The role of enzymatic activities of antiviral proteins from plants for action against plant pathogens

Abstract

Antiviral proteins (AVPs) from plants possess multiple activities, such as N-glycosidase, RNase, DNase enzymatic activity, and induce pathogenesis-related proteins, salicylic acid, superoxide dismutase, peroxidase, and catalase. The N-glycosidase activity releases the adenine residues from sarcin/ricin (S/R) loop of large subunit of ribosomes and interfere the host protein synthesis process and this activity has been attributed for antiviral activity in plant. It has been shown that AVP binds directly to viral genome-linked protein of plant viruses and interfere with protein synthesis of virus. AVPs also possess the RNase and DNase like activity and may be targeting nucleic acid of viruses directly. Recently, the antifungal, antibacterial, and antiinsect properties of AVPs have also been demonstrated. Gene encoding for AVPs has been used for the development of transgenic resistant crops to a broad range of plant pathogens and insect pests. However, the cytotoxicity has been observed in transgenic crops using AVP gene in some cases which can be a limiting factor for its application in agriculture. In this review, we have reviewed various aspects of AVPs particularly their characteristics, possible mode of action and application.

Introduction

The agricultural crop production is always under pressure due to environmental factors including biotic and abiotic stresses. Among biotic stresses, fungi, bacteria, and insect pests are managed to a great extent by employing the integrated disease management strategy, but very little or no effective control method is currently available for plant viruses. This is mainly because viral pathogens depend on host cellular resources, particularly on ribosomes for protein synthesis for their replication and multiplication. Approximately 250 agriculturally important viruses are characterized, which are known to cause significant yield losses world wide (Briddon and Markham 2000). The various viral disease management strategies including host resistance developed through rigorous breeding programme often fail because viruses have ability for quick changes in their genome, develop complex virus–host–vector relationship, and has diversity of infection mechanism (Verma and Varsha 1995). Nevertheless, some of the management practices for plant viruses like control of insect vector population, virus-free propagating material, cultural practices and use of resistant cultivars are adopted as integrated approach, but could not achieve desired success. The gene silencing mechanism of host defense has also been exploited with viral coat protein gene, replicase protein, movement protein and helper component protein to develop virus resistant crops (Lomonossoff 2015). These approaches may also have limitations like potential risk of genetic recombination between a transgene and an infecting virus, emergence of new pathogenic viruses and change in the host preference (Greene and Allison 1994).

Antiviral proteins (AVPs) have been reported as an endogenous, nonstress induced proteins from large number of plants, which are capable of virus inhibition. The AVPs are synthesized as pro-proteins consisting of signal peptide and C-terminal extension to follow secretory pathway that allows plant cells to segregate the cytotoxic proteins into vacuoles or possibly in another extracytoplasmic compartment (VanDamme et al. 2001). The immunolocalization study shows that AVPs are majorly located in cell wall matrix and a small amount is found in vacuoles in leaves of Phytolacca americana (Monzingo et al. 1993; Ready et al. 1986).

The AVPs are classified into three types based on their structure and composition viz. type 1, type 2, and type 3 (Fig. 1). Type 1 are holo-protein that consists only a single chain protein of approx. 30 kDa, whereas type-2 and type-3 are chimero protein consists 2 chain. The type 2 chimero protein of approx. 60 kDa has one α-chain polypeptide with RIP activity and another one β-chain with galactose binding lectin domain, both are linked through a disulfide bond. The type 3 chimero protein also contain one α-chain polypeptide with RIP activity and another domain with unknown function, however β-chain polypeptide must be removed to become active RIP (Nielsen and Boston 2001). These AVPs have rRNA N-glycosidase activity, which depurinate the large subunit of ribosome of host cells upon virus infection and therefore AVPs are also known as ribosome-inactivating proteins (RIPs) (Irvin 1975). Type-1 protein possess multiple enzymes activities like N-glycosidase, RNase, DNase, and antioxidant activities. The rRNA N-glycosidase activity is the main antiviral function of AVPs, which inactivates the host ribosomes and block their participation in protein synthesis (Meng et al. 2014; Zhu et al. 2013). In addition to enzymatic activity, AVPs have nonenzymatic antiviral activity like direct interaction to bind virus particles, interfere with viral infection process and also alter the host cell metabolism (Tumer 2015). Some AVPs are also characterized for their antibacterial, antifungal and insecticidal property (Rumiyati et al. 2014; Hamshou et al. 2016, 2017; Ajji et al. 2016; Zhu et al. 2013, 2018; Lodha et al. 2011; Tumer 2015; Kim et al. 2003). To show the antiviral activity, aqueous crude extract of leaves containing AVP was rubbed on to the leaf of nonhost healthy plants like Nicotiana glutinosa and Cyamopsis tetragonoloba, which were preinoculated with tobacco mosaic virus (TMV) and sunnhemp rosette virus (SRV). These plants showed ~ 95% lesser local lesions as compared to control plants that proved the antiviral property of AVP (Verma and Baranwal 1983; Roy et al. 2006; Stripe et al. 1981; Straub et al. 1986; Dutt et al. 2003). Some AVPs also induce antioxidant molecules that provide systemic resistance in plant upon virus infection (Bhatia and Lodha 2005; Srivastava et al. 2015; Straub et al. 1986). Like native AVPs, the recombinant AVP expressed and purified from E. coli also showed antiviral activity against viruses (Begam et al. 2006; Roy et al. 2006; Choudhary et al. 2008a; Sipahioglu et al. 2017; Shang et al. 2016; Zhu et al. 2013). An AVP can provide resistance to multiple viruses and other plant pathogens at low concentration that promises that AVPs can be a potential candidate in management of plant pathogens (Chen et al. 2002). However, the mechanism of AVPs against plant pathogens is not known completely. The researchers need to conduct depth study to understand the cell entry mechanism, specificity, stability, dosages of AVP for its wide application in agriculture (Puri et al. 2012).

Fig. 1.

Structure and processing of type- 1, 2 and 3 AVPs/RIPs. Type 1 RIP is a single chain holo-RIP and not required enzymatic processing to become active. Type 2 RIP is a chimero-RIPs and consists two parts, one chains has the enzymic properties and other part usually has lectin domain. Type 3 RIP is also chimero-RIPs and contain two parts, one chain has enzymatic properties, but it become active only if the other unknown domain removed

Source of AVPs

AVPs are found in leaves, seeds, flowers, fruits, roots, rhizomes or bark of plants belonging to plant taxonomic families like Amaranthaceae, Chenopodiaceae, Compositae, Cucurbitaceae, Euphorbiaceae, Graminae, Nyctaginaceae, Pinaceae, Rosaceae, Solanaceae, Verbenaceae (Verma and Varsha 1995). Most of the plants express only one type of AVP except for Iridaceae, Euphorbiaceae and Cucurbitaceae family, which can express more than one type of AVP. The source of different AVPs/RIPs with their activities is presented in Table 1.

Table 1.

Source of AVPs and their biochemical properties

Source of AVPs	AVP/RIP	M.W. (kDa)	Biochemical property	Activity against plant pathogens, insect pest and others	References
Type 1 AVP
Amaranthus tricolor (lal chaulai)	AAP	27	N-glycosidase and RNase	Virus [sunnhemp rosette virus (SRV)]	Roy et al. (2006)
Amaranthus viridis	Amaranthin	30	N-glycosidase activity	Viruses (tobacco mosaic virus)	Kwon et al. (1997, 2000)
Beta vulgaris L. (Sugar beet)	Beetins BE27	27, 29	RNA depurination, and SAR	Viruses (TMV, artichoke mottled crinkle virus), Fungi (Penicillium digitatum)	Iglesias et al. (2005), Citores et al. (2016)
Boerhaavia diffusa L.	BDP-30	30	N-glycosidase activity	Viruses (TMV)	Srivastava et al. (2015)
Bougainvillea × buttiana	BAP-28 & BAP-24 BBAP1(recombinant)	28 & 24 35.49	N-glycosidase, RNase, DNase	Viruses (TMV and SRV)	Narwal et al. (2001a, b) Choudhary et al. (2008b)
Bougainvillea spectabilis (Roots, Leaves)	BAP 1, Bouganin	28 26.2	N-glycosidase	Bacteria (Staphycococcus aureus, Bacillus subtilis)	Umamaheswari and Nuni (2008), Hartog et al. (2002)
Celosia cristata	CCP-25 & CCP-27	25 & 27	N-glycosidase, RNase, DNase, and antioxidant activity	Viruses (TMV and SRV)	Begam et al. (2006), Gholizadeh et al. (2004)
Chenopodium album (bathua)	CAP-I and CAP-II (Isoform)	24	N-glycosidase	Viruses (TMV and SRV)	Dutt et al. (2003)
Chenopodium album	CAP-30	30	N-glycosidase	Viruses (TMV) and anti-microbial activity	Park et al. (2004b)
Clerodendrum aculeatum	CA-SRI	34	Induce systemic resistance	Viruses	Kumar et al. (1997)
Clerodendrum inerme	CIP	29	N-glycosidase, induce SAR	Viruses (SRV, TMV, and Papaya ringspot virus)	Prasad et al. (2014)
Dianthus caryophyllus	Dianthin	30 & 32	N-glycosidase	Viruses (TMV)	Stripe et al. (1981)
Elaeis guineensis (Oil palm)	EgRIP-1a and EgRIP-1b	28–30	N-glycosidase	Fungus (Ganoderma boninense)	Sargolzaei et al. (2016)
Mirabilis expansa	ME1 and ME2	27.0 & 27.5	N-glycosidase	Bacteria (Pseudomonas syringae, Agrobacterium tumefaciens), Fungus (Pythium irregulare, Fusarium oxysporum solani)	Vivanco et al. (1999)
Mirabilis jalapa	MAP and MAP-4	27.78 & 29.34	Adenine polynucleotide glycosylase activity	Viruses (TMV), Bacteria (Propionibacterium acnes and Staphylococcus epidermidis)	Bolognesi et al. (2002), Rumiyati et al. (2014)
Momordica charantia	MAP, α-MC	30	N-glycosidase	Viruses (Zucchini Yellow Mosaic virus), Bacteria Anti-cancer activity	Puri et al. (2009), Meng et al. (2014), Wang et al. (2016)
Phytolacca americana	PAP	29	N-glycosidase	Viruses (Potato virus X, Zucchini yellow mosaic virus), Fungus (Rhizoctonia solani)	Tumer et al. (1997), Zoubenko et al. (1997), Sipahioglu et al. (2017)
Phytolacca dioica	PDL	29.40	Polynucleotide: adenosine glycosidases	Viruses	Di Maro et al. (1999)
Salsola longifolia	SLP-32	32	DNase, N-glycosidase	Viruses (Tobacco necrosis virus, Bean yellow mosaic virus)	Torky (2012)
Spinacia oleracea	SoRIP1 and SoRIP2	31 & 29	N-glycosidase activity, systemic resistance	Viruses (TMV)	Straub et al. (1986), Kawade et al. (2008)
Trichosanthes kirilowii	Trichosanthin	30	N-glycosidase activity	Viruses (TMV and CMV)	GuoYong et al. (1999)
Zea mays L.	maize RIP	30	N-glycosidase	Insect pest (cigarette beetle, Lasioderma serricorne (F.), and the tobacco hornworm, Manduca sexta (L.), Helicoverpa zea) and fungus sheath blight (Rhizoctonia solani)	Dowd et al. (2003, 2006, 2012), Kim et al. (2003)
Type-2 AVP
Abrus precatorius	Abrin	63 (30 + 33)	N-glycosidase	Viruses	Singh (2018)
Malus domestica Borkh) Apple	Type II	30	N-glycosidase	Insect pest Pea aphids (Acyrthosiphon pisum) and green peach aphids (Myzus persicae)	Hamshou et al. (2016)
Ricinus communis	Ricin A & B	32 & 34	N-glycosidase	Biomedical application	Polito et al. (2019)
Sambucus ebulus	Ebuin I	56 (26 + 30)	N-glycosidase	Viruses	Citores et al. (1997)
Sambucus nigra	SNA-I	24 + 23.5	N-glycosidase	Insect pests (Acyrthosiphon pisum, Myzus nicotianae) beet armyworm (Spodoptera exigua)	Chen et al. (2002), Shahidi-Noghabi et al. (2009)
Viscum album Himalayan	Hm RIP	65	N-glycosidase	Anti-cancer, antigenicity and pharmacological properties	Mishra et al. (2004)

Type 1 AVP/RIP

The type 1 AVP of 28 kDa and 24 kDa was found in leaves of B. xbuttiana (Narwal et al. 2001a; Bhatia and Lodha 2005) and CAP-I and CAP-II of 24 kDa in leaves of Chenopodium album cv Pusa Bathua 1 (Dutt et al. 2003). The SoRIP1 isolated from the roots of Spinacia oleracea L. cv. Jiromaru, accumulates primarily in pre-embryos stage and peripheral meristem of somatic embryos during early development. Its expression was very low in the callus, but high in the epidermis of somatic embryos suggesting that the expression of spinach RIP genes is differentially regulated in a development‐dependent fashion during somatic embryogenesis in spinach (Kawade et al. 2008). Some AVPs are present in isoforms and may be localized in different plant parts viz. AVP of 26.2 kDa in leaves and 28 kDa in roots of B. spectabilis (Rajesh et al. 2005; Umamaheswari and Nuni 2008). Seven isoforms of saporin was found in seeds, leaves and roots of soapwort (Saponaria officinalis L.) (Ferreras et al. 1993) while two isoforms of PAP have been reported in leaves and roots of Phytolacca americana (Duggar and Armstrong 1925; Irvin 1975, 1983; Barbieri et al. 1982). AVPs may also express differently in different season like PAP-II of 30 kDa was expressed in summer, while 29 kDa was observed in spring (Irvin et al. 1980). Similarly, CCP-25 expresses at pre-flowering stage, whereas CCP-27 expressed at postflowering stage in Celosia cristata (Balasubrahmanyam et al. 2000). Some well characterized type 1 AVPs are presented in Table 1.

Type 2-AVP/RIP

Ricin is the first AVP isolated from seeds of Ricinus communis L. which was characterized as a type 2 AVP. Type 2 protein contain the characteristic N-glycosidase activity and an additional an additional sugar-binding moiety, which helps this protein to cross the cell membrane (Polito et al. 2019; VanDamme et al. 2001; Bertholdo-Vargas et al. 2009). In a study, it was found that one ricin molecule can interfere the function of approximately 1500 ribosome molecules per minute in host plant. Therefore, type 2 AVP characterized as more toxic protein than type 1. Ricin is considered a threat for bioterrorism because no antidote is available to neutralize ricin (May et al. 2013; Tumer 2015). Abrin, a type II RIP isolated from seeds of Abrus precatorius, rosary pea induces the apoptosis process in cells triggering intrinsic mitochondrial pathway. Abrin toxin might inhibit the large subunit of ribosome, which block the protein synthesis and chain elongation in eukaryotes (Singh 2018). Ricin and abrin are a toxic RIP. A nontoxic type 2 RIP, ebulin was isolated from rhizomes of dwarf elder (Sambucus ebulus L.), which only depurinates sensitive ribosomes and interferes protein synthesis in mammal, but not in plant system (Citores et al. 1997). Himalayan mistletoe ribosome-inactivating protein II (HmRIP-II), a disulfide linked toxin and lectin subunits was purified from Viscum album. HmRIP-II occurs in four isoforms, HmRIP 1 and 2 of MW 28 and 34 kDa and HmRIP 3 and 4 of MW 28 and 32 kDa having isoelectric points of 6.6, 6.1, 5.2, and 4.7 (Mishra et al. 2004). Some well characterized type 2 AVPs are presented in Table 1.

Type 3 AVP/RIP

The jasmonate-induced protein (JIP60) is the well-known type 3 AVP found intracellularly in vacuoles, peroxisomes, and in the nucleoplasm of barley crop (Hause et al. 1994). The N-terminal and C terminal of JIP60 protein contain the homologous catalytic domain of ribosome-inactivating protein and eukaryotic translation initiation factor 4E (eIF4E) domain, respectively (Chaudhry et al. 1994). JIP60 synthesized as a precursor, which undergoes proteolytically processing and releases their C-terminal domain. The intact N-terminal domain of JIP function as classical N-glycosidase activity to inhibit the cellular translation. However, RIP function of JIP for plant immune response is dependent on host jasmonate hormones level (Przydacz et al. 2020; Reinbothe et al. 1994; VanDamme et al. 2001).

Enzymatic activities

AVPs possess the multiple enzymatic activities, however, the domain for enzymatic activities and recognition site of AVPs can be different for antiviral activity. Some of the enzymatic activities of AVPs are characterized as: (1) N-glycosidase activity; (2) specific depurination of capped mRNA; (3) depurination of supercoiled double-stranded DNA, and (4) RNase activity of AVPs.

N-glycosidase activity

A specific adenine conserved at nucleotide position 4324 (A⁴³²⁴) in α-S/R loop of large ribosomal subunit, is important for stability and structure of ribosome in plant species. The S/R loop is a critical element and it belongs to GTPase associated center for unidirectional route for translational apparatus by translational GTPases (trGTPase) activity upon GTP hydrolysis. GTP hydrolysis does not get activated by trGTPase enzyme in absence of intact S/R loop and hinders translation process (Grela et al. 2019). Upon virus infection, the basic amino acid present on surface structure of AVP interacts with the C-terminal of S/R loop to activate the N-glycosidase activity (Shi et al. 2016). The N-glycosidase activity releases the adenine (A⁴³²⁴) from S/R loop to damage the ribosome, which do not support the binding of initiation (eIF) and elongation factors (eEF), and thus protein synthesis stops (Wang and Tumer 1999). The PAP function is known to inhibit eEF1 dependent binding of amino acyl-tRNA to acceptor site (A site) and EF-1-mediated GTP hydrolysis. PAP also inhibits formation of EF2-GDP-ribosome complex and stimulates the ribosome-dependent hydrolysis of GTP (Irvin 1995). This is called ‘local suicide model’, which is widely accepted antiviral mechanism of AVP (Gessner and Irvin 1980). In noninfected cells, the ribosomes are protected from endogenous AVP because of compartment and predominant localization in extracellular matrix of mesophyll cells of leaf (Ready et al. 1986). Similar N-glycosidase activity also reported with AVPs like ME, Saporin, MAP, CIP-29, BAP 1 (Balasaraswathi et al. 2001; Barbieri et al. 1996, 1997; Bolognesi et al. 2002; Narwal et al. 2001b; Kwon et al. 1997, 2000; Olivieri et al. 1996; Park et al. 2004a; Roy et al. 2006; Kataoka et al. 1991). The catalytic activities of AVPs are also found at A⁴³²⁴ in 28S rRNA of rat liver, A²⁶⁶⁰ of 23S rRNA in E. coli, A³⁰¹⁷ of 25S rRNA in plants, A⁴³²¹ from large ribosome of yeast and A⁴³²³ from tobacco ribosome (Endo et al. 1987; Endo and Tsurugi 1987; Prestle et al. 1992; Hudak et al. 2000).

The type 2-AVP ricin releases guanine (G⁴³²³) from S/R loop of large subunit of ribosomes of rat liver (Endo and Tsurugi 1987). The AVP-ME1 has been reported to release guanine from wobble base pair from hairpin stems (Park et al. 2004a). In some cases, PAP also releases guanine from ribosomes of rabbit reticulocytes, tobacco, yeast and E. coli (Rajamohan et al. 1999b; Hudak et al. 2000). However, the mechanism of release of guanine from ribosome is unknown and it is different than the N-glycosidase activity (VanDamme et al. 2001).

Depurination of capped mRNA

Viral genome-linked protein (VPg), an analogue of m7G cap structure is covalently linked at 5′ end via a tyrosine residue in many plant virus mRNAs and it is required for infectivity, replication, cell-to-cell movement, and other function of virus. PAP complex specifically binds to translational enhancer (TE) structure of VPg, which interacts with host eIF4E isoform (eIFiso4E) and eIF4F (eIFiso4F) factors at position between 511 and 624 for protein synthesis (Wang and Hudak 2006). A mutant AVP was created by deleting the 25 amino acids from C-terminal end (Tumer et al. 1997; Zoubenko et al. 1997). The depurination of capped viral mRNA was studied with mutant AVP, which do not have N-glycosidase activity. The mutant AVP was found to have four times more binding affinity to viral VPg than rRNA in an in vitro study (Hudak et al. 2002). However, PAPc mutants did not differentiate between viral and own capped RNA (Parikh et al. 2002). ME1 and saporin have different affinities with diverse types of capped viral RNAs, which suggest that specific depurination activity is limited to selective cap of RNA (Vivanco and Tumer 2003). This study shows a powerful antiviral strategy of intact PAP, which may interact with viral VPg to prevent replication of viral mRNA along with ribosome depurination (Domashevskiy et al. 2012; Domashevskiy and Goss 2015).

Depurination of DNA

The active site of AVP has apurinic/apyrimidinic (AP) region for the depurination activity, which removes the adenine nucleotide from dsDNA however, the molecular mechanism is still unknown. It was demonstrated that an active site mutant of PAP, which failed to perform the depurination activity in an in vitro study with PAPx (Wang and Tumer 1999; He and Liu 2004). AVPs-PD-S2 and PD-L4 isolated from Phytolacca dioica shows DNA polynucleotide: adenosine glycosylase activities on supercoiled plasmid DNA. Similarly, AVPs like BBAP1, CCP-27, PAP, tricosanthin AVP, momorcharin, ricin, luffin, cinnamomin, and camphorin having depurination activity, were found to alter the conformation of ds supercoiled plasmid DNA into nicked, circular and linear form at lower and higher concentration of AVPs, respectively. The typical conformation of DNA is essential for depurination activity because no further changes were observed in DNA with extended incubation time (Li et al. 1991; Thomas et al. 1992; Day et al. 1998; Ling et al. 1994; Choudhary et al. 2008b; Gholizadeh et al. 2005a; Bhatia and Lodha 2005; Begam et al. 2006; Torky 2012).

Ribonucleases (RNase) activity

N-terminal amino acids of AVP-figaren were sequenced and analyzed, which revealed that few conserved amino acids have similarity with RNase-like enzymes and were suspected to act on viral RNA genome to release a fragment (Masayuki et al. 2001).

RNase activity of AVPs were demonstrated in a gel assays containing RNA, which ruled out any external contamination of nucleases (Vivanco and Tumer 2003; Hudak et al. 2000). RNase activity of momorcharin-AVP releases the small RNA fragment from naked rRNA but not from polyU nucleotide sequence to supports ribonucleolytic cleavage (Mock et al. 1996). The CCP-25 releases a 360-ntd fragment from ribosomal RNA and inhibits the protein translation from RNA of Brome mosaic virus (BMV) (Baranwal et al. 2002). The recombinant PAP and nontoxic PAP mutant also depurinate BMV-RNA. In an in vivo experiment, decreased level of BMV-RNAs was found in barley protoplast pretreated with BMV-RNA3, which indicated that replicase enzyme stop synthesis at missing base of depurinated template. This depurination activity affects directly the replication of viral RNA and also the formation of subgenomic RNA (Picard et al. 2005). Similarly, BAP-24, BBAP1, CCP-27, PAP isoforms PAP-I, PAP-II, and PAP-III also showed RNase activity against the RNA of Torula yeast, CMV, TMV, and SRV (Masayuki et al. 2001; Choudhary et al. 2008b; Begam et al. 2006; Roy et al. 2006; Rajamohan et al. 1999a).

Mechanism of action of AVP

The antiviral mechanism of AVP has not yet been decoded completely at biochemical and molecular level, which is essential for its application in crop science to enhance quality and production (Bolognesi et al. 2016). Some in vitro and in vivo studies helped to understand the mechanism of antiviral activity of AVP. It believed that AVP expresses as soon as virus infects the plant host and remains active fighting at various stages of virus pathogenesis process to prevent them to establish inside host. AVP might interfered the virus infection process by blocking the entry of virus to host cell. In an experiment, it was demonstrated that the inhibitory response may persist up to 6 days (Verma and Baranwal 1983; Baranwal and Verma 1992). The AVPs-PAP I and PAP II facilitate to adsorb viruses and aggregate to form loose complex, which helped inhibit the pathogenicity of viruses. However, such experiment cannot justify completely the extent of inhibition of virus infection process (Grasso and Shepherd 1978; Kumon et al. 1990). The site-directed mutagenesis experiment shows that 16 amino acids of N-terminal of AVP are required for its toxicity and ribosome depurination. The ribosome depurination activity gradually decreases in case of sequential deletion of amino acids from C-terminal AVP. In another experiment, introducing stop codon at Glu-244 position or mutation at Tyr-123 of active site domain of PAP, interfered the ribosome depurination, but cytotoxicity and systemic acquired resistance (SAR) induction were stopped (Tumer et al 1997; Zoubenko et al. 2000). This study suggests that active site and C-terminal of AVP are essential for cytotoxicity and ribosome depurination activity, and antiviral activity of AVP might be different from ribosome-inactivating property (Hudak et al. 2004). AVPs might induces the apoptosis process for local cell death by probable action of cellular stress integration. AVP may also mitigate the virus induced hypersensitive response of cell death to control virus growth and maintain normal growth of plant (Gholizadeh et al. 2005b; Narayanan et al. 2005).

In another study, it was reported that AVP–BAP modifies the antioxidant level in TMV infected leaves. The level of superoxide dismutase (SOD) and peroxidase (POD) was also found increased, whereas catalase (CAT) level deceased as compared to noninfected leaves. It was believed that AVP may scavenge the reactive oxygen species (ROS) as well as alter host cell metabolism to maintain its antioxidant status to fight against viral pathogens (Bhatia et al. 2004; Zhu et al. 2016; Yang et al. 2016). Bioinformatics study analysis revealed that approx. 15 amino acid of AVP has similarity with Fe-SOD, which is known for strong antimicrobial activity against viruses, fungi and bacteria (Sharma et al. 2004). The systemic acquired resistance (SAR) has also been reported as an antiviral activity by AVP against plant viruses. In an experiment, it was shown that AVP-beetins application interfere the infection of artichoke mottled crinkle virus (ACMV) at local as well as distal leaves by inducing expression of salicylic acid (SA), a plant defense mediator molecule. It is also believed that AVP improves the damaged photosystem upon virus infection and enhances the glutathione disulfide ratio (Iglesias et al. 2005; Verma and Awasthi 1980).

Some AVPs showed antifungal activity by damaging the ribosome, inhibiting the protein synthesis and growth of fungus (Citores et al. 2016; Park et al. 2004a, 2009; Sargolzaei et al. 2016; Vivanco et al. 1999; Kim et al. 2003). The AVPs like BE27, BAP1, have shown antibacterial activity too by unknown mechanism in in vitro study against bacteria like Staphycococcus aureus, Bacillus subtilis, Streptococcus faecalis, Micrococcus luteus, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhii, Klebsiella pneumoniae, Proteus vulgaris, Serratia marcescens, Shigella flexneri and Vibrio cholera (Umamaheswari and Nuni 2008), MAP-4 against Propionibacterium acnes and Staphylococcus epidermidis (Rumiyati et al. 2014). It is suspected that different functions of AVP might play role to minimize the local lesion formation on leaves after infection with pathogenic viruses, such as SRV, TMV, and potato virus X (Noronha et al. 1980).

AVP/RIP possess the insecticidal property and function against crop insect pest in concentration-dependent manner. The different concentrations of SNA-I, a type II RIP from Sambucus nigra, decreased the fecundity and survival of pea aphid Acyrthosiphon pisum (Shahidi-Noghabi et al. 2008, 2010). The transgenic tobacco expressing SNA-I demonstrated to show similar observation against green peach aphids Myzus nicotianae (Shahidi-Noghabi et al. 2009) and similar observation found with type I and type II RIPs of apple (Malus domestica Borkh) against wild type Anticarsia gemmatalis Hübner and Spodoptera frugiperda (Bertholdo-Vargas et al. 2009; Hamshou et al. 2016). In addition, the overexpression of a maize RIP in tobacco plants enhanced resistance against corn earworms, Helicoverpa zea (Dowd et al. 2003, 2012). How different AVPs/RIPs function as anti-insect pest is not yet known. However, some studies suggest that apoptosis process might be induced by RIP in the insect pest midgut through the activation of caspase 3 (Narayanan et al. 2005; Sikriwal and Batra 2010; Das et al. 2012; Shahidi-Noghabi et al. 2010).

Transgenic crop development with AVP gene

The promising antimicrobial and anti-insecticidal property of AVPs with no risk of genetic recombination due to their plant origin, they are better candidate for management of viruses and other pests of agriculture crops (Dasgupta et al. 2003; Akkouh et al. 2015; Chen et al. 2002; Park et al. 2004b; Hassan and Ge 2018). Some of the isolated AVP genes presented in Table 2 have been used or could be used for the development of transgenic crops.

Table 2.

List of isolated genes encoding for AVPs/RIPs

Source	Name of AVPs	MW (kDa)	No. of a.a	References
Adenia volkensii (Roots)	Volkensin-type-2 RIP	29	523	Chambery et al. (2004)
Amaranthus viridis	Amaranthin	30	270	Kwon et al. (2000)
Amaranthus tricolor	AAP	27	297	Roy et al. (2006)
Bougainvillea spectabilis (Leaves)	Bougainin	26.2	250	Hartog et al. (2002)
Bougainvillea spectabilis (Roots)	AP 1	28	315	Balasaraswathi et al. (1998)
Bougainvillea × buttiana	BBAP	28	319	Choudhary et al. (2008a, b)
Clerodendrum aculeatum	CA-SRI	32	302	Kumar et al. (1997)
Celosia cristata	CCP-27	27	283	Begam et al. (2006), Gholizadeh et al. (2005a)
Dianthus caryophyllus	Dianthin 30	29.5	293	Legname et al. (1993)
Dianthus sinensis	Dianthus AVP	23	294	Cho et al. (2000)
Himalayan misteletoe	Hm RIP- a type-2 RIP	65	552	Mishra et al. (2004)
Mirabilis jalapa	MAP	24.2	250	Kataoka et al. (1991)
Mirabilis expansa	ME1	29.5	317	Vepachedu et al. (2003)
Phytolacca americana	PAP	29	291	Poyet and Hoeveler (1997)
	PAP-S PAPII	30	293
Sechium elude	Sechiumin	27	285	Wu et al. (1998)
Trichosanthes kirilowii	TCS	30	293	GuoYong et al. (1999)

NU nucleic acid, Pr protein

The transgenic tobacco crops developed with full-length cDNA of PAP shows resistance against mechanical or aphid-transmitted viruses from their early stage of pathogenesis and antifungal property against fungus Rhizoctonia solani (Lodge et al. 1993; Poyet and Hoeveler 1997; Wang et al. 1998). The constitutive expression of pathogenesis related protein (PR-1) was also reported but the expression of SA was unchanged in transgenic tobacco. The double mutant PAP at position L20R and Y49H was found less toxic than the full-length cDNA of PAP. The transformation efficiency of mutant PAP was also found enhanced by 7–18% with mutant PAP (Di and Tumer 2015). The transgenic creeping bent grass developed with mutant PAPII gene do not show antifungal property against Clerotinia homoeocarpa, a causal agent of dollar spot disease because of unstable and very less protein expression (Dai et al. 2003). The active form of Maize RIP when co-expressed with basic chitinase gene in transgenic rice helped to increase resistance against the fungus sheath blight (R. solani) (Kim et al. 2003). The transgenic crops developed with mutant PAP (amino acid substitution G75D) did not depurinate ribosome and was nontoxic but still provided antiviral resistance. It was suspected that mutant AVP induces the defense proteins like PR proteins, wound-inducible protein kinase and protease inhibitor II for antiviral resistance (Zoubenko et al. 2000).

In another experiment, transgenic tomato transformed with cDNA of trichosanthin (TCS) shows resistance to TMV and cucumber mosaic virus (CMV) (GuoYong et al. 1999). The C. tetragonoloba plants transformed with CIP-29 gene induces the systemic resistance and shows resistance to SRV, TMV, and papaya ringspot virus (Prasad et al. 2014). The tobacco plants transformed with cDNA of type-2 AVP gene, SNA-1, SNA-V, and SNLRP show reduced local lesion upon TMV infection (Venbussche et al. 2004; Chen et al. 2002).

Transgenic grapevine plant expressing chitinase AVP showed resistance to fungus Uncinula necator and Plamopara viticola under field conditions (Bornhoff et al. 2005); transgenic black gram expressing chitinase AVP showed enhanced resistance upto 47% to fungus Corynespora cassiicola (Chopra and Saini 2014); tobacco crop expressing barley AVP provided resistance to soil-borne fungus R. solani (Logemann et al. 1992); transgenic rice expressing α-MC provide resistance to rice blast disease (Wang et al. 2016; Qian et al. 2014); and transgenic potato expressing PAP provided resistant to Botrytis cinerea and R. solani (Gonzales-Salazar et al. 2017). The cDNA encoding PD-S2 was transferred to tobacco crops displays the antipathogenic properties against viruses, fungi and insects (Iglesias et al. 2016).

Conclusion and future prospects

Higher plants contain the antiviral proteins (AVPs) in an inactive state in localized compartment, which become active to protect host cells upon plant virus infection. AVPs are known for their multiple enzymatic activities like N-glycosidase, RNase, DNase, and also induce the PR proteins, SA, JA, SOD, and CAT in host cells. They play an antiviral role and provide protection from viruses at local and distal sites with unexplained mechanism. The N-glycosidase activity is the principal function of AVPs and is widely accepted for the antiviral activity. The N-glycosidase activity depurinates the host ribosome of infected cells and interferes with the host protein synthesis as well as of virus genome. As a result, survival of viruses inside the host cells get disturbed as they are an obligate parasite and dependent on host cells resources for their replication and protein synthesis for multiplication. The transgenics crops generated with single AVP gene have shown expected antiviral activity against several viruses and also displays antifungal, antibacterial and antiinsecticidal activity. However, cytotoxicity is the major concern, and due to this reason AVP genes have not been in use for agriculture crops against plant viruses. AVP isolated from bougainvillea plant was found less toxic among all characterized type 1 AVPs, therefore, it can be a candidate for agricultural uses (Barbieri et al. 1993; Hartog et al. 2002; Tumer 2015). Bougainvillea AVP, a lysine-rich protein of pI ~ 10.0–10.5 having characteristic enzymatic activity and also induces systemic resistance against plant viruses (Baranwal and Verma 1992; Narwal et al. 2001a; Bolognesi et al. 1997; Balasaraswathi et al. 1998; Narwal et al. 2001a; Bhatia and Lodha 2005; Straub et al. 1986). The mechanism of AVP for its antiviral, antibacterial, antifungal, and anti-insecticidal activity is still not fully understood and it requires the integrated approach of molecular biology, biochemistry, genomics and structural biology for better understanding. The detailed study of AVP at structure and function level will reveal the degree of binding, intracellular trafficking, ribosome interactions, and toxin activity. In addition, the complete understanding of AVP should help translate it into spray bioformulation for easy application on agricultural and horticultural crops against plant pathogens to enhance the quality production.

Compliance with ethical standards

Conflict of interest

The author(s) declare that they have no competing interests.