprojectile bombardment, In planta assays	[113, 114, 115, 116]
Carbohydrate oxidases	-	Helianthus Annuus	Pectobacterium cartovorum ssp. cartovorum	Tobacco	Electroporation	[30]
Antimicrobial protein	Sp-AMP	Pinus Sylvestris	Heterobasidion annosum	Tobacco	Agrobacterium-mediated transformation	[31]

Table 2.

Example of transgenic plants over-expressing PR proteins against plant pathogens.

7. Transgenic plants expressing antifungal activities

Fungi are one of the most harmful phytopathogens, resulting in considerable production losses in most agricultural crops [119]. PR proteins have proven effective in preventing fungal diseases in plants as many of these targets or hydrolyze fungal cell walls, resulting in cell death. PR1, PR2, PR3, PR4, PR5, PR8, PR11, PR12, and PR13 have been identified as plants’ most effective antifungal proteins. Transgenic approaches using PR proteins are suitable for developing long-lasting fungal pathogen-resistant crops [64]. Of the various antifungal PR proteins, glucanases and chitinases are most widely used in transgenic technology to provide resistance against fungus.

The transgenic over-expression of glucanase and chitinase genes from different sources has been shown to be effective against pathogens, specifically fungus. It has been reported that overexpression of the tobacco glucanase gene imparted groundnut resistance to Cercospora arachidicola and Aspergillus flavus, demonstrating that fungal resistance is conferred via in planta transformation [120]. Transgenic Arabidopsis plants expressing grapevine b-1,3-glucanase (VvGHF17) confers resistance to Colletotrichum higginsianum and Botrytis cinerea [121]. Furthermore, tea with transgenic overexpression of the endo-1,3-D-glucanase gene, which expresses a potato glucanase, significantly improved tolerance to the blister blight fungus Exobasidium vexans [122]. Recently, oil palm resistance to G. boninense was improved by transgenic overexpression of M. sativa glucanase (AGLU1) [123]. Likewise, transgenic expression of chitinase genes have been reported to be antifungal generated transgenic zoysia grass was generated which overexpressed Zjchi2 via Agrobacterium-mediated transformation and hence showed disease resistance against Rhizoctonia solani [124]. Currently, the overexpression of LcCHI2 gene was identified that increasing the chitinase activity in transgenic tobacco and maize, resulting in improved resistance to Pseudomonas tabaci, Alternaria alternata, Exserohilum turcicum, Curvularia lunata [80].

Some other antifungal PR proteins that have been reported to be used in transgenics are thaumatin-like/osmotin-like proteins, defensin-like proteins, thionin, oxalate oxidase and lipid transfer protein. In fungal cells, thaumatin-like proteins are known to form transmembrane pores, whereas osmotin proteins are known to maintain the osmolarity of suitable solutes in cellular compartments [88]. In Arabidopsis thaliana, overexpression of the TLP29 gene from grape VqTLP29 improved resistance to powdery mildew and the bacteria Pseudomonas syringae [125]. Under in vitro conditions, transgenic poplars overexpressing PeTLP thaumatin genes showed enhanced resistance to Marssonina brunnea [126]. Similarly, in potatoes, overexpression of the osmotin gene (OsmWS) conferred resistance to the early blight fungus A. solani [88]. Many more transgenic plants have been generated that show increased resistance to phytopathogenic fungi by expressing the TLPs and OLPs as listed in Table 2.

The successful developed and characterized transgenic peanut and tobacco plants which overexpress the mustard defenisn gene and Raphanus sativa, RsAFP2 gene for fungal resistance respectively [127]. The late leaf spot diseases Cercospora arachidicola and Pheaoisariopsis personata were more resistant to transgenic peanut plants whereas, Phytophthora parasitica pv. nicotianae and Fusarium moniliforme resistance was higher in transgenic tobacco plants. Similarly, the rDrr230a defensin protein gene suppressed spore germination and growth of both Fusarium tucumaniae and Colletotrichum gossypii var. cephalosporioides in transgenic Pichia pastoris [128]. The antifungal thionin genes (AT1G12660 and AT1G12663) from A. thaliana had been used to produce transgenic potato conferring resistance against pathogenic fungi such as Fusarium solani and Fusarium oxysporum [104]. Furthermore, the overexpression of thionin increased canker resistance and decreased canker bacterial development when transgenic Carrizo plants expressing the modified plant thionin were produced by Agrobacterium-mediated transformation [129]. Peanuts with transgenic expression of the oxalate oxidase expressing gene were more resistant to Sclerotinia blight [130]. Also, overexpression of oxalate oxidase genes has been developed to increase resistance against Sclerotinia sclerotiorum in transgenic Glycine max [108].

Transgenic expression of LTPs has been shown to improve resistance to phytopathogenic fungi in some studies. As an example, antimicrobial protein gene (Ace-AMP1) isolated from Allium cepa has been overexpressed in both Triticum aestivum and Oryza sativa through Agrobacterium-mediated transformation, microprojectile bombardment, in planta assays, conferring resistance against Sphaerotheca pannosa var. rosae [113], Magnaporthe grisea, Rhizoctonia solani and Xanthomonas oryzae [116] respectively. Recently, A. thaliana LTP overexpressing transgenics has been shown to increase resistance toward pathogens Plasmodiophora brassicae and F. graminearum [112, 131]. Some other examples of successfully generated transgenic plants with enhanced production of hydrolytic enzymes and resistance against phytopathogenic fungi are given in Table 2.

8. Transgenic plant expressing bacterial resistance

Numerous bacterial pathogens causing massive yield losses have been isolated and identified from different agriculturally important crops. Pathogenesis-related proteins are well-known weapons to combat resistance against these bacterial pathogens. Many in-vitro studies have shown the antibacterial properties of many PR proteins viz PR10 (Ribonuclease-like proteins), PR12 (defensins), PR13 (thionins) and PR14 (Lipid-transfer protein) [90, 116, 132]. Among these, PR10 shows broad spectrum of antibacterial activity against P. syringae, Agrobacterium tumefaciens, A. radiobacter, Pseudomonas aureofaciens and Serratia marcescens [90, 133]. Overexpression of lipid transfer protein (PR14) in rice plants showed increased resistance to bacterial as well as fungal pathogens (Table 2) [116]. The antibacterial efficacy of additional PR proteins and AMPs against a variety of bacterial diseases in economically significant crops has to be further investigated.

9. Transgenic plant expressing insect resistance

Plants expressing PR genes have been engineered in several experiments, resulting in enhanced pest resistance. The expression of both low and high levels of MTI-2 was reported by using Agrobacterium transformation technique in tobacco and Arabidopsis plants leading to resistance against Spodoptera littoralis [134]. The wound-inducible expression of a Bacillus thuringiensis endotoxin gene which directed significant insecticidal gene expression to protect transgenic rice from Chilo suppressalis Walker [94]. Transgenic rice plants were developed by particle bombardment or Agrobacterium-mediated transformation of mpi gene leading to resistance against C. suppressalis (Table 2) [93].

10. Transgenic plant expressing viral resistance

Apart from their antifungal or antibacterial effects, PR proteins appear to be a promising candidate gene for producing virus-resistant transgenic crops based on different studies of PR proteins, as given in Table 2. Antiviral activities of PR proteins such as defensins, thionins, peroxidase and lipid transfer proteins have been observed in vitro [115]. Antiviral activity has also been observed in ribosome-inactivating proteins (RIPs), which suppress translation by enzymatically damaging ribosomes [115]. Plant resistance to plant viruses was improved by a transformation study involving RIPs. In addition, CaPR10 from Capsicum annuum has been found to have increased ribonucleolytic activity against the Tobacco mosaic virus (TMV) RNA, allowing it to break viral RNAs [116].

11. Synergistic effect of transgenic PR proteins

In transgenic plants, the synergistic action of two or more PR genes reduces susceptibility to various pathogens. Researchers have reported that β-1,3-glucanases and chitinases synergistically inhibited the growth of Fusarium oxysporum by using in planta transformation [135]. Transgenic potato plants co-expressing chitinase (BjCHI1) and β-1,3-glucanase (HbGLU) suppressed Rhizoctonia solani and showed healthier root growth [70]. In another study, transgenes carrying the chitinase gene (chi11) and the thaumatin-like protein gene (tlp) from rice were introduced by co-bombardment, and overexpression of these antifungal chi and tlp proteins provided resistance to fungal infections in barley [136]. Likewise, in transgenic carrots, the synergistic action of three different PR-protein genes such as chitinase, β-1,3-glucanase and peroxidase, conferred disease resistance to necrotrophic pathogens namely, Botrytis cinerea and Sclerotinia sclerotiorum [137]. Amian et al [138] reported the development of transgenic pea plants with stable integration of two genes viz β-1,3-glucanase (Hordeum vulgare) and chitinase gene (Streptomyces olivaceoviridis) via Agrobacterium-mediated gene transformation and hence produced suppression of fungal spore germination. Chhikara et al [139] used Agrobacterium-mediated transformation to co-express the barley antifungal genes chitinase and ribosome-inactivating protein in Indian mustard, protecting against Alternaria leaf spot disease. Furthermore, transgenic potato plants expressing rip30 and chiA genes transformed by A. tumefaciens strain GV3101 showed improved resistance to Rhophitulus solani [140]. In the case of Oriental melon (Cucumis melo Makuwa Group), the fusion of chitinase (CHI) and antifungal protein (AFP) genes confers enhanced protection against Rhizoctonia solani and Fusarium oxysporum [141]. Rice plants co-transformed with chitinase (OsCHI11) and oxalate oxidase (OsOXO4), which are defense-related genes, showed improved resistance to the pathogen that causes sheath blight [142]. Boccardo et al [143] suggested co-expression of PR proteins AP24 and β-1,3 glucanase enhanced resistance against Rhizoctonia solani in greenhouse conditions and Peronospora hyoscyami f.sp. tabacina and phytophthora nicotianae pathogens in field conditions.

12. Challenges faced by transgenic expression with PR proteins

Since the advent of plant genetic engineering, PR proteins have consistently been the top choice among scientists when creating transgenic plants to increase disease resistance against a variety of diseases. PR proteins expressed either singly or synergistically in transgenic plants can provide broader and more effective disease resistance against different pathogens as described above.

Aside from these successful outcomes, many studies have described the challenges of using PR proteins in transgenic technology. In contrast to the above findings, numerous studies have suggested that the transgenic expression of PR proteins did not lead to increased tolerance to pathogens. Szwacka et al [144] reported no relationship between transgenic protein expression level and increased tolerance against the pathogen. Transgenic cucumber plants with stably integrated thaumatin II cDNA under the control of the CAM35S promotor via Agrobacterium did not exhibit tolerance to Pseudoperonospora cubensis. Moravckova et al [145] co-introduced chitinase and glucanase into Solanum tuberosum to increase resistance to R. solani infection, but hyphal extension assay revealed that transformants did not affect R. solani growth in vitro.

Various transgenic plant modifications have been described, with varying degrees of protection against certain fungal and oomycete infections. However, the resulting resistance levels were frequently insufficient for breeding [146]. Furthermore, constitutive expression of PR proteins can lead to the spontaneous production of lesions that look like HR lesions in the absence of a pathogen), which can be an unfavorable outcome [147]. Disease resistance techniques must control specific diseases without affecting crop yield and quality.

Moreover, most researchers have used constitutive promoters to control the expression of PR genes in agricultural plants to enhance resistance, resulting in homology-dependent gene silencing. As a result, unregulated and untimely activation of PR genes or AMPs harms plant growth and development. Human allergenicity is one of the main issues hindering the success of transgenic technology with PR genes. According to the current classification, there are 19 different classes of PR-Proteins, and 8 of them have been confirmed to cause allergic reactions in humans by using in-silco approaches. These proteins have been known to trigger allergenic symptoms such as food allergens depending upon their mode of entry into the human body [148], dermatitis, airborne, asthma, airway allergy etc. and if all these allergens have been consumed in greater amount, the gastrointestinal symptoms are also triggered.

13. Conclusion

The goal of this chapter was to review the role of PR-proteins in plant defense and how transgenic expression of PR-proteins in agricultural plants resulted in increased resistance to stresses. Biotic stress has become a significant concern in modern agriculture and many research institutions are actively researching to generate resistant cultivars using PR proteins. PR proteins have become a highlighted topic between scientists because of their effectiveness against biotic agents. Genetic engineering is considered the best way to develop transgenic resistant plants using PR proteins. To increase agronomic characteristics worldwide, new inventions or novel approaches in PR protein transgenic technology are necessary and will continue to improve plant health in the future. Another future concern is that the formation of virulent phytopathogen strains increases as the global climatic change rate increases. So, to cope with such significant obstacles, it is necessary to define and identify novel PR genes functionally. Advances in genomics, transcriptomics, phenomics, proteomics, metabolomics, and ionomics, will substantially aid our understanding of the complex network of PR genes and the interaction of PR proteins with other proteins from plants and pathogens. Therefore, PR proteins could be utilized to develop crop plants more resistant to various stresses. They could also be employed as candidate genes for genetically engineering crop multi-trait factors. Future research is needed to assess the PR transgenic plants’ responses to various traits, including biotics, plant development and yield.