Plant-derived antifungal proteins and peptides

14 Pages·2005·0.14 MB·English

1001
REVIEW / SYNTHÈSE
Plant-derived antifungal proteins and peptides
A.J. De Lucca, T.E. Cleveland, and D.E. Wedge
Abstract: Plants produce potent constitutive and induced antifungal compounds to complement the structural barriers to
microbial infection. Approximately 250000 – 500000 plant species exist, but only a few of these have been investi-
gated for antimicrobial activity. Nevertheless, a wide spectrum of compound classes have been purified and found to
have antifungal properties. The commercial potential of effective plant-produced antifungal compounds remains largely
unexplored. This review article presents examples of these compounds and discusses their properties.
Key words: antifungal, peptides, phytopathogenic, plants, proteins.
Résumé : Les plantes produisent des composés antifongiques actifs de manière constitutive et induite pour complémen-
ter les barrières structurelles aux infections microbiennes. Environ 250000 – 500000 espèces de plantes existent mais
seulement une petite proportion de celles-ci a été analysées pour une activité antimicrobienne. Malgré cela, un large
spectre de classes de composés ont été purifiés et ont démontré des propriétés antifongiques. Le potentiel commercial
de composés antifongiques efficaces produits par des plantes demeure en grande partie inexploré. Cet article de revue
présente des exemples de ces composés et discutent de leurs propriétés.
Mots clés : antifongiques, peptides, phytopathogènes, plantes, protéines.
[Traduit par la Rédaction] De Lucca et al. 1014
Introduction teins in response to fungal infection (Broekaert et al. 1997;
Garcia-Olmedo et al. 1998; Selitrennikoff 2001). Research
Floristic analyses indicate that approximately 250000 –
continues to discover novel antifungal plant-produced pep-
500000 plant species currently exist (Tivy 1993; Borris 1996).
tides and proteins with potential utility in agriculture,
Though hundreds of plant-produced antifungals have been
medicine, food processing, and other areas where fungal in-
identified,fewarecommerciallyused.However,plantsshould
hibiting and (or) fungicidal compounds are needed.
be an excellent source of potent antifungals, since they are
exposed to a wide array of phytopathogenic fungi present in
Proteinaceous plant antifungals
their environment and have had to develop antifungal com-
Plants respond to infection and stress by synthesizing a
pounds to survive.
variety of host plant proteins. Peptides are generally defined
Phytopathogenic fungi must overcome complex defenses
as proteins of no more than 100 amino acid residues in
(Bell 1981; Borgmeyer et al. 1992; Linthorst 1991). These
lengthwhosemolecularweightisnotgreaterthan10000.In
defenses include a cell wall, composed of compounds such
contrast,theterm“plantprotein”isdefinedasproteinsgreater
as lignin, tannins, phenols, and cellulose, which presents a
than100aminoacidsresidueswithmolecularweightsgreater
physical barrier to inhibit microbial infections (Bell 1981;
than10000.Alargenumberofbothproteintypeshavebeen
BolandLinthorst1990).Thoughplantslackanimmunesys-
purified and studied. Below is a brief review of these pro-
tem, they do produce constitutive and (or) induced pro-
teins.
Received 29 October 2004. Revision received 20 June 2005. Plant peptides
Accepted 24 June 2005. Published on the NRC Research Plants produce a number of cysteine-rich, antifungal pep-
Press Web site at http://cjm.nrc.ca on 6 December 2005. tideclassesthatarebasedonaminoacidsequencehomology
A.J. De Lucca,1,2 T.E. Cleveland. Southern Regional and are commonly found in seeds (see Table 1). They are
Research Center, USDA, ARS, 1100 Robert E. Lee Blvd., both inducible and constitutive and they differ in size and
New Orleans, LA 70124, USA. structure. Plant antifungal peptides include theα-defensins,
D.E. Wedge. Natural Products Center, USDA, ARS, lipid transfer proteins (LTPs), thionins, hevein- and knottin-
University of Mississippi, University, MS 38677, USA. type peptides, the cyclopeptide alkaloids, and other unique
1Corresponding author (e-mail: [email protected]). peptide groups (Broekaert et al. 1997; Garcia-Olmedo et al.
2Current address: Soil and Water Research Unit, USDA, ARS, 1998). Several of these peptide families (defensins, LTPs,
SRRC, FFS, 4115 Gourrier Avenue, Baton Rouge, LA andthionins)areclassifiedaspathogenesis-related(PR)pro-
70808, USA. teins, which are defined as proteins genetically coded for by
Can. J. Microbiol. 51: 1001–1014 (2005) doi: 10.1139/W05-063 © 2005 NRC Canada
1002 Can.J.Microbiol.Vol.51,2005
0 the host plant but are produced only when induced specifi-
Reference Broekaertetal.1992Cammueetal.1995;Tassinetal.1998aTerrasetal.1993 PandayandDevi199Kraghetal.1995Molinaetal.1993Thevissenetal.1997Tailoretal.1997 Nielsenetal.1997 Duvicketal.1992Shaoetal.1999Kooetal.1998 Morenoetal.1994DeLuccaetal.1999Seguraetal.1999bTerrasetal.1993 cmV(cid:1)star-aalαuDnlycy-eDtLunfieenrooenftoespsnnhiancsataihosvnnonesdlt,oaavignpnaitricnienfasuglSentnogrdtriaieslrniuenpll1fari9otsdee9pede9e-dlr)sits.niitekuoseardt(iVolcenayasnsvtaeLensio,ndoehwnsahveiinetchaacl.omtm1rai9ypp9lleo4exr-;
stranded antiparallel β-sheet with only one α-helix (Osborn
etal.1995;Terrasetal.1992,1993a,1995)andarehomolo-
L) gous to those produced by human and rabbit neutrophils
g/m (Fantetal.1997,1998).Plantα-defensinsareconsideredPR
µ proteins and are classified as members of the PR-12 group
(
MIC C)50 C)50C)50 C)50 C)50 ((VTearnraLsoeotnaal.n1d9v9a2n, S19tr9ie3na,11999995).;TOhsebsoernpeepttiadl.es19a9re5)prinesethnet
vitro 2.00.0 0.7(I 5.04.04.05.01.0(I 2.0(I 0.00.00.0(I 0.05.08.00.0(I Rseaepdhsanuosr slaetaivvuess (oRfs-AHFePuchtehrraougshanRgus-inAeFaP )(,HAs-eAscFuPl1u)s,
n 1 72 641 5232 1 4
I 11 11 hippocastanum (Ah-AMP ), Clitorea ternatea (Ct-AMP ),
1 1
and Dahlia merckii (Dm-AMP ). Plant α-defensins do not
1
form ion-permeable pores in artificial membranes but may
m
Activeagainst FusariumculmorumFusariumoxysporum Fusariumculmorum AspergillusnigerCercosporabeticolaFusariumsolaniFusariummoniliformeFusariumculmorum Cercosporabeticola FusariumgraminearuFusariumoxysporumFusariumoxysporum FusariumsolaniFusariummoniliformeFusariumsolaniFusariumsolani a1femeeooctnt9firtv9anahiHl6larv.y.)tosii1p,1an-o9hAa9mn9as9aFl5e7Psan))druf.1t.etegTecrivSgnereheeespelewissotsdtdoeepehe-erdmsib-pdcmoeohebrpnneycnftdtoeui.ftidaunhaαTetngtese-ghadddoaealeilbpsgfmpsrpperuelaolneaarprwsvcnsetihiamtntnohtastiandlnoyweainmsfraaameeenstcnhmdtrssae(oiubtnlTcnprestarhpahetnesrehHaeveeetssdisbess-ei(sAenuTaeaddprFhnlioyeP(nmnTvegsieittsgcraassrreinageaotrldnees--.
1
Rs-AFP are lethal to the germinating conidia but not the
2
nongerminated conidia of Aspergillus flavus and Fusarium
Modeofaction BindschitinPhospholipidbinding* Receptormediated* UnknownInhibitshyphaltipgrowthPoreformation*Receptormediated*Unknown Unknown SporegerminationReceptormediated*Actindepolarization UnknownReceptormediated*UnknownPoreformation ducing50%growthinhibition. mhddcaTilyeehoa.ffTpilnee1olhhinny9nalessi9eifiibn5onntybhs;rssumiTdoit(seienDvn,riraroneealtiedsnpndLudeetocuitprecccmtdaacailtdata.iibenno1ygetn9ftscaa9onmt2a(hnBl,ii.ailnrd1ytoci19aer99aetkh93rhaaee9aaseev))ra..etenineaTbtitrihmlinyaoedil-nc.isinratc1oacg9bgtsi9sietvari7itelte;nydspOgelftvoashoebnf,rlotoetprphpsnlieparnsoneege--tt
o
pr teins containing 45–47 amino acids with two antiparallel
n α-helices and an antiparallel double-stranded β-sheet with
ntifungalpeptidefamilies. Source AmaranthuscaudatusAlliumcepa Aesculushippacastanum ZizyphusxylopyraBetavulgarisHordeumvulgareHeucherasanguineaImpatiensbalsamina Betavulgaris ZeamaizePhytoloccaamericanaPharbitisnil SolanumtuberosumRaphanussativusSolanumtuberosumT.aestivum yconcentration;IC,concentratio50withcertainty. s1fTpcpTiptiiy9lrhiehhxavos9odienettoo7lveetlinr;yiaipisnnissiBensp–deciesremssgPhnichuoRaeipietxcremn-gtrcsc1eeeubay3stfdalrrseu.anatnpnien1ltdntr.ihge9.noia19aseotliAe9er6snneei9)nitmnfdne3botsgsriaeyrr,fam,wmou(cseV1untibtiliten9pageorhmnag9eacnnd3tlsstrdeLbh,oiasa(,eossr,cBcuto1otcanolio9naotpvehint9rhcisgldd5ta,miyaidni)ntna.aeditntgvhrnTeedbeinrvhsstlraaoyiuilcodaenlrtnangteticshvodiesShneeunsatfssAlrrr(tonigosaFeopfuermnafeemdtinlhnst1cbeed1pl9eevai9phpr9tres8oooe9rsao7rcassi)lef---tl;..
Table1.Examplesofplanta Peptidetype(peptidefamily) Ac-AMP(hevein)1Ace-AMP(ns-LTP)1 αAh-AMP(-defensin)1AmphibineH(cyclopeptide)AX1(thionin)Cw18(ns-LTP)αHs-AFP(-defensin)1Ib-AMP(Ib-AMP)1IWF4(hevein) MBP-1PAFP-s(knottin)Pn-AMP-1(hevein-like) Pseudothionin-St1αRs-AFP(-defensin)1Snakin-1βW(thionin) Note:MIC,minimuminhibitor*Modeofactionisnotknown fmW(bpu1spoBtaae9uoaraαrts9rmrihl2on3aebo,aiagy)negn.t(eadtiedFTnwolonl2ednhBoleiaSicerenβvWoaneaef)acrusdsβlka1,nbda)n×guiieadnt1isimnnis,hpo0Thodiiien–anylnb6canSspcioitt,cafleetfuiiendcpeto2ddhkrttifoiehhtennr4htmeegoet×iregmoaa1aFrrcnsan0ouyei1cwtw–nssi9t6fastiiht9enuohrme(i4hnninBau)igoteob.mLwal-fi/elTratLinios,tycpahdorBphrrsolaoe,aLnaspnpnpubLeg1eicem,pTe,rertttaPwmbriih(net,seMiaisds,sortihunnoaBoacoilnnEfnihLlfnddiCsepcaaap)5ibs(wd0cheWafshhtryrvedolaetaaeeooom1allydf---t,.
© 2005 NRC Canada
DeLuccaetal. 1003
the thionins (Blochet et al. 1993; Molina et al. 1993; Terras could be a potential binding site for lipids (Gincel et al.
et al. 1993b). In contrast, the antifungal activity of this pep- 1994; Shin et al. 1995). IWF5 is similar to the antifungal
tide family is inhibited by Ca2+ at concentrations above peptides AX1 and AX2 (also present in sugar beet leaf
5 mmol/L but not by Mg2+ or Ba2+ at levels up to washings and discussed earlier) in that at low concentrations
10 mmol/L or by monovalent cations at concentrations up to it inhibits the growth of C. beticola, as do IWF1 and IWF2
50 mmol/L (Terras et al. 1992). (Nielsen et al. 1996; Kristensen et al. 2000b).
The cysteine-rich antifungal peptides AX1 and AX2 are Ace-AMP is a nonhaemolytic peptide present in onion
1
present in sugar beet leaves. They are monomeric, basic pro- seeds, with 93 amino acid residues and four disulfide bridges
teins consisting of 46 amino acid residues, of which eight (Tassin et al. 1998). This peptide shares 76% of the residues
are cysteines, and are related to the thionins (Kragh et al. conserved among all ns-LTPs and is unusually rich in arginine
1995). Immunoblotting and immunohistology show that the (20% of the amino acids), but as opposed to the ns-LTPs,
AX peptides are present in high concentrations extra- Ace-AMP is unable to transfer phospholipid from lipo-
1
cellularly in the cell walls and in necrotic lesions of sugar somes to mitochondria. This molecule differs from IWF5 in
beet leaves infected with Cercospora beticola, suggesting a that the continuous hydrophobic cavity, which runs through
role in antifungal defense (Kragh et al. 1995). At concentra- the center of IWF5 and is believed to bind lipids, is absent
tions ≤1 µmol/L, AX1 and AX2 prevent the growth of the (Tassin et al. 1998). It also differs from members of the ns-
plantpathogensC.beticolaandBotrytiscinereabuthavelit- LTP group in that it does not bind phospholipids in solution
tle or no activity against bacterial growth. AX2 reduces the but does interact with phospholipids membranes of artificial
longitudinal hyphal growth of C. beticola though the fungus liposomes (Tassin et al. 1998). Ace-AMP inhibits the growth
1
develops side branch growth (Kristensen et al. 1999). of 12 phytopathogenic fungi, including Fusarium oxysporum,
Recombinant AX2, despite being correctly expressed and at concentrations ≤10 µg/mL. Cations near physiological
folded, was much less active than the native form ionic strength do not, or only weakly, affect its antifungal
(Kristensen et al. 1999). The recombinant AX2 contained an properties.
additional N-terminal arginine that introduced an additional
positive charge to the molecule. This additional positive Hevein and hevein-like peptides
chargemayweakentheionicinteractionbetweenthepeptide Hevein, a small 4.7-kDa, cysteine-rich, chitin-binding pep-
and its putative receptor. Another possibility is that the extra tide, is present in the lutoid bodies of rubber tree (Hevea
charge affects the three-dimensional structure of AX2, brasiliensis) latex (Archer 1960; Walujono et al. 1975). This
which in combination with the change in charge, could af- peptide is resistant to heat (90 °C for 10 min) and is found
fect the fungal inhibition properties of AX2 (Kristensen et inthebottomfractionofthelatexandstronglyresemblesthe
al. 1999). chitin-binding lectin from the stinging nettle, Urtica dioica
(Van Parijs et al. 1991). Hevein, wheat germ agglutinin, and
Plant nonspecific LTPs the stinging nettle lectin share amino acid sequence hom-
Plant nonspecific LTPs (ns-LTPs) are basic proteins, ology, so it is not surprising that these three peptides act in
9–10 kDa in size, that are known for their ability to enhance the same way, that is, by binding chitin (Van Parijs et al.
invitrotheintermembraneexchangeand(or)transferofvar- 1991). Hevein inhibits the hyphal growth of fungi by bind-
ious polar lipids, such as phospholipids and glycolipids ing to chitin.
(Arondel and Kader 1990; Douliez et al. 2001). Their Hevein-like peptides are small (43 residues) chitin-binding
antifungalmodeofactionisnotyetknown,thoughtheymay peptides. While hevein is a rather weak antifungal (Van Parijs
insert themselves in fungal membranes and form a pore re- et al. 1991), the hevein-like peptides inhibit the growth of
sulting in an efflux of intracellular ions culminating in cell Alternaria brassicicola, Ascochyta pisi, and Fusarium
death (Selitrennikoff 2001; Van Loon and van Strien 1999). culmorum at doses lower than those for other known chitin-
A common structure for several ns-LTP molecules (except binding proteins. Ac-AMP and Ac-AMP are members of
1 2
Ace-AMP ) believed to play a role in their antifungal prop- the hevein-like peptide family, with 29 and 30 amino acid
1
ertiesisthepresenceofacontinuouscavity,whichisabind- residues, respectively (Broekaert et al. 1992), they share se-
ingsiteforlipids,thatpassesthrougheachmolecule(Gincel quence homology to the cysteine- and (or) glycine-rich do-
et al. 1994; Shin et al. 1995). These compounds, which are main of chitin-binding proteins (see below), and they both
classified as PR-14 proteins (Van Loon and van Strien 1999), reversibly bind to chitin. Ac-AMP and Ac-AMP are strongly
1 2
are much larger than and differ in structure from thionins antagonized by cations.
and plant defensins. Molina et al. (1993) demonstrated that Seeds of Pharbitis nil contain two α-hevein analogs, Pn-
homologousns-LTPs(Cw18,Cw20,Cw21,andCw22)pres- AMP and Pn-AMP (with 41 and 40 amino acid residues,
1 2
ent in barley leaves inhibit the growth of F. solani. respectively), that cause leakage of cytoplasmic materials by
Several related ns-LTP antifungal compounds, IWF1, IWF2, attaching to hyphal tips and septum (Koo et al. 1998). Both
and IWF5, are present in the intercellular washing fluid of are highly basic molecules exhibiting potent antifungal activity
sugar beet leaves (Nielsen et al. 1996; Kristensen et al. against both chitin-containing and non-chitin-containing fungi
2000b). The first two are nearly identical to each other and andareconsideredthefirsthevein-likepeptidestoshowfun-
are basic, monomeric proteins of 91 and 89 amino acid resi- gicidal effects similar to those of theg thionins. The Pn-
dues (Nielsen et al. 1996). IWF5 is a monomer in both its AMPs differ in mode of action from the other members of
reduced and unreduced states, having 92 amino acids (eight the hevein group. The latter bind to chitin, whereas the Pn-
arecyteines),andis47%identicaltoIWF1(Kristensenetal. AMPs cause rapid depolarization of the actin cytoskeleton
2000b). IWF5 has a tunnel-like hydrophobic cavity that within 15 min (Koo et al. 2004). Pn-AMP and Pn-AMP
1 2
© 2005 NRC Canada
1004 Can.J.Microbiol.Vol.51,2005
have been constitutively expressed in tomato and have Knottins
enhanced resistance against both nonchitinous and chitin- The knottin family of peptides include Mj-AMP and Mj-
1
containingfungi(Leeetal.2003).ThePn-AMPsarepresent AMP , which are present in Mirabilis jalapa seeds. They
2
during seed maturation and germination but not in the aerial have37and36aminoacids,respectively,andthreedisulfide
vegetative plant parts (Koo et al. 1995). bridges;theydifferbyonlyfouraminoacids(Cammueetal.
IWF4 is one of a group of antifungal peptides present in 1992). Both inhibit the growth of phytopathogenic fungi,
the interstitial wash fluid of sugar beet leaves. It is consid- suchasB.cinerea,Colletotrichumlindemuthianum,andVenturia
ered a hevein-like peptide because of its homology to the inaequalis. Mj-AMP and Mj-AMP have been expressed
1 2
hevein-type Ac-AMP peptides that bind to chitin (Nielsen et transgenically in tobacco but none of these plants show en-
al. 1997; Koo et al. 1998). IWF4 has six cysteine and seven hanced resistance to infection by B. cinerea or Alternaria
glycine residues and inhibits the growth of C. beticola with longipes. The knottin, PAFP-s, produced by Phytolacca
an IC of ≤2 µg/mL, which is comparable to the Ac-AMP americana, has significant sequence similarities and the same
50
peptides (Koo et al. 1998). The mature IWF4 and Ac-AMP cysteine motif as the Mj-AMP peptides (Shao et al. 1999).
protein families resemble each other in size, pI, amino acid PAFP-s is highly basic, consists of 38 residues with six
sequence, chitin-binding ability, and antifungal potentials cysteines comprising three disulfide bridges, and inhibits the
(Nielsen et al. 1997). However, IWF4 differs from Ac-AMP growth of F. oxysporum and Trichoderma viridae (Shao et
2
in that it is present in vegetative, aerial tissues and is not in- al. 1999). Several hydrophilic and positively charged resi-
duced by pathogens, whereas Ac-AMP is present in mature duessurroundthehydrophobicsurfaceofPAFP-s,givingthe
2
seeds of amaranth only, not vegetative tissues, and is in- molecule an amphiphilic character that is believed to be the
duced by pathogen infection or other stress factors (Nielsen mainstructuralbasisofitsspecificitytothefungalcell(Gao
et al. 1997). et al. 2001).
Two novel antifungal peptides, EAFP1 and EAFP2, con-
tain 41 amino acid residues, are present in the bark of the Impatiens antimicrobial peptides
tree Eucommia ulmoides, and share core amino acid sequences Impatiens balsamina plant seeds contain a group of anti-
with hevein (Huang et al. 2002). This tree produces a latex fungal peptides (Ib-AMP1 through Ib-AMP4) that comprise
similar to that of the rubber tree, Hevea brasiliensis (Huang about 0.5% of the total protein in the mature seed (Tailor et
et al. 2002), which produces hevein. Both peptides show al. 1997). They are short, basic peptides, 20 amino acids in
characteristics of a hevein domain and exhibit chitin-binding length that contain four cysteine residues forming two intra-
properties similar to other hevein-like peptides (Huang et al. molecular disulfide bonds, have no significant homology
2002). They are also are similar to hevein in that they are with any peptides or proteins, and are nontoxic to human,
heatresistant.EAFP1andEAFP2arethefirstplant-produced, plant, and insect cells in vitro (Broekaert et al. 1997; Tailor
antifungal hevein-like peptides isolated that contain 10 cysteines et al. 1997). The Ib-AMP family may include a β-turn but
that are paired and form five disulfide bridges (Huang et al. doesnotshowevidenceforeitherhelicalorβ-sheetstructure
2002). In comparison, hevein and the other hevein-like pep- (Patel et al. 1998). They inhibit up to 50% of the growth of
tideshaveonlythreeorfourdisulfidebridgesthatstabilizea phytopathogens,suchasF.culmorumandAlternarialongipes
chitin-binding domain (Huang et al. 2004). The unique at ≤25 µg/mL (Tailor et al. 1997). Ib-AMP3 is lethal to the
disulfide bond Csy7–Cys37 confers on EAFP2 a more de- germinatingconidiaofAspergillusflavusandF.moniliforme
fined amphipathic character than that of hevein, which also at≤25 µmol/L (De Lucca et al. 1999). Their mode of action
does not have the cationic face and amphipathic topology of isunknown,thoughitisknownthatatevenhighconcentrations
EAFP2 (Huang et al. 2004). EAFP1 and EAFP2 are similar (500 µg/mL) no visible cell lysis occurs (Tailor et al. 1997).
to the Pn-AMP peptides in that they are active against both
chitinous and nonchitinous fungi, whereas hevein is only Cyclopeptide alkaloids
active against chitinous fungi. Structural analysis of EAFP2 Cyclopeptide alkaloids are produced by members of
indicates that (i) its dual activity is dependent on its chitin- Rhamnaceaeandotherplantfamilies(PandayandDevi1990;
binding properties and (ii) its amphiphilic surface renders it Digel et al. 1983; Gournelis et al. 1997). Frangufoline, am-
active against the non-chitin-containing fungi (Huang et al. phibineH,rugosaninesAandB,andnummularinesB,K,R,
2004). These peptides, compared with havein, are three to and S are cyclopeptides whose antifungal properties have
fivetimesmoreactiveagainstfungiandarestronglynegated significant growth inhibition activity against Aspergillus
by calcium ions. Another antifungal peptide of this group, niger but not Candida albicans, as shown by zonal inhibi-
Ee-CBP, present in the bark and leaves of the spindle tree, tion studies (Panday and Devi 1990). Though frangufoline is
Euonymus europaeus, has 10 cysteine residues comprising known to bind to calmodulin (Han 1993), a Ca2+-binding
five disulfide bonds (Van den Bergh et al. 2002). The Ee- proteinthatmediatescalcium-drivenmetabolicreactions,the
CBP precursor and a chitinase are synthesized as similar mode of action of these molecules is unknown.
chimeric precursors consisting of an N-terminal hevein do-
mainlinkedtoaC-terminalchitinase-likedomainbyahinge Other peptides
region (Van den Bergh et al. 2004). However, Ee-CBP un- Other plant peptides include the 5-kDa pseudothionin-St1
dergoes cleavage of the N-terminal hevein domain, resulting present in potato tubers and found active against F. solani
in the formation of the final Ee-CBP molecule. Ee-CBP and (Moreno et al. 1994). Another potato-produced antifungal
the chitinase are active alone and act synergistically as anti- peptide is sankin-1, a 63 amino acid residue, cysteine-rich
fungals against a number of phytopathogenic fungi (Van den molecule (Segura et al. 1999). Sankin-1 is constitutive and
Bergh et al. 2004). active at less than 10 µmol/L against potato fungal patho-
© 2005 NRC Canada
DeLuccaetal. 1005
gens (Segura et al. 1999). Maize (Zea mays) seeds produce lowing infection by a pathogen (Van Loon et al. 1994; Van
theantifungalpeptidesMBP-1andanunnamed22-kDapep- Loon and van Strien 1999; Walton 1997). PR proteins are
tide (Duvick et al. 1992; Huynh et al. 1992). MBP-1 con- grouped into families (PR1–PR17) based on primary struc-
tains 33 amino acids with no free cysteines and is mostly ture, serological relatedness, enzymatic activities, and bio-
ahelical. It shows no homology with thionins, though it is logical activities (Christensen et al. 2002; Linthorst 1991;
antifungal. MBP-1 inhibits the hyphal elongation of several Stintzi et al. 1993; Van Loon et al. 1994; Van Loon and van
phytopathogenic fungi, such as F. moniliforme and Strien 1999). Although PRs are most commonly observed
Fusarium graminearum, while the 22-kDa protein inhibits during HRs of plants to microbial pathogens and appear to
F. oxysporum and F. solani growth. contribute to systemic-acquired resistance, the definition of
The intercellular washings of sugar beet leaves contain thePRgroupdoesnotrequirearoleinresistance(VanLoon
IWF6,whichhasnohomologytoanyknownantifungalpro- and van Strien 1999). PR proteins (see Table 2) also share
tein. However, it has some homology, less than 26%, with common features related to structure, regulation of gene ex-
agelenin,aneurotoxinfromthevenomofthespiderAgelena pression, and antifungal activity (Yun et al. 1997). They are
opulenta that acts as a calcium blocker (Kristensen et al. stableinlowpH,areresistanttoproteaseactivity,aremono-
2000a). Though the IWF6 mode of action is unknown, this mers of low molecular weight, and show apoplastic location
homology suggests that IWF6 may act in a similar manner (Cheong et al. 1997). The stability in low pH and resistance
(Kristensen et al. 2000a). IWF6 is a 37 amino acid peptide to proteases are characteristics of food allergens. In fact,
with six cysteines and has an isoelectric point of 7. As with seven of the PR groups contain proteins with allergenic
the other peptides (e.g., AX1, AX2, IWF1–IWF4) isolated properties, with six of the groups containing food allergens
fromtheintercellularwashingsofsugarbeetleaves,IWF6is (Ebner et al. 2001; Hoffmann-Sommergruber 2002).
active against C. beticola (Kristensen et al. 2000a). PR proteins are widely distributed in healthy plants in
trace amounts but are produced in much higher concentra-
Plant antifungal proteins tions following elicitation caused by infection. They have
The first response to infection by the plant is necrosis typical physicochemical properties that enable them to resist
around the site of infection, the purpose of which is to re- acidic pH and proteolytic cleavage, allowing them to survive
duce the spread of the disease (Matthews 1991). The hyper- the harsh environments of the vascular compartment or cell
sensitive response (HR) is a common response of plants to wall or intracellular spaces (Stintzi et al. 1993). The signifi-
invasionbyaplantpathogen.TheHRischaracterizedbylo- cance of PR proteins lies in their strong antifungal and anti-
calizedandrapidcelldeath(necrosis)andisrestrictedtotis- microbial activity. Some inhibit conidial release and
sue immediately surrounding the site of pathogen invasion germination, while others are associated with strengthening
(Oku 1994; Agrios 1997; Walton 1997). The HR is thought the host cell wall and papillae. Certain PR proteins, such
to be responsible for limiting the growth of the pathogen as β-1,3-glucanase(PR-2)andchitinases(PR-3,-4,-8,and-11),
and, in this way, provides the host plant with a mechanism diffusetowardtheinvadingpathogenandbreakdownchitin-
toresistfurtherinfectionbeyondthepointofinitialinvasion. supported cell wall in certain fungi.
Typically,theHRdevelopswithin24hofinfection,whereas Most PR groups can be subdivided into two classes. Gen-
celldeathduetothedevelopmentofdiseasesymptomstakes erally, class I proteins are localized in the vacuole of the
several days. An effective HR may not always be visible plant cell, whereas class II proteins are extracellular. The
whenaplantremainsresistanttoattackbyapathogen,since class I and II proteins are related both structurally and
it is possible for the HR to involve only a very few cells and immunologicallybutdifferintheirinductionpatterns(Brederole
remain unnoticed. The HR-induced resistance has been de- et al. 1991; Ward et al. 1991). The PR groups listed below
scribed in numerous diseases involving obligate parasites are those reported to have antifungal properties.
(fungi, viruses, mollicutes, and nematodes) as well as non-
obligate parasitic fungi and bacteria (Agrios 1997). The HR PR-1 proteins
is the culmination of plant defense responses initiated by the Members (PR-1a, PR-1b, and PR-1c) of the PR-1 group
recognition of a pathogen-produced elicitor. This response is were first isolated from tobacco leaves after infection by the
followed within hours or a few days by induction of a long- tobacco mosaic virus (Van Loon and Van Kammen 1970;
lasting, broad-based resistance and is considered as a very Gianinazzi et al. 1970). However, they are now known to be
strong defense response induced in the plants by the present in rice, wheat, maize, barley, tobacco, and many
pathogen itself (Stintzi et al. 1993; Shah and Klessig 1996; other plants (Agrawal et al. 2000; Bryngelsson et al. 1994;
Caruso et al. 2001). The long-lasting response, termed Molina et al. 1999; Muradov et al. 1993; Rauscher et al.
systemic-acquired resistance, occurs in both the immediate 1999). PR-1 family members are also present in non-plant
area of infection as well as in the uninfected plant areas systems.AnovelPR-1familymemberlocalizestothecytosolic
(Chester 1933; Ross 1961). site of the endomembrane system in mammalian cells
(Eberle et al. 2002). PR-1 proteins are characterized by their
Pathogen-related proteins acidic or basic nature, their resistance to proteases, and their
An example of systemic-acquired resistance in plants is extracellular secretion (Bol and Linthorst 1990; Linthorst
the production of pathogen-related (PR) proteins that were 1991;VanLoon1985).Theirmodeofactionisyetunknown
first reported in the 1970s (Gianinazzi et al. 1970; Van Loon (Van Loon and van Strien 1999). A protein, P14, was iso-
and Van Kammen 1970). The term PR protein is defined as lated from tomato leaves and determined to be similar in
any protein genetically coded for by the host plant that is in- structuretothePR-1groupfromtobacco(Camacho-Henriquez
duced specifically in pathological or related situations fol- and Sanger 1982, 1984; Granell et al. 1987). Phytophthora
© 2005 NRC Canada
1006 Can.J.Microbiol.Vol.51,2005
1 infestans infection induces tomato leaves to produce three
9
9
1 distinct 14-kDa PR-14 proteins that are members of the PR-
3 ff 1 family (Niderman et al. 1995). These PR-14 proteins
9 o
Reference Nidermanetal.1995 Sela-Buurlageetal.19YeandNg2002Ponsteinetal.1994RobertsandSelitrennikFloresetal.2002Melchersetal.1994bTerrasetal.1993 Xuetal.1998 Parkashetal.2002 bTerrasetal.1993 Broekaertetal.1989 (apmPinPglRoat1aToys-i4h2tntaedhsa,ptircfPPerPtfoeiR1ev.t4rce-eei2blinnan,bftspseieasasrineotlnsdatgfenouPisPnnn.11sg4tT4ihc(coceβ)ibd-b(ea1ataxco,l3shcmia-oisgbcatloiatiutonfvc)idianatamyntnvoaidaimsntgeroPaasot)iRaonac-hsni1PtaddgRvPsei-(e.n1tqboiunvpebefieraevonncsotcctegeaoairnn)c(o.ssBtu,ivedptfiihetfsydae-
and Meins 1996; Cote et al. 1991; Leah et al. 1991;
InvitroMIC µ100.0g/mL(IC)90µ1.0g/wellµ60.0g/mLµ6.7g/mLµ0.5g/mLµ25gµ5–10g/wellµ90.0g/mL(IC)50 0.36mg/mL µ0.06mol/L(IC)50 µ45.0g/mL(IC)50µ40.0g/mL(IC)50 i3LeaFinntp2iitfvnp-eaeektrrlchosD.tPtoxii1aoRrti9ismna-t9m2lab10-etty9t)iemys.9lspyRPb1uehe;Re3risMp-z42rooooe,cffkittnceboDtlisanhanaresieslasteaayaPnI(rl,BReld.ege-1ap2rla9vruaro9ecepsd2aarun;ncopcdoNatreeesniiPdestenaesulbinsrnageyktrnraPeoriyrnieu-cabtp3esapa2tshlli,(i.anZacan1nart19per99aresvon9i7paett7;oeci)fnPiueu.nsaltnsoeTygeanhotasloeefl..
2002). Though active alone, Pr-32 is more active in the
s
Targetorganism Phytophthorainfestan FusariumsolaniFusariumoxysporumTrichodermaviridaeCandidaalbicansFusariumoxysporumAlternariaradicinaFusariumculmorum Rhizoctoniasolani Fusariumoxysporum Verticilliumdahliae Botrytiscinerea ptec(cdhJxururieaetmcrausaeasebmdncenedearcrliteelniKlusneiolu-asβalfcvtir-ak,e1dn1es,aic39fceupf-9iegpdr5aroloi)guectn.enac,iiAati3nnnnc2ssfatot-eiskammctelDhtsbieeopao-irnncfPpouoardRbntueiuygr-oscs2uenCetsnispoots(rnoltJoilufinetdietaasoiytnnhtcadreshisihscsKilhotoepiuwuacnrcfimoasadse1tetelux9hadct9aagre5tnaewd)cdnt.ithaitsnther.hicruAiienunmne---
PR-3 proteins
Modeofaction Unknownβ-1,3-GlucanaseChitinaseChitinaseLysisInhibitshyphalextensionEndochitinaseTrypsininhibitor;inhibitshyphalgrowthMannosebinding;inhibitsgerminationRibosome-inactivatingprotein;inhibitproteinsynthesisPermeabilizemembranes Inhibitsconidialgermination producing50%growthinhibition. ttAlWft((hheaiuBSavhiranahvPonelttgii.lslmannl1aa)anesPgnl9arrahalets8cbmasises9eneetco;lepatsolaslterVael-etw.rboocakte1aruaglnkd.tl9olleown.lai8fl,1nwori1e39awtt9enme)hht8n,9tenue7altw9asyos)2)tlH.sho.r6eipaeeRT1nsnanrpw9adotdodrtn8udooo(fed8cMaudr2)eiccpcu.n8d4otehacgP3ltiebaahtwRPnyintoknhRc-diogt3Daehch-es3el3iaceetnct2ighasenpa(,vlikliaulNtrlni.DisosPncnfie1taeghaeaoes9nc,lasiss7ttanesrm(ieh9seessoPneesio)nRnh,spitcnesia-ea(ch3tv(ccnMaPiAletdtulaiRpilevldmnytri.-eaoeno2lonibs1sytnleedba,9epinace9cantrnscahBour7siodnee)---;,
o) on presentinPhaseolusvulgaris(YeandNg2002a).PhaseinA
ntifungalproteinfamilies. Source Nicotianatabacum NicotianatabacumPhaseolusvulgarisNicotianatabacumZeamaysOxalistuberosaNicotianatabacum(tobaccHordeumvulgare Nicotianatabacum,GastrodiaelataLuffacinerea Brassicanapus UrticadioicaRhizomesof yconcentration;IC,concentrati50 A(PmadaaVnngeRnuaadmdPt-ciniR4nPhofauDsn-hrpst4nesyamrtgPsmorapcaaa.tlmllreltalpoeoiescnesistrrsehpseiipi(iocfntt1oioriesa3tnaledl–aa.nbps1t1iieit4nrnh9sa.itda95nconh3otkica)lftDaFwvhua,.einanotwg)iodnaaxhtglllhyeseraasooarepnnecukaodtpbtis(rvhseVuhPioetme(anshyrFevagrte(siaoioYseeufwoidmewnltarahoiearBtcednlenheddPiceFsRute.fNmtlaro-ao3glxora.myrlp12e.smr9p0oa1g9oa0tca98ser2tr9su)iialnv.e1im)cess;.
Table2.Examplesofplanta Protein(proteinfamily) PR-1b(PR-1) Pr-32(PR-2)Phasein(PR-3)CBP-20(PR-4)Zeamatin(PR-5)Ocatin(PR-10)Tobaccochitinase(PR-11)Hv-Ti3(thionin) Gastrodianin(lectin) Luffacylin(ribosomeinacti-vatingprotein)2Salbumin Urticadioicalectin(lectin) Note:MIC,minimuminhibitor H1hedlbH1faut9eoa9eetnvr9m9eajjlgeggdl99ea.aiaay))nil..1aa,fnrrr9TcC(aoddF9e(nhmlSl9raedeleies)tet,clswtmdaiolwtalaraIrma.liselh.cnlP1sna,ihy19lRtnIeor99iee-gpk924tts(rlho2;Buaoaefp;lnPulrftr.atiopcKosn2v1ln,tgao0hoe9sso0aiit9snienes1n1tcise)Ina;l.ItaiumbnKlePpdai.htinorRiln2in.oadobo-0gtl41ie.0ttea9oii13mtcnop99;isnrβa8da9oFl-i;l4.toczsare;eheifVc1ieq,Vikn9adgtus9inaprranen8ioohcnDw;ctiahDcanhvVatethiaeoemsatdm;ti,nensmbahmvi-temDloobee.eewbilainn1oelaemead9ptctivri9ismcnneaaot1orllogge-;..,,
© 2005 NRC Canada
DeLuccaetal. 1007
the mechanism of action of the class II PR-4 proteins is not cytoplasmic fluid and inhibits the growth of C. albicans,
known (Borman et al. 1999; Selitrennikoff 2001). One ex- N. crassa, and T. reesei (Roberts and Seliltrennikoff 1990;
ample of a PR-4 protein is CBP20, which is 20 kDa in size Vigers et al. 1991). Proteins homologous to zeamatin are
(Ponstein et al. 1994) and is induced by pathogens or presentinbarley(Hejgaardetal.1991)andoat(Vigersetal.
wounds in tobacco (Nicotiana tabacum) leaves. It is active 1991).Antizeamatinserumcrossreactswithproteinsofsim-
in vitro against T. viridae and F. solani by causing lysis of ilar size from a wide range of cereals, which suggests that
the germ tube and (or) growth inhibition. CBP20 also acts these proteins are ubiquitous and structurally related (Vigers
synergistically with a tobacco class I chitinase against et al. 1992).
F. solani and with a β-1,3-glucanase against F. solani and
Alternaria radicina (Ye and Ng 2002a). Maize produces a PR-10 proteins
class II chitinase, ZmPR4, which is accumulated in plant PR-10 proteins are a family of small homologous, primar-
cells that first establish contact with the pathogen (Bravo et ily acidic molecules present in a variety of angiosperms,
al. 2003). ZmPR4 production is induced by wounding of the monocots, and dicots (Ostmark et al. 1998). Several mem-
plant and by several compounds, such as abscisic acid, methyl bers of this group possess ribonuclease properties and are
jasmonate, and moniliformin (a mycotoxin produced by the antifungal. One such protein is CaPR-10 that is present in
fungus F. moniliforme, a maize pathogen). hot pepper (Capsicum annuum). It is an intracellular protein
(17.3 kDa) located in the cytosol and is known to inhibit
PR-5 proteins hyphal extension of Phytophthora capsici, possibly by inhibi-
The PR-5 proteins are considered thaumatin-like proteins tion of translation activity (Lam and Ng 2001; Park et al.
(TLP) because their amino acid sequence is highly homolo- 2004). PR-10 proteins share similarity with the allergen Bet
gous to that of thaumatin, an intensely sweet protein from v 1, known to have ribonuclease activity in white birch and
Thaumatococcus danielli (Cornelissen et al. 1986; Richard- in major latex proteins found in arabidopsis, bell pepper,
son et al. 1987). The majority of these proteins are approxi- mellon, strawberry, and tobacco (Bufe et al. 1996; Ostmark
mately 22 kDa and are stabilized by eight disulfide bonds et al. 1998). A group of inducible, small molecular weight
(Selitrennikoff 2001). This structure allows the PR-5 pro- proteins with acidic natures and N-terminal amino acid se-
teins to be resistant to protease degradation (Roberts and quences similar to the PR-10 proteins are present in leaves
Seliltrennikoff 1990). PR-5 proteins can be categorized into from Lupinus albus (Pinto and Riccardo 1995). These pro-
three subclasses based on their isoelectric point, as well as teins, PR-p16.5, PR-p16.5b, and PR-p16.5c are induced
their acidic, neutral, and basic properties (Koiwa et al. 1994). upon infection by Colletotrichum gloeosporioides.
In vitro, the PR-5 proteins inhibit hyphal growth and medi- Ocatin is a 18-kDa storage protein in the Andean tuber,
atehyphalandsporelysis(Hejgaardetal.1991;Robertsand oca (Oxalis tuberosa), and is classified as an antifungal of
Seliltrennikoff 1990; Vigers et al. 1991, 1992; Woloshuk et the PR-10 group. This protein constitutes between 40% and
al. 1991), which is correlated with plasma membrane per- 60% of the total soluble oca tuber proteins and, in vitro, in-
meabilization and dissipation of the plasma membrane po- hibitsthegrowthofseveralphytopathogenicfungi,including
tential (Abad et al. 1996). The primary role of PR-5 proteins F. oxysporum and Rhizoctonia solani (Flores et al. 2002).
in planta is not known, but current models suggest that they The concentration of ocatin is highest at harvest time and
form lethal transmembrane pores in fungi (Skadsen et al. decreases during storage. The amino acid sequence of ocatin
2000). A PR-5 protein from pumpkin, PLPT, rapidly hy- is very similar to proteins of the Bet v 1/PR-10 families and
drolyzes the hyphal tips of Neurospora crassa and signifi- major latex proteins (Ostmark et al. 1998).
cantly inhibits growth of F. oxysporum (Cheong et al. 1997).
Barley seeds contain the antifungal proteins R and S, which Other antifungal PR proteins
arehomologoustothaumatinandotherPR-5familyproteins Cucumber plants (Cucumis sativus) produces a chitinase
(Hejgaard et al. 1991). These proteins, in vitro, inhibit the found in both leaves infected with Colletotrichum lagenarium
growth of C. albicans and T. viridae and act synergistically and in uninfected leaves up to five leaves above the infected
with barley grain chitinase C (Hejgaard et al. 1991). To- one (Métraux et al. 1988). This chitinase has a molecular
bacco cells produce PR-5d, a hydrophobic, neutral protein mass of 28 kDa and is classified as an example of PR-8 pro-
that inhibits phytopathogenic and nonphytopathogenic fungi teins. Tobacco plants produce a number PR proteins, among
(Koiwa et al. 1997). Another PR-5 protein present in to- them are chitinases belonging to the PR-11 group (Melchers
bacco is osmotin (Singh et al. 1987), which is active against et al. 1994). These two chitinase groups are related and have
P. infestans, C. albicans, N. crassa, and Trichoderma reesei molecular masses of 41 and 43 kDa. Both have endo-
(Woloshuk et al. 1991; Vigers et al. 1992). Osmotin is a ba- chitinase activity toward T. viridae and Alternaria radicina
sic PR-5 protein that interacts with cell surface phospho- and show synergy with a tobacco β-1,3-glucanase against
mannan, suggesting that fungal cell surface polysaccharides F. solani germlings (Melchers et al. 1994). Barley produces
control target specificity of plant PR-5 proteins (Ibeas et al. a germin-like oxalate oxidase, classified as a PR-15 protein,
2000). Overexpression of a heterologous cell wall glyco- which produces hydrogen peroxide in response to infection
proteininF.oxysporumincreasesvirulenceandresistanceto by the causative agent of powdery mildew, Erysiphe graminis
osmotin, which indicates that the fungal cell wall compo- (Hang et al. 1995; Zhang et al. 1995). The oxalate oxidase
nents can increase resistance to plant defense proteins and appears 1–3 days earlier than does a PR-1 protein.
affect virulence (Narasimhan et al. 2003). Zeamatin is a The PR-17 group contains two protein families, WCI-5
22-kDa PR-5 protein present in Z. mays seeds (Roberts and from wheat and HvPR-17 and HvPR-17b from barley, with
Selitrennikoff 1990). Zeamatin causes the rapid release of antifungal properties (Christensen et al. 2002; Görlach et al.
© 2005 NRC Canada
1008 Can.J.Microbiol.Vol.51,2005
1996). The two PR-17 proteins from barley are extracellular tains at altitudes between 6000 and 14000 feet (1 foot =
and may affect cell wall metabolism or signal transduction 0.3048 m). Two type 1 RIP proteins, ME1 and ME2, com-
and are active against the fungus Blumeria grammis, which prise approximately 20% of the total storage root proteins
causes powdery mildew disease on barley fungus. WCI-5 in (Vivanco et al. 1999). These proteins are active, with an ad-
wheat is an induced lipoxygenase active against Ery- ditive affect observed, against a number of root-rot fungi,
siphe graminis. includingPythiumirregulaire,Alternariasolani,andF.oxy-
sporum (Vivanco et al. 1999).
Trypsin inhibitors
Corn genotypes resistant to Aspergillus flavus express high 2S albumins
levels of a 14-kDa trypsin inhibitor (Brown et al. 1999; 2S albumins are low molecular weight storage proteins
Chen et al. 1998, 1999a, 1999b), which affects conidial ger- rich in glutamine with similar physical–chemical properties
mination,hyphalextension,andfungalα-amylases.Bankset presentinmonocotyledonousanddicotyledonousseeds(Youle
al. (2002) showed this trypsin inhibitor can cross the blood– and Huang 1981). They are composed of two different sub-
brain barrier in mice, indicating a possible role for this units that are derived from a single precursor polypeptide
protein in the treatment of fungal infections in the central and are linked together by disulfide bridges (Krebbers et al.
nervous system of mammals. Barley leaves also produce a 1988). The smaller of the two subunits of radish 2S albumin
trypsin inhibitor that in vitro, enhances (2- to 55-fold) the appears to contain the antifungal properties of the molecule
antifungal activity of wheat and barley thionins (Terras et al. (Terras et al. 1993b). 2S albumins from radish and rape
1993b),whichsuggeststhatasynergisticeffectoccursinthe seedsinhibitfungalgrowthbyincreasingthepermeabilityof
plant when fungal infection occurs. Recently, a new trypsin– the fungal plasma membrane (Terras et al. 1993b, 1995).
chymotrypsin inhibitor was found present in broad bean (Vicia Radish and rape seed 2S albumins enhance by 2- to 73-fold,
faba)seeds(YeandNg2002b).Thisprotein,withamolecu- depending on test medium, the antifungal properties of wheat
lar mass of 13 kDa, not only exhibits antifungal properties and barley thionins (Terras et al. 1993b). However, 2S albu-
against Mycosphaerella arachidicola and P. piricola but also mins only enhance activity of thionins towards cell types
inhibits the activity of human immunodeficiency virus-1 re- that are affected by both proteins separately (Terras et al.
verse transcriptase. 1993b). Passion fruit (Passiflora edulis f. flavicarpa) seeds
also contain a 2S protein that inhibits F. oxysporum growth
Ribosome-inactivating proteins (Aggizzio et al. 2003).
Ribosome-inactivating proteins (RIPs) are widely distrib-
uted among higher plants (Mehta and Boston 1998) and Lectins
inhibit protein synthesis as a result of their N-glycosidase Lectins are proteins or glycoproteins that recognize and
activity (Endo and Tsurugi 1988). These proteins inactivate bind reversibly to carbohydrate moieties of complex glyco-
ribosomes by cleaving a single adenine from the large conjugates, inhibit fungal conidial germination, alter germ
rRNA, thus arresting protein synthesis at the translocation tubes, and inhibit hyphal growth (Allen et al. 1973; Lotan
step (Barbieri et al. 1993; Sharma et al. 2004). It is possible and Sharon 1973; Mirelman et al. 1975). Plants are known
that as little as one RIP molecule per cell is capable of turn- to produce lectins that protect the plant from fungal infec-
ing off protein synthesis (Strirpe and Barbieri 1986). In tion(Keen1992).Theyproduceawidevarietyoflectinsthat
vivo, RIPs may act synergistically with chitinases and either specifically bind to oligomers of N-acetylglucosamine
β-1,3-glucanases against invasive fungi (Broekaert et al. and (or) chitin (Van den Bergh 2004). The great majority of
1989; Kombrick et al. 1988; Mauch et al. 1988). RIPs are them belong to the ubiquitous family of chitin-binding pro-
composed of several categories: single-chained type 1 RIPs, teins composed of hevein domains (Van den Bergh 2004).
double-chained type 2 RIPs, and small RIPs. The types 1 For example, wheat germ agglutinin (WGA) lectin, present
and 2 are 30 and 60 kDa, respectively, while the small RIPs in wheat germ, has an affinity for N-acetylglucosamine and
are approximately 10 kDa. Type 2 RIPs have two subunits, binds to chitin-containing fungal walls (Ciopraga et al. 1999).
an “A” (Gal/GalNAc-binding subunit) chain and a “B” chain WGA binds to the chitin in the fungal cell wall thus disrupt-
having lectin properties. Ricin, an extremely toxic molecule ingthesteadystatebetweenhydrolysisandsynthesisofwall
because of its lectin subunit, is a type 2 RIP. In contrast, polymers (Ciopraga et al. 1999). This binding of WGA to
type 1 RIPs have only an “A” chain present and, as a result, the fungal cell wall polymers affects the inner compartment,
are much less cytotoxic because of the lack of the lectin as observed in fungal morphological changes, namely the
chain. A few antifungal RIPs have been reported in plants. swelling and vacuolation of cellular content and lysis of
Tobaccoleavesproduceatype1RIP(TRIP),whichisactive hyphal tips (Ciopraga et al. 1999).
against Trichoderma reesei (Sharma et al. 2004), while an Thestingingnettle,Urticadioica,producesUDA(Urticola
antifungal RIP present in asparagus (Asparagus officinalis) dioica agglutinin), a small, single-chain (8.5 kDa) fungistatic
is active against B. cinerea (Wang and Ng 2001b). Luffacylin lectin that is unusually heat and acid resistant and is very
is a small RIP (7.8 kDa) from sponge gourd seeds (Luffa similartotheaminoacidsequenceofWGA(Broekaertetal.
cylindrica) with activity against F. oxysporum and Myco- 1989). UDA, which consists of two cysteine-rich chitin-
spaerella arachidicola (Parkash et al. 2002). Luffacylin binding domains (Beintema and Peumans 1992), inhibits the
inhibited translation in a rabbit reticulocyte lysate system hyphal growth of several phytopathogenic fungi and is three
with an IC of 140 pmol/L and reacted positively in the to five times more active against fungi than hevein (Broekaert
50
N-glycosidase assay for ribosome inactivating proteins. et al. 1989; Van Parijs et al. 1991). The growth-inhibiting
Mirabilis expansa, a relatively disease-resistant crop that effect of UGA appears to be based on a specific phase of
produces an edible storage root, grows in the Andean moun- fungal growth and is temporal, suggesting that the fungi
© 2005 NRC Canada
DeLuccaetal. 1009
have an adaptation mechanism (Dies et al. 1999). UGA, by soil or plant fungal pathogens (e.g., Alternaria, Curvularia,
though similar in amino acid homology to hevein, aggluti- andRhizopus)formerlyconsideredasfungioflowvirulence
nates red blood cells whereas hevein does not (Van Parijs et but now considered as emerging pathogens. Plants have
al.1991).TheseantifungalsalsodifferinthattheCterminal been defending themselves against fungal infection for mil-
of hevein shows homology to chitinases, whereas the C ter- lions of years, so it is not surprising that they produce a
minal of UGA shows homology to tobacco PR-4 chitinases wide range of effective, antifungal compounds. Plants offer
(Broekaert et al. 1990; Linthorst et al. 1991). an enormous source of potent, novel antifungal compounds
The lectin STA (Solanum tuberosum agglutinin) is a with potential utility in agriculture, medicine, food process-
hydroxyproline-rich glycoprotein (HRGP) present in potato ing, and other areas.
tubersthatoccurspredominatelyatthecellsurfaceorassol-
ublemucin-likemolecules(Kieliszewskietal.1994).STAis
the best characterized HRGP lectin, which binds to chitin
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