chitosan-induced antiviral activity and innate immunity in plants

RESEARCH ARTICLE

Chitosan-induced antiviral activity and innate immunity in plants

Marcello Iriti & Elena Maria Varoni

Received: 15 April 2014 /Accepted: 5 September 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract Immunity represents a trait common to all livingorganisms, and animals and plants share some similarities.Therefore, in susceptible host plants, complex defence ma-chinery may be stimulated by elicitors. Among these, chitosandeserves particular attention because of its proved efficacy.This survey deals with the antiviral activity of chitosan, fo-cusing on its perception by the plant cell and mechanism ofaction. Emphasis has been paid to benefits and limitations ofthis strategy in crop protection, as well as to the potential ofchitosan as a promising agent in virus disease control.

Keywords Plant activators . Plant innate immunity . PAMPs/MAMPs . PRRs . Viral diseases . Plant viruses . Cropprotection . Environmental safety

AbbreviationsABA Abscisic acidAIDS Acquired immunodeficiency

syndromeAMV Alfalfa mosaic virusTBSV Bean/tomato bushy stunt virusCaMV Cauliflower mosaic virusCERK1 Chitin elicitor receptor-like kinaseCEBiP Chitin elicitor-binding proteinCHT ChitosanCC Coiled-coilDD Deacetylation degreeHAMPs or DAMPs Host- or damage-associated molecular

patterns

HR Hypersensitive reactionISR Induced systemic resistanceTIR Interleukin-1 receptorJA Jasmonic acidLRR Leucine-rich repeatLAR Local acquired resistanceMAPK activation Mitogen-activated protein kinaseMW Molecular weightNPR1 Non-expressor of PR-1NB Nucleotide bindingPAMPs or MAMPs Pathogen- or microbe-associated

molecular patternsPR-10 Pathogenesis-related protein 10PRRs Pattern recognition receptorsPD Polymerization degreesPSTV Potato spindle tuber viroidPVX Potato virus XPCD Programmed death of plant cellsRaMV Radish mosaic virusRNS Reactive nitrogen speciesROS Reactive oxygen speciesRLKs Receptor-like kinasesRLPs Receptor-like proteinsSA Salicylic acidSAR Systemic acquired resistanceTMV Tobacco mosaic virusTNV Tobacco necrosis virusTuMPV Turnip mosaic virusBABA-IR β-Aminobutyric acid-induced

resistance

Virus biology, infection process and disease control

Avirus is a nucleoprotein consisting of nucleic acid (RNA orDNA) and protein, the latter forming the capsid, a protectivecoat around the former. This nucleoprotein multiplies only in

Responsible editor: Philippe Garrigues

M. Iriti (*)Department of Agricultural and Environmental Sciences,Milan StateUniversity, Via G. Celoria 2, 20133 Milan, Italye-mail: [email protected]

E. M. VaroniDepartment of Biomedical, Surgical and Dental Sciences, MilanState University, Via G. Celoria 2, 20133 Milan, Italy

Environ Sci Pollut ResDOI 10.1007/s11356-014-3571-7

living cells and may cause a multitude of diseases in allorganisms. Some viruses attack humans, animals or both,causing diseases such as influenza, polio, rabies and acquiredimmunodeficiency syndrome (AIDS); others infect higherplants and other microorganisms as well, such as fungi andbacteria. The total number of viruses known to date exceeds2000, and nearly half of these have the ability to causediseases in plants (Agrios 2005).

The nucleic acid of most plant viruses consists of RNAprotected by the protein shell. Plant viruses enter cells onlythrough wounds, or by vectors, or by deposition into an ovuleby an infected pollen grain. They are transmitted either in apersistent or nonpersistent manner by insects (mostly aphids).Persistent transmission means that once the insect vectorbecomes infectious, it remains in this condition for the restof its life cycle. Nonpersistent transmission implies that theinsect can acquire the virus by a brief probe on an infectedplant, afterwards it can transmit the virus directly: the vectorwill lose viral particles after probing only one or two times onhealthy plants. The mode of transmission has implications onthe way a virus develops in the field and its management.Once inside the leaf, they move from one cell to anotherthrough the plasmodesmata connecting adjacent cells, multi-plying in them. Then, viruses reach the phloem, spread sys-tematically throughout the plant, and finally, re-enter the pa-renchyma cells adjacent to the phloem through the plasmo-desmata. Therefore, local and/or systemic symptoms mayarise and result from cell-to-cell or phloematic translocation,respectively (Stange 2006; Fraile and García-Arenal 2010). Ingeneral, virus infections are sporadic and their levels dependheavily on seasonal conditions, differing greatly in the yearsand according to locations. Early infections can lead tostunting, reduced tillering and plant death, and losses can behigh. Late infections have fewer impacts, but can still affectseed quality (Stange 2006; Fraile and García-Arenal 2010).

Plant viruses differ from fungal pathogens since no curativetreatment is available, and therefore, viral disease control aimsat prevention through integrated management practices tocontrol the virus source, aphid populations and virus trans-mission into crops. Alternatively, the use of virus-resistantcultivars and stimulation of the plant’s own defence mecha-nisms by elicitors may represent, in some cases, two effectivestrategies (Faoro and Iriti 2009).

Plant innate immunity

Disease is a rare outcome in the spectrum of plant-microbeinteractions and plants have (co)evolved a complex set ofdefence mechanisms to hinder pathogen challenging and, inmost cases, prevent infection. The battery of defence reactionsincludes physical and chemical barriers, both preformed (orconstitutive or passive) and inducible (or active), depending

on whether they are pre-existing features of the plant or areswitched on after challenging (Table 1). When a pathogen isable to overcome these defences, disease ceases to be theexception. Three main explanations support this rule: (i) plantis not a substrate for microbial growth and does not supportthe lifestyle of the invading pathogen; (ii) constitutive barriersprevent colonization of plant by pathogen; and (iii) plantrecognizes pathogen, by its innate immune system, and thenactivates inducible defences (Iriti and Faoro 2003).

The host ability of responding to an infection is determinedby genetic traits of both the plant itself and the pathogen.Some resistance mechanisms are specific for plant cultivarsand certain pathogen strains. In these cases, plant resistance(R) genes, encoding for receptors, recognize pathogen-derived molecules (specific elicitors) resulting from the ex-pression of avirulence (avr) genes (Table 2).

This gene-for-gene relation, also known as host resistance,triggers inducible barriers, i.e. a cascade of events leading tosystemic acquired resistance (SAR). In addition, another typeof resistance is activated through the recognition, by plantreceptors, of general (race-nonspecific) elicitors, pathogen-or microbe-associated molecular patterns (PAMPs orMAMPs) including mainly lipopolysaccharides, peptidogly-cans, flagellin, fungal cell wall fragments, lipid derivatives(sterols and fatty acids), proteins, double-stranded RNA andmethylated DNA (Table 2). This non-host or basal resistancecan also be induced by endogenous, plant-derived, generalelicitors (host- or damage-associated molecular patterns,HAMPs or DAMPs), such as oligogalacturonides releasedfrom the plant cell wall by fungal hydrolytic enzymes(Table 2) (Sanabria et al. 2010; Głowacki et al. 2011; Henryet al. 2012). In any case, the spectrum of defence reactionstriggered by both types of resistance, which collectively rep-resent the plant innate immune system, is rather similar (Iritiand Faoro 2007). Immunity may be expressed locally (localacquired resistance, LAR), in the infected cells, or in uninfect-ed distal tissues (SAR), probably because of one or moreendogenous systemically translocated (or volatile) signals thatactivate defence mechanisms in plant organs distal from theinitial site of infection (Kumar and Klessig 2008).

Recognition of a biotic stress by cell entails physical inter-action of a stimulus (elicitor) with a receptor. According to thereceptor/ligand model, the constitutively expressed plant Rgenes encode proteins that possess domains characteristic oftypical receptors responsible for the innate immunity in mam-mals and Drosophila. These proteins, also known as patternrecognition receptors (PRRs), can be grouped into differentclasses according to certain common structural motifs. ManyR proteins contain a leucine-rich repeat (LRR) domain in-volved in recognition specificity. Some receptor-like proteins(RLPs) possess an extracellular LRR anchored to a transmem-brane domain, whereas other R genes encode receptor-likekinases (RLKs) with extracellular LRR and a cytoplasmatic

Environ Sci Pollut Res

kinase domain. Another group of receptors possesses aminoacid sequences with strong similarity to nucleotide binding(NB) sites. These NB-LRR proteins are likely localised incytoplasm, with a putative coiled-coil (CC) domain or aregion with similarity to the Toll and interleukin-1 receptor(TIR) at the N terminus. In particular, CC (in CC-NB-LRR)can be a leucine zipper (LZ), whereas TIR (in TIR-NB-LRR)plays a central role in the immune and inflammatory responsesof mammals and Drosophila (Sanabria et al. 2010; Głowackiet al. 2011; Henry et al. 2012).

Among MAMPs, chitosan (CHT), a deacetylated chitinderivative, is worthy of special attention because of its use inchemical-induced resistance and efficacy against virus dis-eases (Iriti and Faoro 2009). Like a general elicitor, CHT isable to prime an aspecific, long-lasting and SAR, possibly bybinding to a specific receptor in the plant cell surface (Chirkov2002; Chen and Xu 2005; Iriti and Faoro 2009; El Hadramiet al. 2010; Silipo et al. 2010; Yin et al. 2010a; Falcón-Rodríguez et al. 2012; Delaunois et al. 2014).

Chitosan chemistry

CHT is a linear, polycationic heteropolysaccharide discoveredby Rouget in 1859. It consists of two monosaccharides,N-acetyl-D-glucosamine (2-acetamido-2-deoxy-β-D-glu-copyranose, C8H15NO6, molecular weight (MW)=221.2),the repeat unit of insoluble chitin, and D-glucosamine(2-amino-2-deoxy-β-D-glucopyranose, C6H13NO5, MW=179.17) linked together by β-(1→4) glycosidic bonds(Fig. 1). CHT is low-cost produced by exhaustive alkaline orenzymatic deacetylation from chitin, widely distributed innature, mainly as the structural component of the arthropod

exoskeleton (crustaceans and insects) and fungal cell wall.Therefore, the relative amount of the two monosaccharidesin this partially N-deacetylated chitin derivative may vary,with samples of different deacetylation degree (DD), MW,polymerization degrees (PD), viscosities and pKa values,and the term ‘chitosan’ does not refer to a uniquely definedcompound, but describes a heterogeneous family of copoly-mers, commercially available from a number of suppliers.Noteworthy, all these chemical properties greatly affect theCHT physicochemical characteristics, which, in turn, governalmost all its biological applications.

In nature, CHT is also found in fungal cell wall, even if itdiffers from invertebrate CHT: whereas the acetyl groups inCHT produced from crustacean chitin are uniformly distrib-uted along the polymer chain, a CHT of similar DD isolatedfrom fungal cell wall would possess acetyl residues that aregrouped into clusters (Pochanavanich and Suntornsuk 2002).

CHT also possesses several favourable biological proper-ties, above all biodegradability and low toxicity. It is suscep-tible to degradation by both specific and nonspecific enzymes,including chitinases, chitosanases, lysozymes, cellulases,hemicellulases, proteases, lipases and glucanases, and itslow toxicity has been also documented in human studies(Ylitalo et al. 2002).

Chitosan antiviral activity

CHT possesses a well documented, broad-spectrum, direct,antimicrobial activity against filamentous fungi, yeasts, Gram-positive and Gram-negative bacteria (Kong et al. 2010;Henández-Lauzardo et al. 2011; Badawy and Rabea 2011).Conversely, its activity against plant viruses is due to thecapability of the polymer to stimulate the plant immune re-sponse (Iriti and Faoro 2009). In this regards, a direct antiviralactivity of CHT was ruled out in tobacco necrosis virus(TNV)-inoculated bean leaves, and the protective effects oftreatment were attributed to the elicitation of the plant defencemechanisms (Iriti et al. 2010).

The elicitor activity of CHT was first demonstrated in theinteraction between pea (Pisum sativum) and the fungal path-ogen Fusarium solani (Hadwiger and Beckman 1980).Similarly, the capacity of inducing resistance against viraldiseases by CHT is known since years. Initially, it was shown

Table 1 Plant defence mechanisms

Structural Chemical

Constitutive(passive preformed)

Anatomical barriers (trichomes cuticle cell wall) Preformed inhibitors (phytoanticipins: glycosides saponins alkaloids)antifungal proteins (lectins) and ribosome inactivating proteins (RIP)

Inducible (active) Cell wall strengthening (callose lignin and suberinappositions; oxidative extensin cross-linking)

Oxidative burst hypersensitive response (HR) phytoalexins(phenylpropanoids) pathogenesis-related proteins

Table 2 Plant innate immunity

Type of resistance Elicitors

Host (specific)resistance

Specific elicitors encoded by the avr genes ofcertain pathogen strains (gene-for-gene theory)

Non-host (basal)resistance

General exogenous (race-nonspecific MAMPs) andendogenous (plant-derived oligogalacturonides)elicitors

MAMPs microbe-associated molecular patterns


that treatment of bean (Phaseolus vulgaris) leaves with thepolymer decreased the number of local necrotic lesionscaused by alfalfa mosaic virus (AMV) by triggering SAR(Pospieszny and Atabekov 1989; Pospieszny et al. 1991). Inparticular, treatment of the lower surface of a leaf stimulatedresistance in its upper surface, treatment of one half of a leafinduced resistance on the other untreated half, and treatmentof lower leaves elicited defence mechanisms in upper leaves(Pospieszny and Atabekov 1989; Pospieszny et al. 1991). Thisfinding prompted further studies aimed at demonstrating thatCHT is able to induce resistance against a number of local andsystemic viral infections in different host plants belonging todifferent botanical families (Chirkov 2002). Noteworthy, CHTprevented infection with viruses characterized by differentstructures and genome expression mechanisms, thus suggest-ing that it may suppress infection irrespective of the type ofvirus, by stimulating the plant’s own defence machinery(Chirkov 2002). The polymer also inhibited infection withpotato spindle tuber viroid (PSTV), when sprayed on tomato(Solanum lycopersicum) leaf prior or not later 1–3 h afterinoculation or added to the inoculum (Pospieszny 1997).

Even if viral infections cannot be directly controlled byconventional agrochemicals, unfortunately, the reliable use ofCHT in crop protection is still hampered by a series of intrinsicand extrinsic factors. The formers are related to the physical-chemical properties of the polysaccharide, whereas the latterare due to the genetic traits of both host plant and pathogen aswell as to environmental factors.

Factors affecting antiviral activity of chitosan

MWof CHT greatly affects its ability to suppress viral infec-tions by eliciting the host defence response. In bean plants, thedegree of CHT-induced resistance to the systemic pathogenbean mild mosaic virus (BMMV) increased as its MW de-creased (Kulikov et al. 2006). After CHT depolymerisationobtained by enzymatic hydrolysis, the fractions with the low-est MW, 1.2 and 2.2 kD, exhibited a higher antiviral activitythan that of the fractions with 10.1, 30.3 and 40.4 kD, whereas

monomers glucosamine and N-acetylglucosamine did notshow any activity. Intriguingly, with a DD of 85%, in sampleswith MW 1.2 and 2.2 kD, on average one or two monomersare acetylated, possibly located at the end of the polymericchain (at least in CHT derived from crustacean chitin), thussuggesting a spatial interaction between particular CHT con-formations and putative receptor sites on plant cell (Kulikovet al. 2006). These results were corroborated by Davydovaand colleagues, who obtained CHTs with different MW andDD after depolymerisation by enzymatic and chemical hydro-lysis. CHT derivatives from 2.0 to 17.0 kD inhibited theformation of local necrotic lesions by systemic tobacco mo-saic virus (TMV) in tobacco plant (Nicotiana tabacum) by50–90 %. These authors also demonstrated that the antiviralactivity of their samples only marginally depended on DD(Davydova et al. 2011). Similarly, DD of krill and crab CHTswithin the range of 60–98% caused no significant effect on itsactivity against AMVon bean plants (Pospieszny et al. 1995).These authors also reported that glucosamine exhibited asignificant antiviral activity, and a considerably lower yetsubstantial activity was also shown for N-acetylglucosamine(Pospieszny et al. 1995). Enzymatic degradation of highMW-CHTs by fungal chitinases from Aspergillus fumigatus signif-icantly increased their ability in suppressing local necroticlesions caused by TMV inoculation on tobacco plants(Pospieszny et al. 1996; Struszczyk et al. 1998). In general,an increase of antiviral activity in low MW-CHTs may be dueto their better penetrating ability across the leaf epidermaltissues. Noteworthy, stomatal uptake experiments carried outon bean leaves showed that CHT entering stomata is determi-nant for the induction of resistance to TNV (Iriti et al. 2010).

Despite these results, the data on the dependence of CHTantiviral activity on it structure, mainly MW, are still incon-sistent and controversial, since it has been reported that high-polymeric CHTs possess higher antiviral properties, too. Inpotato (Solanum tuberosum) plants, 120-kD CHT obtainedfrom krill was more effective than crab CHTs of 3 and36 kD against systemic infection of potato virus X (PVX),and similar result was documented by the same authors inbean plants inoculated with AMV (Chirkov et al. 1998, 2001).

Fig. 1 Chitin N-deacetylationthrough alkaline hydrolysis:acetamido [–NHCOCH3] groupsare deacylated in chitosanmolecule


This contrasting results may be due to the different sourcesof the polymer, for instance, crab or krill CHT, and to theheterogeneity of derivatives (oligomers) arising from differentdepolymerisation processes. Depolymerisation can be obtain-ed by many enzymes including chitinases, chitosanases,glucanases, cellulases, hemicellulases, pectinases, proteasesand lipases or by chemical hydrolysis, for instance, withhydrogen peroxide. However, independently from the poly-mer source, it has been suggested that, in contrast to chemicalhydrolysis, enzymatic depolymerisation excludes the forma-tion of bioactive oxidized groups in the oligosaccharides.Therefore, in the case of chemical hydrolysis, the higherantiviral activity of lowMWCHTs is determined not so muchby the change of the polysaccharide PD as by the appearanceof oxidized groups in the oligomers (Rhoades and Roller2000; Cabrera and Van Cutsem 2005; Davydova et al.2011). At the same time, lowMWCHTs obtained by chemicalor enzymatic hydrolysis from the same source may differ inthe distribution of acetate residues, which, in turn, may affectthe antiviral properties of derivatives (Cabrera and VanCutsem 2005).

Though CHT inhibited virus infection in plant speciesbelonging to different botanical families, its activity wasshown to be higher in Fabaceae than in other families(Pospieszny and Atabekov 1989; Pospieszny et al. 1991;Chirkov 2002), possibly because this activity seems to bemediated by the plant’s own defence response. Generally,bean and pea plants were more responsive to treatment, ac-quiring high levels of long-lasting resistance to different viralor fungal infections after one application with low concentra-tions of CHT (Hadwiger and Beckman 1980; Pospieszny andAtabekov 1989; Pospieszny et al. 1991). Differently,Solanaceae, such as tobacco, potato, tomato and stramony(Datura stramonium) plants proved to be more refractoryeven to repeated treatments with high CHT concentrations(Chirkov et al. 1994; Pospieszny 1995; Surguchova et al.2000). On the other hand, CHTwas not able to elicit resistanceto systemic infections with cauliflower mosaic virus (CaMV),turnip mosaic virus (TuMPV) or radish mosaic virus (RaMV)in cabbage (Brassica campestris, Crucifereae) (Chirkov et al.1994).

Mechanisms of chitosan antiviral activity

Sensing of chitosan by plant cell

Perception of elicitors by cell represents the first step to triggeran effective plant immune response. As previously intro-duced, PRRs are able to recognize invariant and conservedstructures usually originating from microbial surfaces, essen-tial for microbial metabolism and not present in the host (withthe exception of HAMPs/DAMPs). Although the role of CHT

as SAR inducer has been convincingly demonstrated, little isknown about the sensing of CHT by plant cell. To date, only aputative receptor for CHT has been described by Chen andXu, who isolated a 78-kD CHT-binding protein from cabbageleaves (Chen and Xu 2005). In addition, in calli of Cocosnucifera, CHT induced the expression of genes with highsimilarities to DNA sequences of RLKs (Lizama-Uc et al.2007).

For chitin, high-affinity binding sites were reported at thesurface of suspension-cultured cells of different species: to-mato (Baureithel et al. 1994), rice (Oryza sativa) (Shibuyaet al. 1996), soybean (Glycine max) (Day et al. 2001), wheat(Triticum aestivum), barley (Hordeum vulgare) and carrot(Daucus carota) (Okada et al. 2002). More recently, a plasmamembrane receptor for chitin was identified and purified inrice cells, both at gene and protein levels (Kaku et al. 2006).This chitin elicitor-binding protein (CEBiP) structurally dif-fers from the two major classes of PRRs in plants, RLKs andRLPs, both groups containing extracellular LRRs (Kaku et al.2006). The mature glycoprotein CEBiP harbours two extra-cellular lysine motifs (LysMs) of approximately 40 aminoacids and a transmembrane domain at the C-terminus, but itlacks any cytosolic domain for signal transduction, such asintracellular kinase domains normally present in RLKs, thussuggesting that additional factors for downstream chitin sig-nalling through the plasma membrane into the cytoplasm arenecessary (Kaku et al. 2006). Furthermore, knocking downCEBiP expression compromised the plant immune response(Kaku et al. 2006). LysM domains generally occur in a varietyof peptidoglycan- and chitin-binding proteins, suggesting thatthey may be directly involved in glycan perception (Hameland Beaudoin 2010). In Arabidopsis thaliana, a second chitinreceptor was identified by the same authors, a chitin elicitorreceptor-like kinase (CERK1) (Miya et al. 2007). The KOmutants for CERK1 completely lost the ability to activate animmune response, in particular mitogen-activated protein ki-nase (MAPK) activation, reactive oxygen species (ROS) gen-eration and defence gene expression (Miya et al. 2007; Wanet al. 2008). Similarly to CEBiP, CERK 1 is a plasma mem-brane protein with three LysMs in the extracellular domain,but, differently from CEBiP, CEPK1 harbours an intracellularSer/Thr kinase domain (Miya et al. 2007).

In any case, it is still under debate whether CHT isperceived by chitin receptors. In in vitro studies,CERK1 ectodomain was found to bind chitin polymerswith higher affinity than chitin oligomers (PD 4-8), butCHT very weakly bound to this receptor (Iizasa et al.2010; Petutschnig et al. 2010). Differently from chitin,CHT is a polycationic molecule able to interact with theoutward-facing, negatively charged hydrophilic groupsof phospholipids bilayers, and these perturbations maybe sufficient to elicit the plant defence response (Hameland Beaudoin 2010).


Defence reactions triggered by chitosan in plant cell

Early events following CHT perception consist of changes inion fluxes and plasma membrane depolarization. Transientlyincreased cytosolic Ca2+ concentration was demonstrated byZuppini et al. (2003) in soybean cells, with a maximal peakreached after about 3 min upon CHT administration andfalling back to the basal level after about 5 min.More recently,Amborabé and colleagues reported that plasma membranedepolarization peaked transiently 10–15 min after CHT treat-ment in Mimosa pudica motor cells, with a correlated cyto-plasm acidification and inhibition of plasma membraneH+-ATPase activity (Amborabé et al. 2008). Inhibitory ef-fects on the proton-pumping activity of this enzyme alsocompromised the uptake of co-transported carbohydratesand amino acids as well as other H+-mediated processes(Amborabé et al. 2008).

The activation of a Ca2+-dependent callose synthase repre-sents another rapid, effective and specific cell response toCHT elicitation (Köhle et al. 1985; Kauss et al. 1989), asshown in monocotyledonous and dicotyledonous species(Iriti et al. 2006; Faoro et al. 2008). Moreover, CHT-inducedcallose apposition was mechanistically correlated to the anti-viral activity of the polymer in different pathosystems, namelybean/TNV, bean/tomato bushy stunt virus (TBSV) andtobacco/TNV (Faoro et al. 2001; Faoro and Iriti 2006; Iritiet al. 2006). In particular, the efficacy of CHTs with differentMWs (6–735 kD) to stimulate callose synthesis in bean leaffragments was evaluated and correlated with their capabilityin inducing resistance to TNV (Faoro and Iriti 2007).Polymers with 76, 120 and 139 kD were the most effectivein stimulating callose apposition in comparison with thosehaving lower or higher MW, and, interestingly, the intensityand pattern of callose deposition in leaf tissues positivelycorrelated with CHT-induced resistance to TNV, with the 76-kD polymer reducing by 95 % the virus necrotic lesions(Faoro and Iriti 2007). The role of callose, a β-1,3-D-glucan,in limiting virus spreading is well established (Levy et al.2007; Guenoune-Gelbart et al. 2008). Extracellular callosedeposition, around plasmodesmata (callose collar), may con-strain the cell-to-cell transport of viral particles; similarly, theirlong-distance transport, along the phloem vessels, may berestrained because of callose apposition in pores of the phloemsieves (Carrington et al. 1996).

One of the largest families of Ser/Thr protein kinases isrepresented by MAPKs, and MAPK-mediated phosphoryla-tion of transcription factors represents a key step in controllingdefence gene expression (Fiil et al. 2009). Activation ofMAPK cascade by CHT was documented (Hu et al. 2004;Lizama-Uc et al. 2007). With relation to antiviral activity, anovel CHT-induced Ser/Thr protein kinase gene was isolatedin tobacco plants and designated as oligochitosan-inducedprotein kinase (oipk) (Feng et al. 2006). Antisense expression

of oipk decreased phenylalanine ammonia-lyase (a key en-zyme in phytoalexin biosynthesis) activity and resistance toTMV in transformed plants (Feng et al. 2006), and, morerecently, in the same host, other authors reported a positivecorrelation between this transduction protein and TMV resis-tance, PAL and peroxidase activities andmRNA levels of PALand two pathogenesis-related (PR) proteins, chitinase andβ-1,3-glucanase (Yafei et al. 2009).

The increased production of ROS by plant, including su-peroxide anion (O2

−), hydrogen peroxide (H2O2) and hydrox-yl radical (OH) is an important early-induced defence re-sponse to pathogen attack. Among these by-products of mo-lecular oxygen, H2O2 plays a fundamental role due to itsmobility (it possesses neither negative charges nor unpairedelectrons), relatively low reactivity and broad-spectrum activ-ity. In fact, it exerts a direct antimicrobial activity, besidesbeing involved in cell wall strengthening by monolignol po-lymerization and lignin apposition, and oxidative cross-linking of hydroxyproline-rich glycoproteins (extensins),though these processes do not seem to be relevant in plant-virus interaction (Mandal and Mitra 2007; Faoro and Iriti2009; Buonaurio et al. 2009). Hydrogen peroxide can alsocross the plasma membrane and affect cell signalling, byinteracting with reactive nitrogen species (RNS) (Zaninottoet al. 2006). Production of H2O2 and nitric oxide (NO) afterCHT treatment was observed in epidermal cells of tobacco,rapeseed (Brassica napus) and pea leaves (Zhao et al. 2007; Liet al. 2009; Srivastava et al. 2009), in pearl millet (Pennisetunglaucum) seedlings (Manjunatha et al. 2008) and in rice cellsuspensions (Lin et al. 2005).

A fine-tuned orchestration of molecular signals, particular-ly Ca2+, H2O2 and NO is responsible for the onset of hyper-sensitive reaction (HR), a rapid and programmed death ofplant cells (PCD) at sites of attempted pathogen penetration(Zaninotto et al. 2006; Zhang et al. 2012). This reaction mayinvolve just a single cell (invisible HR) or extensive andvisible tissue areas, and it is particularly effective againstviruses, because these disease agents necessitate of the bio-synthetic machinery of the healthy plant cell (Faoro and Iriti2009). A CHT-induced, calcium-mediated PCD, in soybeancells, was studied by Zuppini and co-workers (Zuppini et al.2003). They observed cytoplasm shrinkage, chromatin con-densation andan increased activity of caspase-like proteases,and morphological and biochemical features of PCD(Zuppini et al. 2003). Similar morphological hallmarks werereported in tobacco BY2 cells, besides inter-nucleosomalDNA fragmentation with a distinct DNA-laddering pattern(Fig. 2) (Iriti et al. 2006). Interestingly, cell death kineticinduced by CHT was delayed by treatment with a calciumchannel blocker, and HR was correlated to reduced TNVinfection on tobacco plants (Iriti et al. 2006). On theother hand, an H2O2-independent form of PCD was demon-strated in tobacco cell cultures (Wang et al. 2008). Cell death


phenomena triggered by CHT administration were also docu-mented on epidermis of pea seedlings (Vasil’ev et al. 2009)and on sycamore (Acer pseudoplatanus) cultured cells(Malerba et al. 2012).

The role of hormonal signalling and cross-talk in plant-virus interaction is somewhat controversial (Iriti et al. 2010).In general, SAR is associated with the accumulation ofsalicylic acid (SA) and PR proteins and is dependent on theregulatory protein non-expressor of PR-1 (NPR1) gene. Onthe contrary, induced systemic resistance (ISR), which can bestimulated by beneficial rhizobacteria and the fungusTrichoderma spp. colonising the root system, does not requireSA, can occur without the production of PR proteins and isdependent on ethylene and jasmonic acid (JA) as well asNPR1. Finally, β-aminobutyric acid-induced resistance(BABA-IR) involves both SA- and abscisic acid (ABA)-de-pendent defence mechanisms, depending on the challenging

pathogen (Buonaurio et al. 2009). The importance of JA forsignal transduction after elicitation of tomato plants with CHTwas demonstrated since years by Doares and colleagues(Doares et al. 1995). Stimulation of octadecanoic pathwayand accumulation of 12-oxo-phytodienoic acid (the precursorof JA) and JA were further documented in CHT-treated riceleaves (Rakwal et al. 2002). On the contrary, after CHTadministration, methyl-salicylic acid (the methyl ester ofSA) accumulated in the same tissues of the same plant species(Obara et al. 2002), and SA biosynthesis increased in TMV-infected tobacco leaves (Ogawa et al. 2006). In bean leaftissues, it was shown that CHT-induced callose appositionwas regulated by ABA, whose accumulation was in turncorrelated with resistance to TNV (Iriti and Faoro 2008). Infact, an inhibitor of ABA biosynthesis administered beforeCHT treatment reduced both callose deposition and plantresistance to the virus, whereas exogenous ABA application

Fig. 2 Morphological features ofprogrammed cell death (PCD)induced by 0005 % chitosantreatment for 6 h in tobacco BY2cells visualized by fluorescencemicroscope (Olympus BX50Tokyo Japan). a Viability ofcontrol cells treated with 005 %acetic acid (the solvent ofchitosan) for 6 h and stained byfluorescein diacetate (FDA); bar30 μm. b Viability and cytoplasmshrinkage (arrows) in chitosan-treated cells stained by FDA; N,nucleus; CW, cell wall; PM,plasma membrane; bar 30 μm. cChitosan-induced chromatincondensation; nuclei were stainedby Hoechst 33258; bar 20 μm. dArrows indicate chitosan-inducedchromatin condensation; nucleiwere stained by Hoechst 33258;bar 10 μm. e Terminaldeoxynucleotidyl transferasedUTP nick end labelled (TUNEL)assay (In Situ Cell Death KITTMR red Roche BaselSwitzerland); TMR red-negativecontrol cells treated with 005 %acetic acid for 6 h; bar 40 μm. fChitosan-treated cells containingDNA stand nicks characteristic ofPCD detected by TUNEL assay(terminal deoxynucleotidyltransferase adds labelled dUTP tothe 3′OH ends of either single- ordouble-stranded DNA); TMRred-positive apoptotic nuclei co-stained with the DNA-bindingdye DAPI; bar 40 μm


induced a significant resistance to TNV, thus indicating theinvolvement of ABA in these processes (Iriti and Faoro 2008).Intriguingly, the transcription levels of BnOIPK, a MAPKgene cloned in B. napus, rapidly increased in leaf tissues afterCHT and JA treatments, but only slightly after SA andABA exposure (Yin et al. 2010b). More recently, it wassuggested that ethylene pathway does not seem to beinvolved in the antiviral activity of CHT (Iriti et al. 2010).An ethylene-independent, CHT-induced resistance was dem-onstrated by a pharmacological approach with inhibitors ofthe main enzymes of ethylene biosynthesis, a donor and aprecursor of the hormone, and an inhibitor of its perception(Iriti et al. 2010).

Finally, other events associated to SAR establishment, suchas phytoalexin biosynthesis and PR protein translation, whileeffective against bacterial and fungal pathogens, do not seemto exert a significant role in preventing virus replication orspreading, even if the effects of CHT on these late steps ofSAR are known since decades in plant-fungus interaction(Hadwiger and Beckman 1980; Walker-Simmons et al.1984). In particular, the role of antimicrobial secondary me-tabolites seems to be of little importance, at least in terms ofantiviral defences, though their relevance against fungal andbacterial diseases has been demonstrated (Buonaurio et al.2009). Similarly, among the PR proteins up to dateisolated, antiviral agents are not included, with the excep-tion of ribonuclease (Faoro and Iriti 2009). This enzyme candepolymerise viral RNA, thus inhibiting its replication andtranslation, and CHT-induced increase in ribonuclease activitywas documented in potato plants and correlated with theirresistance to PVX infection (Chirkov et al. 2001). An18-kDa ribonuclease (pathogenesis-related protein 10, PR-10) able to degrade TMVRNAwas also isolated in hot pepper(Capsicum annuum) (Park et al. 2004).

Epilogue

Despite the benefits of CHT in crop protection and its limita-tions in plant disease control were previously illustrated in thissurvey, emphasis should also be paid to some ‘positive sideeffects’ which may derive from the polymer application(Uthairatanakij et al. 2007). These include the possibility ofrising the levels of bioactive secondary metabolites in plantand produce healthier foods, as shown in bean and grapevine(Iriti et al. 2010; Iriti et al. 2011; Vitalini et al. 2011; García-Mier et al. 2013). In particular, CHT increased polyphenol andmelatonin contents in grapes and the corresponding experi-mental wines, as well as their antiradical activity (Iriti et al.2011; Vitalini et al. 2011). Interestingly, the new CHT formu-lation used in one of this study efficiently controlled grapevinepowdery mildew (Erisyphe necator), but not downy mildew

(Plasmopara viticola) (Iriti et al. unpublished). The polymerwas also effective in reducing mycotoxin contamination offungus-infected cereal grains (Khan and Doohan 2009). Inaddition, in open-field trials on bean plants, CHT treatmentsdid not incur fitness costs, a potential detrimental trait ofinduced resistance (Iriti et al. 2010). Finally, the activity ofCHT as anti-transpirant agent, via an ABA-mediated stomatalclosure, may contribute to relieve the physiological stress dueto water deficit in drought conditions (Iriti et al. 2009).Therefore, an approach based on CHT application may reallyrepresent a sustainable, and possibly effective, strategy tocontrol (virus) diseases in plant, by reducing the environmen-tal impact of agrochemicals and their economic costs,possibly with higher crop yields and healthier plant prod-ucts (Mejía-Teniente et al. 2010; Delaunois et al. 2014).

Acknowledgments We apologize to the colleagues whose excellentstudies have not been cited for brevity. MI is grateful to Dr. AndreaKuthanova and Prof. Zdenek Opatrny (Department of Plant Physiology,Charles University in Prague, Czech Republic).

Conflict of interest The authors declare no conflict of interest.

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