Essay: Effects of Pesticides on the Enviroment

Introduction
With the ever increasing global population projected to touch 9.7 billion by 2050, combined with the reducing arable land area, crop productivity has become an important concern. Plants are continuously under the attack of wide range of pathogens namely fungi, bacteria and viruses. These pathogens have numerous ways of adversely affecting the growth of development of plants some of which include (a) expressing a multitude of degradative enzymes like cellulases, hemicellulases, pectinases to destabilize the plant structural framework and facilitate its invasion (Walton,1994) (b) secreting toxins which adversely affects the metabolic homeostasis in plants by inhibiting enzymes and modulating membrane permeability (Quiggly and Gross,1994) (c) promoting hormonal imbalance in the plant leading to abnormal growth and development (Suckstorff and Berg,2003).
Now, plants over the course of evolution have developed two-tier defense against these pathogen attacks. The first constitutive layer is characterized by readymade structures and compounds synthesized during the normal development of plant and the second inducible layer being triggered by pathogen infection. This inducible defence mechanism again branches into PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI).Plants consists of pathogen recognition receptors on the cell surface which on encountering the slowly evolving but essential for survival pathogen-associated molecular patterns (PAMPs), trigger the PTI. PTI involves the activation of multiple processes, like mitogen-activated protein kinase (MAPK) cascades, generation of reactive oxygen species (ROS), altering hormone signaling pathways and expression of defense related genes. But unbeaten pathogens overcome the PTI by secreting virulence related effectors. These effector proteins encoded by the avirulent (avr) genes, on being recognized by intracellular plant receptors encoded by the resistance (R) gene products, initiates an even more robust effector-triggered immunity (ETI) that eventually ends in programmed cell death (Jones and Dangl 2006).
The modus operandi of both these immune responses involve both localized or systematic developments namely, a) rapid production of ROS which not only kills the microbes but also strengthens the plant cell wall against hydrolytic attacks (Lamb and Dixon,1997); (b) build up of antimicrobial secondary metabolites known as phytoalexins (Hammerschmidt 1999); (c) hypersensitive response (HR) leading to cell death thereby further restricting the spread of the pathogen (Hammond-Kosack and Jones 1996; Alvarez et al. 2000). (d) expression of defense related proteins like pathogenesis related (PR) proteins and antimicrobial peptides (AMPs) (Dixon and Harrison,1990).
Besides plants, AMPs have been discovered and characterized from different organisms and have occurred through the course of evolution as an important defense response against pathogen invasion. Study of the natural counterparts coupled with the use of bioinformatics tools has enabled us to generate customized synthetic peptides which are also toxic and effective against the various phytopathogens.
But pathogens with the passage of time have managed to successfully infect the host, inspite of the multilayered defense and lead to crop yield losses. Decades of pesticide usage have a major contribution in protecting against these pathogens, but their heavy usage is responsible for increased environmental issues. In addition, conventional breeding strategies due to lack of sufficient resistance in the germplasm and also being a laborious process have not been successful enough in generating durable resistance. Therefore AMPs due to their direct antimicrobial effect, coupled with genetic engineering have great potential in the development of robust disease resistant crops.
This review will be highlighting the different antimicrobial peptides from both plant and non-plant sources and how they have been utilized in providing resistance in against the different plant pathogens.
Overview of Antimicrobial Peptides
Antimicrobial peptides (AMPs) are small, structurally diverse peptides produced as an integral component of the evolutionarily conserved innate immune system of a wide variety of organisms ranging from insects to humans in order to protect against a broad spectrum of pathogens (Zasloff 2002). They are usually cationic in nature interspersed with hydrophobic residues giving it the ability to interact with the cell membranes of the pathogens. These are an expansive group consisting of peptides and small proteins containing less than 100 amino acids. More than 5500 AMPs of both natural and synthetic origins has made way for the creation of numerous databases, namely
1. PhytAMP: Plant AMP Database (http://phytamp.pfba-lab-tun.org/main.php)
2. CAMP: Collection of Anti-Microbial Peptides (http://www.camp.bicnirrh.res.in)
3. APD: Antimicrobial Peptide Database (http://aps.unmc.edu/AP/main.php)
4. LAMP (http://biotechlab.fudan.edu.cn/database/lamp/index.php)
5. DAMPD: Dragon Antimicrobial Peptide Database (http://apps.sanbi.ac.za/dampd/)
6. RAPD: Recombinantly produced Antimicrobial Peptides Database (http://faculty.ist.unomaha.edu/chen/rapd/index.php)
7. DRAMP: Data Repository of Antimicrobial Peptides (http://dramp.cpu-bioinfor.org/)
8. Defensins knowledgebase (http://defensins.bii.a-star.edu.sg/)
9. YADAMP: Yet Another Database of Antimicrobial Peptides (http://www.yadamp.unisa.it/default.aspx)
10. DBAASP : The Database of Antimicrobial Activity and Structure of Peptides (http://www.biomedicine.org.ge/dbaasp/)
Plant Antimicrobial peptides
The general features of plant AMPs are small molecular size, net positive charge, amphipathic properties, and rich in cysteine residues conferring a high termostability. Recent analyses suggest that plant genomes are rich in genes encoding cysteine-rich peptides resembling AMPs, which might account for up to 2-3% of the predicted genes, suggesting that plant possess a formidable defense arsenal (Silverstein et al. 2007). They are expressed constitutively and also induced by pathogen attack and help in modulating the plant immune response (García-Olmedo et al. 1998). Expression of plant AMPs is also localized to different organs as evidenced by the fact that they been found in leaves, roots, stems, flowers and seeds. The plant AMPs contains 4–12 conserved cysteine residues which stabilize their 3D structure by disulfide bonds. Antimicrobial peptides from plants share similarities in pattern of disulfide bridging and structural characteristics (Odintsova and Egorov 2012) and accordingly have categorized into different classes, namely thionins, defensins, lipid transfer proteins, heveins, snakins, knottin-like, puroindolines and cyclotides .Plant AMPs not only have diverse structures, but also have activity against a wide range of phytopathogens via various mechanisms involving membrane permeabilization, impairment of intracellular process and regulation of the plant immune machinery (Rahnamaeian et al. 2011).
Mode of action of plant antimicrobial peptides
Antimicrobial peptide families possess unique and conserved structures and amino acid composition allow them to distinguish host tissue and selectively act against the pathogens. This selectivity is also a result of the structure and composition of host and pathogen membrane surface. The AMPs on interacting and binding with the surface, affect pathogens in mainly two ways: a) Permeabilization of the cell membrane b) Incapacitating the intracellular machinery
AMPs due to their cationic nature and distinctive structural features enable to interact with the negatively charged lipids of the cell membrane and change the membrane topology. The electrostatic interactions between the peptide and the membrane lipids coupled with the buildup of the AMPs above the threshold level on the membrane surface, sets into motion the collapse of the pathogen membrane (Pelegrini and Franco 2005). This occurrence is explained by three different models:
• Barrel-stave model: The peptides act as monomers and undergo oligomerization upon aggregation on the membrane surface. This leads to alignment of the hydrophobic residues with hydrophobic core of the membrane and polar residues to form inner lining of the pore.
• Toroidal or wormhole model: The peptide intercalates itself into the membrane interior and disrupts the hydrophobic interior by favoring the realignment of the phospholipid heads. The polar head groups curve into and forms the lumen of the pore unlike in the barrel-stave model.
• Carpet model: This non-pore forming mechanism involves ‘carpeting’ of the membrane surface with the peptide molecules. Here the peptides orient themselves via electrostatic interactions and upon reaching threshold levels, upsets the membrane stability. In addition, the peptides can act as ‘detergent’ molecules and interact with membrane lipids to form micelles which further breaks down the membrane eventually causing cell death.
Sometimes in addition to membrane pore formation, antimicrobial peptides have been found to act intracellularly and affect the pathogen. They cross the membrane and act via a wide variety of mechanisms: depolymerization of actin (Koo et al. 2004), binding of the fungal cell wall chitin (Fujimura et al. 2005), generation of ROS and subsequent cell death (Aerts et al. 2007), cell cycle inhibition (Lobo et al. 2007), cytoplasm granulation (van der Weerden et al. 2008), impairment of DNA synthesis (Haney et al. 2013), disruption of cell signaling (Nanni et al. 2014).
Plant antimicrobial petide families
Thionins
Thionins belong to a family of low molecular weight antimicrobial peptides (approximately 5kDa), with conserved cysteine residues. Due to the abundance of basic amino acids like arginine and lysine, they are predominantly positively charged at physiological pH. Their basic structure consists of an antiparallel double-stranded β-sheet along with two antiparallel α- helices with three to four conserved disulfide bridges. They are widely distributed in plant kingdom, both in monocots and dicots along with rosids (Stec 2006) and were the first peptides from plants whose antimicrobial properties were recognized and characterized. The fact that thionins can act against a wide range of pathogens and are widely distributed in different parts of plant makes them integral part of the plant defense system. Thionins share similarity in both gene and protein structure and are expressed as inactive pre-proteins. An N-terminal leader peptide gets cleaved and activates the pro-peptide with an acidic C-terminal tail (Stec 2006).
The thionins can be distinctly divided into two main groups: α-/β-thionins and γ- thionins. But since γ-thionins share structural similarities with both insect and mammalian defensins, they were subsequently named as ‘plant defensins’ (Terras et al. 1995). α-/β-thionins can be further be subdivided into five classes all of which share protein sequence homology (Bohlmann and Apel, 1991):
1. Type I thionins are highly basic in nature with 8 cysteine residues involved in four disulphide linkages .They are found in the monocot family Gramineae (Egrov et al. 2005).
2. Type II thionins were found in leaves of the parasitic plant Pyrularia pubera (Vernon, 1992) and barley (Bohlmann and Apel, 1987). They have the same disulfide linkage pattern as Type I thionins but comparatively less positively charged.
3. Type III thionins includes the viscotoxins and phoratoxins from mistletoes (Mellstrand and Samuelsson, 1973; Samuelsson and Pettersson, 1970). In contrast to Type I and Type II, they have three disulphide bridges.
4. Type IV thionins represent crambins from Abyssinian cabbage of the Cruciferae family contain three disulfide bonds and are neutral in charge. (Schrader-Fisher and Apel 1994).
5. Type V thionins are very different from the rest of the thionins and has been isolated from the cDNA library of hexaploid wheat (Castagnaro et al. 1994)
Antimicrobial activity against both bacteria and fungi has been documented (De Caleya et al. 1972; Giudici et al. 2004) and involve an increase in cell membrane permeability. The interaction of the basic thionins with the negatively charged phospholipid membrane leads to two effects (1) Primary effect involves formation of ion pore channels in the cell membranes (Hughes et al. 2000) and (2) secondary effect results in membrane depolarization and increase in Ca2+ permeability (Evans et al. 1989).
Constitutive expression of the α-thionin from barley endosperm conferred transgenic tobacco enhanced resistance to two important pathovars of the bacterium Pseudomonas syringae (Carmona, María José, et al, 1993). The endogenous thionin Thi2.1 on being overexpressed in Arabidopsis developed resistance against the fungus Fusarium oxysporum (Eppel et al. 1997). Transgenic Arabidopsis thaliana expressing viscotoxin A3 from Viscum album showed resistance against the the clubroot pathogen Plasmodiophora brassicae (Holtorf et al. 1998). Rice engineered to express the oat cell wall bound thionin demonstrated protection against two vital seed-transmitted phytopathogenic bacteria, Burkholderia plantarii and Burkholderia glumaein (Iwai et al. 2002). Arabidopsis thionin (Thi2.1) under the control of a fruit-inactive promoter on being expressed in tomato provided enhanced protection against the diseases of bacterial wilt and fusarium wilt (Chan et al. 2005). Tobacco plants constitutively expressing the barley β-hordothionin were conferred protection against the grey mold causing fungus Botrytis cinerea and the bacterial wilt causing Pseudomonas solanacearum (Charity et al. 2005). β-purothionin expressing Arabidopsis demonstrated enhanced resistance against the bacterium Pseudomonas syringae and the fungus Fusarium oxysporum (Oard and Enright, 2006). Elite apple cultivars expressing the barley hordothionin showed resistance against the fungal disease scab caused by Venturia inaequalis (Krens et al. 2011). Potato plants constitutively expressing thionin genes from the Brassicaceae species showed enhanced resistance against the against gray mold fungi, Botrytis cinerea (Hoshikawa et al. 2012). Sweet potato modified to express the barley α-hordothionin developed resistance against the black rot causing pathogenic fungus Ceratocystis fimbriata (Muramoto et al. 2012).
Defensins
The earliest defensins from plants were found in bread wheat (Triticum aestivum) and barley (Hordeum vulgare) and were termed as γ-thionins, since they have a size of about 5kDa and four disulphide bridges, features which they share with α- and β-thionins. But due to the structural similiarities with both insect and mammalian defensins, they were renamed as plant defensins (Bruix et al. 1993). The defensins are an integral part of the innate immune system which has been found to be conserved across the plant and animal kingdoms (Thomma et al. 2002). Defensins are 45-54 aa long cationic peptides which are rich in cysteine residues. The disulfide bridging formed by the conserved eight cysteine residues supports the structure containing well-defined triple-stranded antiparallel β-sheets with one α-helix. Two disulfide bridges join the α-helix with the β-sheet forming the distinctive Cys-stabilized α-helix β-sheet (CSαβ) motif (Cornet et al. 1995). The plant defensin family members are omnipresent in both monocots and dicots. The wide range of distribution in the different plant tissue along with its localization in the outer cell layers, the stomata and the phloem underlines its role in plant defense (Terras et al. 1995). The defensins have been categorized into two distinct classes namely (a) The first class which forms the majority, contains a defensin domain with a secretory N-terminal endoplasmic reticulum (ER) signal peptide while (b) The minor second class of defensins containing a C-terminal 33aa long prodomain (Lay and Anderson, 2005).
Defensins are known to have broad spectrum action against both fungi and bacteria with negligible toxicity against the host tissues (Stotz et al. 2009). Their mode of action against fungi seems to involve binding with membrane bound sphingolipids (Thevissen et al. 2004) and subsequent penetration into fungal membrane. This leads to disruption and destabilization of membranes coupled with leakage of ions (Pelegrini and Franco 2005). Based on this aspect, defensin functions can be grouped into (a) Morphogenetic which inhibits hyphal elongation with hyperbranching of the hyphae and (b) Non-morphogenetic which only causes a reduction in hyphal elongation without the associated morphological distortions (Lay and Anderson, 2005).
Rs-AFP2 isolated from from the seeds of radish (Raphanus sativus) (Terras et al. 1992) has been found to cause cell death in Candida albicans through the induction of endogenous ROS generation (Aerts et al. 2007).It also induces apoptotic death via caspase dependent pathway by an unknown caspase in Candida albicans (Aerts et al. 2009).On further investigation through the screening of Candida albicans deletion mutants, it was deduced that Rs-AFP2 affected cell wall integrity by causing increased ceramide accumulation in the fungal cell membrane leading to septin mislocalization. These inhibit the growth of the filamentous fungi (Thevissen et al. 2012). DmAMP1 obtained from seeds of dahlia (Dahlia merckii) (Osborn et al. 1995) was posited to interact with membrane sphinolipids and ergosterol and cause membrane depolarization associated with elevated K+ efflux and Ca2+ uptake, leading to cellular death (Thevissen et al. 1996; Thevissen et al. 2003). VvAMP2, a grapevine flower specific defensin was very specific for Botyritis cinerea and is internalized by the fungus and affects intracellular targets like Rho-type GTPase, G-protein coupled receptor, transcription factors, thereby altering cellular health and finally leading to membrane disruption and fungal cell death (Nanni et al. 2014). PvD1, a novel defensin from the seeds of Phaseolus vulgaris (Games et al. 2008) had inhibitory activity against a wide range of fungi by affecting the membrane H+-ATPase, generating reactive oxygen species and NO in the cell and causing structural changes in both the cytoplasm and the plasma membrane, ultimately leading to cell death (Mello et al. 2010). A newly characterized seed defensin of Adenanthera pavonina, ApDef1 (Soares et al. 2012) was found to cause membrane depolarization and permeabilization, chromatin condensation, inhibition of the cell cycle and leading to caspase dependent apoptosis (Soares et al. 2017). NaD1, a potent defensin from the flowers of Nicotiana alata (Lay et al. 2003) unlike other defensin family members not only can permeabilize the membrane via aperture formation and interaction with an unknown protein receptor (Van Der Weerden et al. 2008, 2010) but also disrupting it by forming a complex with the PI(4,5)P2, a negatively phospholipid rich in the inner leaflet of the membrane (Payne et al. 2016). It also enters the fungal cytoplasm and causes the formation of ROS thereby generating oxidative stress and ultimately cellular death (Hayes et al. 2013).NaD1 antimicrobial activity has been reported to have been augmented by dimerization (Lay et al. 2012).Psd1 obtained from Pisum sativum acts intracellularly by interacting with the cyclin F and blocking its activity thereby preventing the progression of the cell cycle from S to G2 (Lobo et al. 2007). HsAFP1 extracted from seeds of coral bells (Heuchera sanguinea) (Osborn et al. 1995) has broad spectrum antifungal activity and affects fungal growth through membrane permeabilization by interacting with high specificity membrane binding sites and causes ROS mediated apoptosis by affecting the mitochondrial respiratory function (Thevissen et al., 1997; Aerts et al. 2011).Like RsAFP2, MtDef4 from Medicago trunculata utilizes ceramides for its antifungal activity (Ramamoorthy et al. 2007).Unlike other defensins, it surprisingly has varied mode of actions for different fungi with different entry points but affecting intracellular targets (El‐Mounadi et al. 2016).
Tobacco brown spot causing fungus Alternaria longipes was inhibited when it was expressing the radish defensin Rs-AFP2 driven by the constitutive 35S promoter (Terras et al. 1995). Canola constitutively expressing the pea defensin DRR230 developed resistance against the blackleg fungus Leptosphaeria maculans (Wang et al. 1999). Oilseed rape hybrids and tomato cultivars engineered to express the defensin Rs-AFP2 inhibited growth in vitro and provided resistance in planta against a mutltiude of fungal phytopathogens (Parashina et al. 2000). Expression of the alfAFP peptide from Medicago sativa in transgenic potato plants provides robust resistance fungal pathogen Verticillium dahliae (Gao et al. 2000).Constitutive over-expression of the BSD1 (Brassica stamen specific plant defensin 1) gene under the control of the 35S promoter conferred enhanced tolerance against the Phytophthora parasitica in the transgenic tobacco plants (Park et al. 2002). Tobacco plants expressing the different members of pea defensin DRR230 family displayed both in vitro and in vivo suppression of a wide range of phytopathogenic fungi and bacteria (Lai et al. 2002). The Dahlia merckii defensin, DmAMP1 on being engineered into the eggplant, Solanum melongena inhibited the growth of the fungi Botrytis cinerea and Verticillium albo-atrum while the arbuscular mycorrhizal fungus Glomus mosseae was completely unaffected (Turrini et al. 2004). The same peptide on being ectopically expressed in papaya provided protection against the disease caused by Phytophthora palmivora (Zhu et al. 2007). Mustard defensin (BjD) on being expressed in tobacco provided resistance against multiple fungal pathogens and resistance against the late leaf spot disease in peanut plants (Anuradha et al. 2008). SE60, a γ-thionin from soybean expressed constitutively in tobacco suppressed the growth of the bacterium, Pseudomonas syringae (Choi et al. 2008). Tomato plants expressing the radish defensin Rs-AFP2 showed enhanced resistance against the devastating gray mold and fusarium wilt diseases (Kostov et al. 2009). Transgenic tomato plants expressing the chili defensin gene under the control of a 35S promoter generated resistance against Fusarium sp. and Phytophthora infestans (Zainal et al. 2009). Ovd, a floral defensin obtained from the Chinese violet cress (Orychophragmus violaceus) on being expressed in rapeseed plants led to reduction in the symptoms caused by the Sclerotinia stem rot fungi (Wu et al. 2009). Single peptide expressing the two different defensins Dm-AMP1 and Rs-AFP2 in transgenic rice developed robust resistance against the phytopathogens Magnaporthe oryzae and Rhizoctonia solani (Jha and Chattoo, 2009). Transgenic tobacco lines expressing the NmDef02, a defensin isolated from Nicotiana megalosiphon revealed enhanced resistance against Phytophthora parasitica var. nicotianae, Peronospora hyoscyami f.sp. tabacina, Alternaria solani and Phytophthora infestans (Portieles et al. 2010). Transgenic rice plants expressing the radish defensin RsAFP2 generated defense against two economically important fungal diseases, fungal blast and sheath blight (Jha and Chattoo, 2010). Tomato plants expressing the Medicago sativa defensin gene MsDef1 were protected against fusarium wilt pathogen Fusarium oxysporum f. sp. Lycopersici (Abdallah et al. 2010). Transgenic wheat expressing RsAFP2 displayed resistance against two devastating diseases, Fusarium head blight and wheat sharp eyespot (Li et al. 2011).Maize seed specific defensin ZmDEF1 constitutively expressed in transgenic tobacco provided resistance against the pathogenic Phytopthora parasitica (Wang et al. 2011). Transgenic banana plants expressing the PhDef1 and PhDef2 led to significant resistance against infection of Fusarium oxysporum (Ghag et al. 2012). MtDEF4.2 was able to impart transgenic Arabidopsis with protection against two pathogens with varied lifestyles when fused with two different targeting signal peptides (Kaur et al. 2012). Tobacco expressing the defensin TvD1 from Tephrosia villosa exhibited strong antifungal activity against the pathogen Rhizoctonia solani (Vijayan et al. 2013). A fusion protein containing the Trigonella foenum-graecum defensin (Tfgd2) and Raphanus sativus antifungal protein (RsAFP2) generated resistance against the fungi Rhizoctonia solani and Phytophthora parasitica var. nicotianae, in transgenic tobacco (Vasirama and Kirti, 2013). Bell pepper defensin J1-1 on being constitutively overexpressed developed transgenic pepper plants resistant against the causal agent of anthracnose disease, Colletotrichum gloeosporioides (Seo et al. 2014). Cotton plants expressing the defensin NaD1, from Nicotiana alata had potent resistance against two important fungal pathogens namely Fusarium oxysporum and Verticillium dahliae (Gaspar et al. 2014). The seed defensin (Sm-AMP-D1) from Stellaria media after being expressed in banana plants showed enhanced resistance against the Fusarium wilt disease (Ghag et al. 2014). Winter wheat constitutively overexpressing the cold inducible defensin TAD1 led to resistance against snow mold and Fusarium head blight (Sasaki et al. 2016). Peanut expressing a fusion of two defensins namely RsAFP2 and Tfgd2 fungal diseases like early leaf spot (ELS) and late leaf spot (Bala et al. 2016). Homozygous wheat lines engineered to express the antifungal plant defensin MtDEF4.2 from Medicago truncatula in the apoplast, was able to differentiate and provide resistance against the pathogenic fungus Puccinia triticina without affecting the mycorrhizal fungus Rhizophagus irregularis (Kaur et al. 2017). Black medic plants expressing the pea defensin showed better survival rates during seedling stage when infected with a mixture of highly virulent isolates of Fusarium fungi in two successive generations (Agafodorova et al. 2017). Transgenic rice transformed with the NmDef02 were able to ward off the sheath rot causing fungus Sarocladium oryzae (Pérez-Bernal et al. 2017). AtPDF1.1 expressing Arabidopsis plants were able to generate resistance against the necrotrophic bacterium Pectobacterium
carotovorum subsp. carotovorum in a unique way by restricting the iron bioavailability required for bacterial growth and infection and also activating the ethylene mediated defense signaling (Hsiao et al. 2017).
Lipid Transfer Proteins
Lipid transfer proteins or rather non-specific Lipid Transfer Proteins (nsLTPs) are cationic peptides derive their name from their ability to transfer lipid molecules between different membranes. They were discovered while searching for lipid carrier proteins in different plants. These proteins can carry and transfer a wide range of lipids like phospholipids (phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol and galactolipids), fatty acids and sterols. The lipid transfer proteins were first identified in the tubers of potato, following which their presence was found in both monocots and dicots. The tertiary structure of LTPs possess four distinctive α-helices which come together to form a hydrophobic space where they bind the lipids. LTPs are synthesized with N-terminal signal peptide which guides them to the cell wall, while the presence of a C-terminal peptide mandates post-translational modification thereby enabling it to be transferred to the extracellular side.
LTPs based on their disulfide bridging pattern can be categorized into two different families which share low sequence similarity of 30% (a) The LTP1 family consist of members which contain 8 conserved cysteine residues which are 90-95 aa long (21-27 aa signal peptide) with mass of about 10 kDa while the (b) LTP2 members are comparatively smaller with a length of 70 aa (27-35 aa signal peptide) and mass of about 7kDa and contain 4 conserved cysteine residues (de Oliveira Carvalho et al. 2007). LTP1 is found primarily in aerial organs, whereas LTP2 is expressed in roots. Interestingly, both classes are found in seeds. They are involved in a wide variety of processes like biosynthesis of membranes, cutin synthesis, maintenance of the fatty acid pool, somatic embryogenesis and as part of the defense response against phytopathogens. Their mode of action against fungi has not yet been deciphered yet, but it has been postulated that the LTPs penetrates and forms pore in the fungal membranes leading to ion efflux and ultimately cellular death (Selitrennikoff 2001).
Arabidopsis thaliana and tobacco expressing the barley LTP2 was granted protection against the attack of the bacterium Pseudomonas syringae pv. tomato and Pseudomonas syringae pv. tabaci (Molina and Garciá Olmedo, 1997). Scented geranium engineered to express the antimicrobial protein Ace-AMP1 from Allium cepa, were protected against Botrytis blight (Bi et al. 1999). The Ace-AMP1 on being expressed in rose plant provided protection against the powdery mildew disease caused by Sphaerotheca pannosa (Li et al. 2003). The pepper lipid transfer protein gene (CALTPI) generated enhanced resistance in Arabidopsis against both Pseudomonas syringae pv. tomato and Botrytis cinerea (Jung et al. 2005). Ace-AMP1 from Allium cepa conferred resistance to transgenic rice against the three economically important phytopathogens namely Magnaporthe grisea, Rhizoctonia solani and Xanthomonas oryzae (Patkar and Chattoo, 2006). The same lipid transfer protein on being introduced in wheat made it resistant against two devastating diseases, Karnal bunt and powdery mildew (Roy-Barman et al. 2006). Tobacco expressing the wheat Ltp 3F1 were conferred resistance against three different fungi viz. Alternaria sp, Cylindrocladium scoparium and Bipolaris oryzae (Kirubakaran et al. 2008). When the CALTPI and CALTPII genes from pepper were constitutively expressed in tobacco plants , it led to enhanced resistance to the fungi, Phytophthora nicotianae and bacterium, Pseudomonas syringae pv. tabaci (Sarowar et al. 2009). Transgenic poplar expressing the motherwort (Leonurus japonicus) nsLTP LJAMP2 developed substantial resistance against the fungi Colletotrichum gloeosporioides and Alternaria alternata (Jia et al. 2010). Wheat consitutively overexpressing the endogenous TaLTP5 were conferred with enhanced protection against both the common root rot and Fusarium head blight diseases (Zhu et al. 2012). Oilseed rape plants expressing the LJAMP2 developed enhanced resistance to stem rot caused by Sclerotinia sclerotiorum (Jiang et al. 2013). Transgenic Arabidopsis expressing TdLTP4 gene showed enhanced fungal resistance against Alternaria solani and Botrytis cinerea (Safi et al. 2015).
Puroindolines
The puroindolines are small 13kDa proteins with five conserved disulfide bonds which are cationic at physiological pH and unlike other plant AMP families consists of an exclusive tryptophan-rich domain. These were first isolated from wheat endosperm and are help regulate grain hardness and kernel texture (Giroux et al. 2003; Xia et al. 2008). The loop containing the Trp- rich region forms the lipid binding site and is the determining factor of its antimicrobial activity. Puroindolines additionally have both antibacterial and antifungal action via interactions with the membranes (Dhatwalia et al. 2009; Dubreil et al. 1998).They were able to cause loss of membrane organization causing cellular lysis via pore formation, but also postulated to be able to affect nucleic acid related vital processes by virtue of their nucleic acid binding capability (Alfred et al. 2013)
Transgenic rice expressing the wheat puroindoline genes pinA and pinB demonstrtated robust resistance against both the rice blast and sheath blight causing fungal pathogens (Krishnamurthy et al. 2001). The wheat puroindoline-b on being heterologously expressed in transgenic apple plants reduced the damage caused by the deadly fungal scab disease (Faize et al. 2004).Constitutive overexpression of the PINA protein in wheat helped provide tolerance against the devastating rust disease of wheat (Luo et al. 2008). When the wheat puroindolines, Puroindoline a and b were expressed constitutively in corn, they were conferred enhanced protection against the southern leaf blight (SLB) pathogen, Cochliobolus heterostrophus (Zhang et al. 2011). Wheat overexpressing the puroindoline PINA was found to have enhanced tolerance against the Penicillium seed rot (Kim et al. 2012).
Hevein-like Peptides
More than five decades ago hevein, a 43aa long peptide was isolated from the latex of the rubber tree, Hevea brasiliensis (Archer et al. 1960). Hevein-like peptides share a common chitin binding domain with hevein, but vary in their amino acid composition. The tertiary structure of hevein-like peptides constitutes antiparallel β-sheets with intermittent short helices stabilized by three to five disulfide bridges (Porto et el., 2012).By virtue of their chitin binding capability, hevein –like peptides are able to bind to and block the fungal cell wall synthesis and confering protection against fungal infection (Van Parijs et al. 1991). Pn-AMP1 isolated from Pharbitis nil were found to be cause actin depolarization by interacting with fungal cell wall mannoproteins, ultimately leading to osmotic stress and cell death (Koo et al. 2004). Wheat antimicrobial hevein like peptides WAMPs have been reported to protect plant chitinases from degradation by fungal Zn-metalloproteases, thereby directly preventing fungal hyphal elongation (Slavokhotova et al. 2014).
Transgenic rice overexpressing the wasabi defensin gene generated effective against the rice blast fungus, Magnaporthe grisea (Kanzaki et al. 2002). Poplar expressing the Amaranthus caudatus seed coat peptide Ac-AMP1 were successful in generating resistance against the fungi Septoria musiva (Liang et al. 2002). The hevein-like antimicrobial peptide Pn-AMP2 from Pharbitis nil when constitutively expressed in tobacco conferred resistance against black shank disease (Koo et al. 2002). When it was expressed in tomato it provided resistance against it’s the two economically important pathogens, Fusarium oxysporum and Phytophthora capsici (Lee et al. 2003). Potato cultivars expressing the wasabi defensin peptide WjAMP-1 displayed resistance against the gray mold disease caused by Botrytis cinerea (Khan et al. 2006). Moth orchids (Phalaenopsis) also expressing it was granted substantial protection against the soft rot causing bacterium Erwinia carotovora (Sjahril et al. 2006). Transgenic ‘Egusi’ melon engineered with the wasabi defensin was conferred with resistance against two fungi Alternaria solani and Fusarium oxysporum (Ntui et al. 2010). When Arabidopsis and tobacco were transformed with Stellaria media seed specific hevein-like peptides they had developed enhanced resistance against Bipolaris sorokiniana and Thielaviopsis basicola, respectively (Shukurov et al. 2012). Wasabi defensin genes under the control of root-specific promoters in both transgenic tomato and tobacco developed resistance against Fusarium oxysporum (Kong et al. 2014).
Snakins
Snakins have the distinction of having the highest number of disulfide bridges among all the plant antimicrobial peptide families. They have 12 conserved cysteine residues. The name is derived from the structural resemblance with the hemotoxic desintegrin-like snake venoms. (Segura et al. 1999). The snakins have also been found to share similarity in structure with both the GASA (giberellic acid stimulated in Arabidopsis) and GAST (giberellic acid stimulated transcript) protein family members. The three dimensional structure of the snakin family has not yet been deciphered, but in silico has provided a tentative molecular model (Porto et al. 2013). These were first identified in potato and found to have both antibacterial and antifungal activity (Berrocal-Lobo et al. 2002), but their mode of action has not been elucidated yet.
Potato plants constitutively expressing snakin-1 were protected against the fungus Rhizoctonia solani and the bacterium Erwinia carotovora (Almasia et al. 2008).When snakin-1 was heterologously expressed in wheat it provided enhanced resistance against the devastating fungal diseases of powdery mildew (Faccio et al. 2011) and take-all (Rong et al. 2013). Alfalfa overexpressing its endogenous snakin-1 provided considerable resistance against caused by the fungi Colletotrichum trifolii (anthracnose disease) and Phoma medicaginis (spring leaf spot) (García et al. 2014). Tomato plants engineered to express its own snakin-2 were able to successfully restrict the growth of bacterium Clavibacter michiganensis (Balaji and Smart, 2012). Snakin-2 overexpressing potato plants were conferred protection against the destructive black leg disease (Mohan et al. 2014).The snakin-1 homologue from soybean GmSN1 surprisingly possessed antiviral activity, on being constitutively expressed provided significant resistance against the turnip mosaic virus and soybean mosaic virus in Arabidopsis and soybean plants respectively (He et al. 2017).
Knottin-like
The knottin peptide was first discovered over a decade ago (Le Nguyen et al. 1990). They are small cysteine-rich peptides with a characteristic knot formed by three disulfide bridges and the peptide backbone, from where they derive their name (Gao et al. 2001). Knottins are usually 40 aa long and its tertiary structure is composed of three antiparallel β-strands joined by loops. The special disulfide bridging confers knottins with exceptional stability against degradation (Kolmar, 2009).
Mj-AMP1 from Mirabilis jalapa introduced into transgenic tomato against the early blight causing fungal pathogen Alternaria solani (Schaefer et al. 2005). Indica rice expressing the Mj-AMP2 developed sufficient resistance to inhibit the growth of the devastating rice blast fungus, Magnaporthe oryzae (Prasad et al. 2008).
Cyclotides
Cyclotides are a major group of plant AMPs with a unique cyclic backbone having a length 28-37 amino acids and a characteristic ‘cyclic cystine knot’ motif formed by three conserved disulphide bonds. This specific arrangement bestows cyclotides with extraordinary thermal, proteolytic and chemical stability against degradation (Craik et al. 1999).They are major presence in the Violaceae and Rubiaceae families, but also have been found in Cucurbitaceae, Fabaceae, Solanaceae and Poaceae families (Gruber, 2010). Cyclotides are expressed as precursor proteins which include an ER signal peptide and a pro-region consisting of the mature cyclotide domain with conserved N-terminal repeat region (NTR) and a short C-terminal tail (Craik et al. 2010). Cyclization of the peptide is achieved by cleavage of precursor polypeptides and subsequent cyclization by asparaginyl endopeptidases (Saska et al. 2007).
Cyclotides due to ultrastability are good candidates for generating disease resistance against plant pathogens, but the transgenic approaches have hit a roadblock since the cyclization process is inefficient leading to formation of linear precursor (Gillon et al. 2008).A probable solution would be to co-express the genes encoding for the cyclotide and its native cyclization enzyme.