Plant pathogenesis-related (PR) proteins: A focus on PR peptides

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Plant Physiology and Biochemistry 46 (2008) 941–950

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Plant Physiology and Biochemistry

journal homepage: www.elsevier .com/locate/plaphy

Review

Plant pathogenesis-related (PR) proteins: A focus on PR peptides

Jan Sels, Janick Mathys, Barbara M.A. De Coninck, Bruno P.A. Cammue*, Miguel F.C. De BolleCentre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Heverlee, Belgium

a r t i c l e i n f o

Article history:Received 25 April 2008Received in revised form 11 June 2008Accepted 14 June 2008Available online 26 June 2008

Keywords:Expression profileGenevestigatorLipid transfer proteinPlant defensinProteinase inhibitorThionin

* Corresponding author. Tel.: þ32 16329682; fax: þE-mail address: [email protected]

0981-9428/$ – see front matter � 2008 Elsevier Masdoi:10.1016/j.plaphy.2008.06.011

a b s t r a c t

The novel classes of plant pathogenesis-related (PR) proteins identified during the last decade also in-clude novel peptide families. This review specifically focuses on these pathogenesis-related peptides,including proteinase inhibitors (PR-6 family), plant defensins (PR-12 family), thionins (PR-13 family) andlipid transfer proteins (PR-14 family). For each family of PR peptides, the general features concerningoccurrence, expression and possible functions of their members are described. Next, more specifically theoccurrence of each PR peptide family in the model plant Arabidopsis thaliana is discussed. Single-genestudies performed on particular gene members of a PR peptide family are reported. In addition, ex-pression data of yet undescribed gene members of that particular PR peptide family are presented byconsultation of publicly available micro-array databases. Finally an update is provided on the potentialrole of these PR peptides in A. thaliana, with a focus on their possible involvement in plant defense.

� 2008 Elsevier Masson SAS. All rights reserved.

1. Introduction

Although micro-organisms are omnipresent in the vicinity ofplants, disease development is a sporadic event. General resistanceis accomplished by preformed efficient structural and chemicalbarriers and inducible defense mechanisms in the plant (reviewedin [38]). Among these preformed structural barriers are the cuticlelayer consisting of cutin, a complex polymer of esterified fatty acidscoated with waxes, and the rigid lignin depositions in the cell wall.Preformed chemical barriers include the presence of antimicrobialcomponents, called phytoanticipins [82,121]. Together, these bar-riers form a first line of defense and can prevent successful invasionby most of the micro-organisms (e.g. [20,63]). However, this is notadequate against all pathogens and, in addition, plants possessinducible defense mechanisms that are activated upon pathogenattack [33,112]. These include cell wall cross-linking, the hyper-sensitive response and concomitant generation of reactive oxygenspecies (ROS), accumulation of secondary metabolites like phyto-alexins, tannins and phenolic compounds, and the productionof so-called pathogenesis-related (PR) proteins (reviewed in[118,119,120]).

The term PR proteins is defined to indicate ‘‘those proteins thatare not or only at basal concentrations detectable in healthy tissues,but for which accumulation at the protein level has been demon-strated upon pathological conditions and related situations in at

32 16321966.(B.P.A. Cammue).

son SAS. All rights reserved.

least two or more plant–pathogen combinations’’ [119,120]. Theterm pathological conditions is used here to indicate direct path-ogen attack of different origins including fungi, bacteria, viruses,insects and herbivores. Related situations include (i) the applicationof chemicals that mimic the effect of pathogen attack (e.g. the planthormones ethylene (ET), jasmonate (JA) and salicylic acid (SA)) and(ii) wound responses that give rise to proteins which also accu-mulate during infections. However, this proposed definition causedconfusion in the past as the term PR proteins was often used todesignate all microbe-induced proteins, including enzymes likephenylalanine ammonia lyase which are constitutively present butalso increase during most infections [120]. This collective of pro-teins was never meant to be designated as PR proteins as there arenumerous enzymatic activities that are increased upon pathogenattack, and Van Loon et al. [120] recently introduced the term‘‘inducible defense-related proteins’’ referring to the originallyintended definition of PR proteins. Although some of these PRproteins exhibit potential in vitro antimicrobial activities and theiraccumulation in the plant is related to plant resistance responses,a direct functional role in defense could not be demonstrated for all.To be complete, the main properties of all the hitherto classified PRproteins are summarized in Table 1. It is clear that different mo-lecular sizes are represented by the different PR proteins, typicallyranging from 5 to 75 kDa. During the last decades, a number ofclasses were added, consisting of proteins with a molecular sizebelow 10 kDa. These so-called PR peptides are the focus of thisreview, more specifically PR classes 6, 12, 13 and 14, as well as theiroccurrence and putative role in the disease response of the modelplant Arabidopsis thaliana.

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Table 1Main properties of classified families of PR proteins

Family Type member Typical size (kDa) Properties Proposed microbial target Original reference

PR-1 Tobacco PR-1a 15 Antifungal Unknown [3]PR-2 Tobacco PR-2 30 b-1,3-Glucanase b-1,3-Glucan [3]PR-3 Tobacco P, Q 25–30 Chitinase (class I,II, IV,V,VI,VI) Chitin [117]PR-4 Tobacco ‘R’ 15–20 Chitinase class I,II Chitin [117]PR-5 Tobacco S 25 Thaumatin-like Membrane [117]PR-6 Tomato Inhibitor I 8 Proteinase-inhibitor –a [43]PR-7 Tomato P69 75 Endoproteinase –a [123]PR-8 Cucumber chitinase 28 Chitinase class III Chitin [77]PR-9 Tobacco ‘lignin-forming peroxidase’ 35 Peroxidase –a [62]PR-10 Parsley ‘PR1’ 17 ‘Ribonuclease-like’ –a [95]PR-11 Tobacco ‘class V’ chitinase 40 Chitinase class I Chitin [74]PR-12 Radish Rs-AFP3 5 Defensin Membrane [102]PR-13 Arabidopsis THI2.1 5 Thionin Membrane [34]PR-14 Barley LTP4 9 Lipid-transfer protein Membrane [41]PR-15 Barley OxOa (germin) 20 Oxalate oxidase –a [130]PR-16 Barley OxOLP 20 ‘Oxalate oxidase-like’ –a [127]PR-17 Tobacco PRp27 27 Unknown –a [81]

Table was adjusted from http://www.bio.uu.nl/wfytopath/PR-families.htma No in vitro antimicrobial activity reported.

J. Sels et al. / Plant Physiology and Biochemistry 46 (2008) 941–950942

2. Proteinase inhibitors (PR-6)

2.1. General aspects

The peptides belonging to the PR-6 family are defined as a sub-class of serine proteinase inhibitors (PIs) related to the ‘‘tomato/potato inhibitor I’’ [42], and have a typical molecular size of �8 kDa[118]. The larger group of serine PIs, which are the best studied PIs,are sub-divided, next to this PR-6 family, in tomato/potato class IIPIs, Bowman–Birk PIs and Kunitz-type PIs (reviewed in [27,47]. Thisproposed original definition and classification of the PR-6 family isdebatable, since it is limited to a sole sub-class of serine PIs, whileall types of PIs can interact with proteinases from plant-attackingorganisms and could have a role in plant defense (reviewed in[21,47]). Therefore, in the literature the general term PI is often usedinstead of PR-6. This section also presents some examples of studiesconcerning PIs, which are not all strictly classified as PR-6-typeproteins.

PIs have the property to bind proteinases and control proteinaseactivity, a general function involved in many biochemical processes,and therefore could have multiple functions in planta (e.g. theregulation of endogenous proteinases during seed dormancy, re-serve protein mobilization; reviewed in [47]), apart from theirproposed defensive role (reviewed in [21,47,115]). Concerning theirrole in defense, PIs may act by reducing the ability of the attacker to(i) use its lytic enzymes necessary for pathogenicity (for fungi; e.g.[32]), (ii) complete its replication cycles (for viruses; e.g. [45]), or(iii) obtain nutrition through digestion of host proteins and therebylimit the released amount of amino acids (for nematodes, insects;e.g. [114,125]).

Different studies have reported that PI genes can be inducedupon inoculation with micro-organisms. For instance in tomato,induction of PI genes was shown upon inoculation with Phy-thophtora infestans [86] and Pseudomonas syringae pv. tomato [84].Terras et al. [100] showed an in vitro antimicrobial effect, albeitrelatively weak, of barley trypsin inhibitors to four importantfungal plant pathogens including Alternaria brassicicola, Ascochytapisi, Fusarium culmorum and Verticillium dahliae. Interestingly, thisactivity increased synergistically in combination with other PRproteins, more specifically thionins (PR-13) [100]. As the role ofspecific microbial proteinases in microbial pathogenicity is notalways clear, the effect of plant PIs on the activity of these enzymeshas not been studied intensively. Yet, Dunaevskii et al. [32] showedthat buckwheat trypsin inhibitors were able to inhibit in vitroproteinases of B. cinerea, likely to be necessary for pathogenesis

[99]. Also, Lorito et al. [68] suggested that PIs could block thesynthesis of chitin in fungal cell walls and consequently fungalgrowth, through inhibition of endogenous trypsin necessary foractive chitin synthase [69]. The in vivo effect of PIs on defenseagainst microbial pathogens was also shown as there are reports ofenhanced resistance in plants overexpressing PI genes. For in-stance, heterologous overexpression of Nicotiana alata PI genes intransgenic tobacco resulted in enhanced resistance againstB. cinerea [19]. Recently, it was shown that endogenous over-expression of two A. thaliana PI genes, including one PR-6-typegene, in transgenic A. thaliana resulted in enhanced resistanceagainst B. cinerea [20].

Nevertheless, hitherto most of the studies on PIs have focussedon their role in plant–insect interactions. It was found that PI genesare induced upon insect attack and mechanical wounding (e.g.[26,42]). The wounding, leading to activation of PI genes, is believedto mimic the chewing action of herbivorous insects. These insect-and wound-induced responses have been extensively studied,especially the wound-activated systemin signaling pathway intomato (e.g. [25,49,66]), and are generally linked to the octadecanoidpathway and JA signaling networks. In A. thaliana, insect attack orwounding activates JA and ET signaling pathways [28,29,50] givingrise to systemic induced resistance (e.g. [22,28]. However, generallyboth wounding and microbial pathogen attack activate JA signalingpathways in A. thaliana. Hereby, the AtMYC2 and the ethylene re-sponse (ERFs) transcription factors play essential roles to discrim-inate between either wounding- or microbial pathogen-related JAresponses and to regulate the activation of the appropriate set ofgenes [2,67]. More specifically, while AtMYC2 transcription factorsactivate wound responsive genes (i.e. PI genes), ERFs activatepathogen-responsive genes (i.e. the PDF gene AtPDF1.2a) [67]. Re-cently it could also be demonstrated that insects try to reducewound-induced expression of PI genes in the plant, i.e. by com-ponents in their regurgitants [64] or, by making use of a decoymechanism which activates antagonizing signal pathways in theplant [129].

With respect to their anti-insect activity, PIs could act againstthe digestive proteases (e.g. trypsin, chymotrypsin) used by her-bivorous insects. This was demonstrated in several in vitro exper-iments by a reduced growth rate of insects fed on artificial dietscontaining PIs compared to the growth rate of insects fed on dietswithout PIs (e.g. [48,72,98]. The effect of PIs on insect attack in vivowas also shown as there are several reports of transgenic plants,expressing a heterologous PI gene and making them more resistantto insect attack (reviewed in [47]). For instance, it was shown that

http://www.bio.uu.nl/%7Efytopath/PR-families.htm

J. Sels et al. / Plant Physiology and Biochemistry 46 (2008) 941–950 943

overexpression of a PI gene from Nicotiana alata in transgenic appleplants inhibits the normal development of light-brown apple moth,Epiphyas postvittana [70]. Duan et al. [31] showed that transgenicrice plants, harboring an introduced potato PI gene, were moreresistant to the pink stem borer (Sesamia inferens). Vila et al. [125]demonstrated that expression of a maize PI gene in rice plantsenhanced resistance against the striped stem borer (Chilo sup-pressalis). No reports were found on increased insect resistanceupon overexpression of a PI gene in the plant from which theyoriginated.

2.2. Occurrence in Arabidopsis thaliana

Using the potato/tomato inhibitor I mature peptide sequence asa template for an A. thaliana BLASTP (available at http://www.arabidopsis.org) 6 PI genes encoding PR-6-type proteins (potato in-hibitor I-type like proteins) were found to be present in theA. thaliana genome (Fig.1). Two gene pairs appear in a cluster (clusterA, At2g38900–At2g38870; cluster B, At5g43570–At5g43580) (Fig.1).The calculated isoelectric point (pI) of the putative proteins is widelyranging from 4.6 to 11.3.

Regarding single-gene expression studies done on these 6 PIgenes, it was demonstrated by qRT-PCR that At2g38870 is inducedin leaves by B. cinerea [20]. Hitherto, no other single-gene expres-sion studies of the genes described in Fig. 1 were found. Publiclyavailable expression data of these genes, based upon micro-arrayanalyses, were checked with Genevestigator [132] (Fig. 1). No ex-pression data were available for At3g50020 and At5g43570 as theyare not represented on the ATH1 Affimetrix gene chip (Fig. 1).At2g38900 was induced by Pseudomonas syringae while At2g38870was induced by B. cinerea. At5g43580 seemed to be up-regulated byboth microbial pathogens (Fig. 1). Although PI genes are consideredto be induced upon JA, no significant induction in leaves uponmethyljasmonate (MeJA) treatment could be observed for the 4 PIgenes represented (Fig. 1). Considering their expression in plantorgans, At2g38870 was expressed in roots, leaves, seeds and thestem, while At2g38900 and At5g43580 were expressed in seedsand roots, respectively. At3g46860 had a very low basal expression

Literature C

Chassot et al., 2007

At2g38900

At2g38870

At 3g46860

At3g50020

At5g43570

At5g43580

93

Fig. 1. Phylogenetic relationships and gene expression data from genevestigator of predictegene products. Signal peptide cleavage sites were determined by SignalP at http://www.ClustalW and a phylogenetic tree was constructed by bootstrap neighbor-joining using MEGmanuscripts are referred to in which the particular gene was (originally) studied. Genes bchromosome) are indicated by the same letter. The isoelectric point (pI) of the mature peptidanalyzed in leaves 2, 6 and 24 h and 18, 48 h after infection by Pseudomonas syringae pv tomacid (SA: 10 mM) or methyl jasmonate (MeJA: 10 mM) or the ethylene precursor 1-aminocycloleaves, inflorescence, seeds, stem). Red squares indicate significant (ratio >3) up-regulatioexpression in roots, leaves, flowers, seeds, stem. Delineation of these organs and their corresgene was considered as significantly expressed in a certain plant organ if the absolute signspecified treatment (left panel) or no significant expression in the specified plant organ (rigand Analysis Toolbox Genevestigator [132]. Gray squares indicate the gene is not represent

in all plant parts tested (i.e. mean absolute signal intensity value<1000, as given by the Gene-Atlas sub-database of Genevestigator;Fig. 1). As a general remark it should be noted here that publiclyavailable micro-array data (Genevestigator) are only indicative andthat they need to be validated by detailed expression analysis (qRT-PCR, Northern blotting) before definitive conclusions can be drawn.Regarding the possible function of the PIs in A. thaliana, Chassotet al. [20] demonstrated that high-level expression of theAt2g38870 gene in transgenic A. thaliana resulted in enhanced re-sistance to B. cinerea. This study was the first to describe a pro-tective effect in A. thaliana against the fungal pathogen B. cinerea byoverexpression of an endogenous PR-6 gene. There are no otherreports on studies performed with any of the other PR-6 A. thalianagenes presented in Fig. 1.

3. Plant defensins (PR-12)


In 1990 the first plant defensins, isolated from wheat [24] andbarley [75], were originally categorized as a novel type thionin (seefurther) because of their similar molecular size (5 kDa) and similarnumber of cysteines (8). However, as the structure (including thepositions of the disulfide bonds) was not related to known a- and b-thionins (see further) [9], they were grouped apart as g-thionins.Later, because of their antimicrobial properties and their structuralsimilarity to mammalian and insect defensins, g-thionins wererenamed ‘‘plant defensins’’ [102]. PDFs were taken up in the groupof PR proteins and classified as the PR-12 family when Terras et al.[102] discovered two antifungal radish (Raphanus sativus) defensins(Rs-AFP3, Rs-AFP4) that were barely detectable in healthy un-infected leaves but accumulated at high levels after fungalinfection.

Hitherto, numerous PDFs could be purified from several plantspecies and plant tissues such as seeds, stems, roots, leaves and floralorgans and show a wide range of in vitro biological activities in-cluding a-amylase activity, ion-channel blocking and antibacterialactivity (reviewed by [65,85,111]. However, their best characterized

Pseud

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11.3B

5.33B

8.16

8.25

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4.61A

d PR-6 encoding genes of Arabidopsis thaliana and calculated pI of the correspondingcbs.dtu.dk/services/SignalP/. Predicted mature PR-6-type proteins were aligned using

A3.1 software [61]. Bootstrap values above 75% are shown. In the ‘‘Literature’’ columnelonging to the same cluster (i.e. homologous genes in close proximity on the same

e was calculated at http://www.expasy.org/tools/pi.tool.html. PR-6 gene expression wasato and Botrytis cinerea, respectively, 1 and 3 h after exogenous application of salicylicpropane-1-carboxylic acid (ACC: 10 mM), or in different (untreated) plant organs (roots,n compared to non-treated controls. Green squares indicate significant organ-relatedponding growth stages (as specified by Genevestigator) was based on Boyes et al. [8]. Aal intensity was >1000. White squares indicate no significant up-regulation upon theht panel). Gene expression data are derived from the Arabidopsis micro-array databaseed on the ATH1 Affimetrix genechip.

http://www.arabidopsis.org

http://www.arabidopsis.org

http://www.cbs.dtu.dk/services/SignalP

http://www.expasy.org/tools/pi.tool.html


activity is the ability to inhibit the growth of a broad range of fila-mentous fungi and yeasts in vitro. For instance, Terras et al. [101,102]described a broad spectrum antifungal activity of several plantdefensins isolated from Brassicaceae species, including radish,against several filamentous fungi including B. cinerea, A. brassicicolaand F. culmorum. The presence of inorganic salts and especially di-valent cations generally antagonized the antifungal activity of plantdefensins, although the cation sensitivity varied with the defensinand the fungus used (e.g. [101]). Recently, insight was gained in themode of antifungal action of two different plant defensins fromradish and dahlia (Dahlia merckii), Rs-AFP2 and Dm-AMP1, re-spectively [104–106]. Both peptides bound on distinct sphingolipids(glucosylceramide and manosyldiinositolphosphorylceramide forRs-AFP2 and Dm-AMP1, respectively) in fungal membranes and,consequently, showed a different specificity against fungal andyeasts species, including the human pathogen Candida albicans[106].

The precise in vivo function of PDFs remains unclear and dif-ferent roles have been attributed to them. Many PDFs are expressedabundantly (e.g. in seed [102]) whereas others are developmentallyregulated [122] or induced by different abiotic and biotic stressfactors, including cold [59], drought [30], heavy metals [78], po-tassium starvation [4], or microbial pathogens (e.g. [87,102,131]).The latter, in addition to their in vitro antimicrobial activity, in-dicates a role of PDFs in the plant defense response, which is furtheremphasized by enhanced disease resistance phenotypes observedin different plant species heterologously overexpressing PDF genes.For instance, Terras et al. [102] showed that transgenic tobaccooverexpressing the Rs-AFP2 gene was more resistant to the fungalpathogen Alternaria longipes. Another study described enhancedresistance of transgenic potato to Verticillium dahliae by over-expression of an alfalfa PDF gene [39]. So far, there are no reports ofenhanced resistance by overexpression of a PDF gene in the plantfrom which it originated.

AtPDF2.4

AtPDF2.1 Thomma et al., 1998

AtPDF2.2 Thomma et al., 1998

AtPDF2.5

AtPDF2.3 Epple et al., 1997a

AtPDF2.6

AtPDF3.2

AtPDF3.1

AtPDF1.4

AtPDF1.5

AtPDF1.1 Terras et al., 1993a

AtPDF1.3

AtPDF1.2b

AtPDF1.2c

AtPDF1.2a Penninckx et al., 199

LiteraturePDF name

At5g44420

At5g44430

At2g26020

At2g26010

At1g75830

At1g55010

At1g19610

At5g38330

At4g30070

At2g02140

At1g61070

At2g02130

At5g63660

At2g02100

At2g02120

100

83

94

96

99

96

Fig. 2. Phylogenetic relationships and gene expression data from Genevestigator of predictegene products. The study was done analogously as described in Fig. 1. To improve sequencecorresponding PDF name for each gene is indicated as specified by Thomma et al. [111]. Th


Hitherto, three A. thaliana PDFs (AtPDFs) were purified andrelatively well studied. Terras et al. [101] purified AtPDF1.1 fromseeds and showed in vitro antifungal activity against a broad rangeof fungi. Penninckx et al. [87] purified AtPDF1.2 from leaves infectedwith A. brassicicola and showed in vitro antifungal activity againstthis fungus and F. culmorum. Sels et al. [92] obtained significantamounts of AtPDF1.3 from transgenic plants and described a com-parable antifungal activity as for AtPDF1.2. Thomma et al. [111]described 15 putative PDF genes in A. thaliana, resulting froma TBLASTN search using the AtPDF1.2a sequence as a template, anddivided them into three families. Here, we focused on these so-called AtPDF1-, AtPDF2- and AtPDF3- families and performed ananalogous study as described in Fig. 1.

The AtPDF1- family contains seven genes encoding defensins(AtPDF1.1 to AtPDF1.5), including the up till now purified plantdefensins AtPDF1.1, AtPDF1.2 and AtPDF1.3 [87,101,92], re-spectively). Five genes show high sequence similarity (AtPDF1.1 toAtPDF1.3). Three genes (AtPDF1.2a, b and c) encode the same maturepeptide (AtPDF1.2), only showing a difference in their corre-sponding signal peptide sequences. The AtPDF1.2a and AtPDF1.2cgene sequences are located in tandem repeat on chromosome 5,while AtPDF1.2b and AtPDF1.3 are clustered in tandem repeat onchromosome 2 [111] (Fig. 2). AtPDF1.1 and the other two members,AtPDF1.4 and AtPDF1.5, are located on chromosome 1. Remarkably,of all classified AtPDF1-family members, AtPDF1.5 has an excep-tional acid character (pI 5.61) while the other members are clearlybasic (Fig. 2). The putative protein sequences of the AtPDF2-familyshow relatively more variation at the amino acid level [111]. TheAtPDF2.1, AtPDF2.2, AtPDF2.3 and AtPDF2.6 genes are organized ina cluster on chromosome 2, while AtPDF2.4 and AtPDF2.5 are lo-cated on chromosomes 1 and 5, respectively. All AtPDF2-familymembers have basic pIs. The AtPDF3-family constitutes the two

8.23

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d PR-12 encoding genes of Arabidopsis thaliana and calculated pI of the correspondingalignment, the C-terminal defensin domains of AtPDF3.1 and AtPDF3.2 were used. Thee three AtPDF-families are divided by a horizontal line.


AtPDF3.1 and AtPDF3.2 genes that encode larger proteins witha carboxy (C)-terminal ‘‘plant defensin domain’’ [111]. Possiblythese proteins are fusion proteins or precursors [111]. Interestingly,the predicted gene products of both these members have acidic pIs,comparable to the pI of AtPDF1.5 (Fig. 2). Concerning the hypothesisthat these proteins are fusion proteins, the pIs of the C-terminaldefensin domains were also calculated and were 7.64 and 5.51 forAtPDF3.1 and AtPDF3.2, respectively. Single-gene expression stud-ies (done by Northern blotting and RT-PCR) showed that AtPDF1.1and AtPDF2.1 were abundantly expressed in seeds [101,108], whileAtPDF2.2 and AtPDF2.3 were expressed in several organs ([35,108].The AtPDF1.2a gene was shown to be inducible in leaves uponpathogen inoculation and ET/JA treatment [35,87,88] and is by farthe most known PDF gene from A. thaliana. Nowadays it is usedglobally as a marker gene to study ET- and/or JA-mediated defenseresponses coupled to necrotrophic pathogens such as A. brassicicolaand B. cinerea [2,88,124]. Moreover, research on this marker genehas co-initiated the idea that the plant disease response is tailoredby the attacking pathogen: resistance against necrotrophic patho-gens (e.g. B. cinerea) appears to be related to the ET/JA-mediatedpathways, leading to the activation of PR genes such as AtPDF1.2a.On the other hand, SA-dependent pathways, activating effector PRgenes like PR-1, seem to be effective primordially against biotrophicpathogens such as Pseudomonas syringae [73,110]. Further testinghas revealed that these ideas are generally true, though the actualsituation is more complicated [28,42]. Next, consultation of thepublicly available micro-array data largely confirmed the single-gene expression studies performed on AtPDF1.1, AtPDF1.2a,AtPDF2.1, AtPDF2.2 and AtPDF2.3 (Fig. 2). Induction of AtPDF1.2a byB. cinerea was observed, however, despite the fact that AtPDF1.2a isfrequently used as a marker gene for ET/JA-mediated signal path-ways, no significant induction ratio upon ACC or MeJA treatmentwas observed consulting Genevestigator data. The latter discrep-ancy points out the importance of single gene expression studies.The AtPDF1.2b gene, encoding the same mature protein asAtPDF1.2a, was expressed in leaves and seeds but shows no sig-nificant induction upon B. cinerea inoculation (Fig. 2). The AtPDF1.2cgene, together with AtPDF1.3 and AtPDF1.5, were not represented onthe ATH1 Affimetrix gene chip. The AtPDF1.1 and AtPDF1.4 geneswere expressed in seeds. Remarkably, the AtPDF1.1 gene seemsinducible in leaves upon B. cinerea inoculation (Fig. 2). Concerningthe AtPDF2-gene family, the AtPDF2.2 and AtPDF2.3 genes areexpressed in all organs, while the other genes are expressed in atleast one plant organ (Fig. 2). No significant induction ratios forAtPDF2-family genes were observed with any of the treatmentsstudied (Fig. 2). AtPDF3.1 and AtPDF3.2 showed very low basal ex-pression in all plant parts (absolute signal intensities <1000),however the latter gene is inducible by SA treatment (Fig. 2).

The three purified AtPDFs, namely AtPDF1.1, AtPDF1.2 andAtPDF1.3 showed in vitro antifungal activity [87,92,101]. Regardinga possible role in defense in vivo, there are several A. thalianamutants, with altered AtPDF1.2a expression levels, which oftenshow altered pathogen sensitivity. For instance, there are severalreports on mutants with diminished AtPDF1.2a expression levelswhich show a higher sensitivity against necrotrophic pathogenslike B. cinerea, including the JA signaling mutant coi1 and the ETsignaling mutant ein2 [109] and bos2, bos3 and bos4 mutants [124].However, these effects are not considered to be solely caused byaltered AtPDF1.2a expression, as for example the bos1 mutantshows increased sensitivity against B. cinerea while having normalAtPDF1.2a expression levels [76]. Altogether, there are many argu-ments to state that AtPDF1.2 as an effector protein is not sufficientfor plant defense against B. cinerea [37,76,116,124]. AlthoughAtPDF1.2 shows in vitro antifungal activity [87] its precise role inplant defense remains unclear, as it is for the rest of the A. thalianaPDFs. Yet, no direct evidence for a role in defense in vivo has been

shown for any AtPDF member. Concerning other putative functionsfor plant defensins in vivo, the role of plant defensins in relation toabiotic stress was studied. Overexpression of PDF genes from Ara-bidopsis halleri, a zinc hyper-accumulating plant closely related to A.thaliana, resulted in transgenic A. thaliana plants which were moretolerant to zinc than wild type plants [78]. It was also shown thatPDFs in A. halleri constitutively accumulated at higher levels than inA. thaliana [78]. Overexpression of the A. thaliana defensin geneAtPDF1.2c in the yeast Saccharomyces cerevisiae resulted in en-hanced metal tolerance compared to wild type yeast [78]. Theseresults support the proposition that defensins, possibly apart froma putative function in plant defense, could be involved in zinc tol-erance. More recently, using motif models developed by a bio-informatics approach, as much as 317 putative ‘‘plant defensin-like’’genes, many of which were un-annotated, were described in theA. thaliana genome and based on their amino acid sequences weredivided into 46 families [93]. These AtPDF-like genes are currentlystudied at our laboratory, using a customized micro-array con-taining most of these gene sequences. Future research will indicatewhether the unknown AtPDF-like genes will have similar expres-sion profiles as the known AtPDFs.

4. Thionins (PR-13)


Like PDFs, thionins are small (5 kDa), usually basic, cysteine-richpeptides and were originally isolated from cereals [6]. Hitherto,around 100 thionin gene sequences have been identified in 15different plant species (reviewed by [96]). Induced expression ofleaf thionins could be shown upon fungal infection in barley and A.thaliana [6,34] and consequently they were classified as the PR-13family of PR proteins.

The main characteristic of thionins is their broad in vitro anti-fungal and antibacterial activity (e.g. [6,11] and like plant defensins,their antimicrobial effects lead to the permeabilization of cellmembranes [96,103]. Antimicrobial activity was demonstratedagainst phytopathogenic bacteria (see references in [17]), phyto-pathogenic fungi [11,100], yeast (see references in [17]), andmammalian cell lines [14]. Combination of thionins and LTPsresulted in synergistically antifungal activity, suggesting theseproteins may cooperate in membrane binding and/or per-meabilization ([79], reviewed in [96]).

There are also several reports on transgenic plants expressinga thionin gene and making them more resistant to fungal or bac-terial pathogen attack. For example, Chan et al. [18] showed thatheterologous overexpression of the A. thaliana Thi2.1 gene intransgenic tomato made these plants more resistant to bacterialwilt and Fusarium wilt. Also, high-level expression of a hordothio-nin gene from barley in transgenic tobacco conferred resistance toPseudomonas syringae [13] and enhanced resistance to bacterialdiseases was described in transgenic rice plants overproducing anoat cell wall-bound thionin [51]. Interestingly, Epple et al. [36]described that overexpression of the endogenous Thi2.1 thioningene enhanced resistance of A. thaliana against Fusarium oxy-sporum). The latter study reports on homologous overexpressionand proves in this way a direct role in defense for this thionin genein A. thaliana. No studies on transgenic plants with RNAi or knock-out of thionin genes and their possible implication on plant defensewere found.

Apart from their putative defensive function based upon thesedata, other in vivo functions have been proposed for thionins. Thereare indications for a regulatory role as thionins have thioredoxinactivity and hereby could act as secondary messengers in the redoxregulation on enzymes [53]. Thionins found in seeds, could alsofunction as storage proteins, especially as a source of sulfur [17].



Using BLASTP with the Thi2.1 sequence as template, 6 putativethionin genes in the A. thaliana genome were found, including therelatively well-studied Thi2.1 and Thi2.2 genes, originally identifiedby Epple et al. [34] (Fig. 3). The At1g12660 and At1g12663 genes areclustered at chromosome 1 (cluster A). The isoelectric points of thepredicted mature peptides range from acidic (for At1g66100 andAt1g12660 gene products) over almost neutral (for At5g36910 andAt2g15010 gene products), to basic (for At1g72260 and At1g12663gene products). Single-gene expression studies showed that theThi2.2 gene is expressed constitutively in seedlings whereas theThi2.1 gene is expressed at high levels in flowers and siliques, andmarginally in leaves [34]. Moreover, Thi2.1 was induced in seedlingsby MeJA, silver nitrate and phytopathogenic fungi as shown byNorthern blotting [34]. Later Bohlmann et al. [7] showed thatwounding and several jasmonate related compounds like corona-tine were also able to induce Thi2.1 and that this induction couldnot be observed in the coi1 mutant background. No other singlegene studies have been performed on the other A. thaliana thioningenes presented in Fig. 3. The micro-array data presented in Fig. 3correlate with the expression studies done on Thi2.1 as expressionin flowers and seeds, and MeJA-mediated activation in leaves werefound. In contrast to the expression study done by Epple et al. [34],relatively high Thi2.2 expression levels in leaves were observed(Fig. 3). The At1g66100 gene was expressed in leaves and, like thewell studied Thi2.1 gene, activated upon MeJA treatment (Fig. 3).The At2g15010 gene was seed-expressed while the At1g12660 genehad a very low basal expression in all plant organs (absolute signalintensities <1000) (Fig. 3). The At1g12663 gene is not representedon the ATH1 Affimetrix gene chip (Fig. 3). Concerning the role of A.thaliana thionins in planta, one study by Epple et al. [36] showed anactive role in plant defense for Thi2.1. No other reports on func-tional characterization of A. thaliana thionins were found.

5. Lipid transfer proteins (PR-14)


Lipid transfer proteins (LTPs) are small, cationic, cysteine-richpeptides found in various plant species (reviews in [15,57,58,128],including barley [79], grapevine [40], wheat [97], A. thaliana andspinach [91] and onion [12]. Plant LTPs are sub-divided into twofamilies, LTP1s and LTP2s, which present molecular masses ofaround 9 and 7 kDa, respectively. Apart from these differences inmolecular size, the other characteristics of members of these twofamilies, such as high pI and the pattern of four conserved disulfidebridges, are similar [15]. LTPs were named because of their ability to

Literature C

Thi2.1 Epple et al., 1997b

Thi2.2 Epple et al., 1995

At1g66100

At5g36910

At1g72260

At2g15010

At1g12663

At1g12660100

Thionin name

Fig. 3. Phylogenetic relationships and gene expression data from Genevestigator of predictegene products. The study was done analogously as described in Fig. 1. The thionin name is

facilitate the transfer of phospholipids between membranes in vitro[56]. They are able to transfer various types of lipids includingphosphatidylinositol, phosphatidylcholine and galactolipids [17].Due to this low specificity for the lipid substrate, plant LTPs are alsonamed ‘‘non-specific lipid transfer proteins’’ [57]. As LTP gene ex-pression was also found to respond to infection with pathogens [41]they were classified as PR proteins.

LTPs possess a signal peptide, targeting them to the cell secre-tory pathway [15]. Various LTPs have been shown to be localized atthe cell wall, as was demonstrated for the A. thaliana LTP1 geneproduct [107]. This extra-cellular location is not a general rule asLTPs have also been found in glyoxosomes where their activity waslinked to lipid catabolism [113] and in protein storage vacuoles ofseeds [16].

LTP genes are generally expressed in leaves and in flowers, andrarely in roots [5]. In leaves, LTP genes are usually expressed at highlevels in young expanding leaves and hereby LTPs were suggestedto play a role in the transport of monomers of cutin and found to beassociated with cutin and wax assembly [20,89]. Though, the ma-jority of LTP genes seem to be expressed in flowers or flower organs(e.g. [5,83,89]. Park et al. [83] showed that an LTP is necessary forpollen adherence to the stigma during pollen elongation in theLilium longiflorum. Next to their inducibility upon pathogen in-fection (e.g. [41,54]), LTP genes are also responsive to abioticstresses like drought, cold and salt [52,54].

Based on these findings in expression patterns, many biologicalactivities have been suggested for LTPs [128], including roles incutin synthesis [89], b-oxidation [113], plant defense signaling[10,71], and plant defense (e.g. [40,60,79]). Concerning their role inplant defense, various LTPs have been shown to have in vitro an-timicrobial activities against fungi and bacteria [12,79,126]. Thisobserved antimicrobial activity could result from the interaction ofLTPs with biological membranes, possibly leading to membranepermeabilization [57]. There are also several reports on transgenicoverexpression of LTP genes resulting in enhanced tolerance topathogen infection. For instance, transgenic tobacco expressinga barley LTP gene showed enhanced resistance against Pseudomo-nas syringae pv. tabaci [80]. Transgenic A. thaliana, overexpressinga barley LTP gene, showed enhanced resistance against Pseudomo-nas syringae pv. tomato and B. cinerea [55]. Concerning homologousoverexpression experiments, Chassot et al. [20] described that en-dogenous overexpression of three LTP-like genes in A. thalianaresulted in enhanced tolerance to B. cinerea.


Arondel et al. [5] described 15 LTP genes present in A. thaliana.These 15 genes were identified by searching EST databases and

Pseud

omon

assy

ringa

e

Botryti

s cinere

aSA MeJ

AACC

Roots

Leav

esFlow

erSee

dStem

luster Core pI

5.3A

8.5A

7.6

8.3

7.6

4.4

d PR-13 encoding genes of Arabidopsis thaliana and calculated pI of the correspondingindicated as specified by Epple et al. [34].


performing BLASTN. Of these obtained sequences only ‘‘typical’’LTPs were retained, that is, LTP1-like proteins with a size of around10 kDa [5]. Larger LTP-like proteins and 7 kDa LTPs (LTP2 family-type proteins) were excluded from the analysis [5]. Here, we fo-cused on these 15 genes (LTP1 to LTP15) identified by Arondel et al.[5] and performed a study analogously as described before for PIs,PDFs and thionins (Fig. 4). Three times two genes appear in tandemrepeats in the A. thaliana genome (LTP1 and LTP2, cluster A; LTP5and LTP12, cluster B; LTP3 and LTP4, cluster C; Fig. 4). The pI of mostLTPs is basic, but nearly covers the whole range from 4.2 to 11.4(Fig. 4).

By performing single-gene expression studies, the expressionpattern was characterized for the LTP1, LTP2, LTP3, LTP4, LTP5 andLTP6 genes by Northern blotting [5]. All six genes were found tobe expressed in flowers and developing seeds, but not in roots [5].In addition, LTP1, LTP2 and LTP5 are expressed significantly inleaves, while LTP6 was only detected in 2–4-week-old leaves. LTP3and LTP4 were reported to be up-regulated by ABA [5]. Clark andBohnert [23] also described LTP1, LTP2 and LTP3 being expressed inflowers and developing seeds. In contrast to the epidermis-specific expression of LTP1 [126], both LTP2 and LTP3, were notrestricted to the epidermis but were also expressed in sub-epidermal layers [23]. No expression patterns for LTP7 to LTP15 aredescribed thus far.

From the micro-array results represented in Fig. 4 it is clear thatabsolute signal intensity values for most LTP genes, in most organs,were above the threshold set at 1000. The LTP1 to LTP6 genesseemed to be expressed in flowers and seeds, as was shown inplanta by Arondel et al. [5], but were also significantly expressedin other organs (Fig. 4). In addition, LTP7 and LTP12 were expressed inleaves and stems and flowers and stems, respectively, while LTP5,LTP8 and LTP10 were significantly expressed in roots, not a typicalorgan for LTP gene expression. The LTP4 gene showed significantinduction in leaves upon P. syringae and B. cinerea inoculation. Noother LTP genes seem to be significantly up-regulated upon path-ogen inoculation or plant hormone treatments consulting the

Literature

LTP11

LTP15

LTP14

LTP13

LTP9

LTP8

LTP10

LTP6 Arondel et al. 2000

LTP12


LTP3 Clark and Bohnert 1

LTP7

LTP2 Clark and Bohnert 1


LTP1 Wang et al. 2005 At2g38540

At3g51600

At2g38530

At2g15050

At5g59320

At5g59310

At3g51590

At3g08770

At5g01870

At2g18370

At4g33355

At2g15325

At5g44265

At5g62065

At4g0853072

57

88

53

98

80

42

85

42

81

3843

LTP name

Fig. 4. Phylogenetic relationships and gene expression data from Genevestigator of predictegene products. The study was done analogously as described in Fig. 1. The LTP name for ea

Genevestigator database (Fig. 4). The LTP11, LTP13 and LTP14 genesare not represented on the ATH1 Affimetrix gene chip.

There are some specific reports on the putative functions of A.thaliana LTPs in planta. Wang et al. [126] identified LTP1 as a cal-modulin-binding protein and the calmodulin-binding regionseemed to be conserved among LTPs, suggesting the involvement ofCa2þ and calmodulin signaling in functions of LTPs. Maldonadoet al. [71] showed the involvement of a putative LTP (gene locusAt5g48485) in systemic resistance signaling in A. thaliana. Theseauthors discovered the dir1-1 mutant which fails to develop SAR tovirulent Pseudomonas syringae or Peronospora parasitica. Theyfound DIR1 to encode a putative apoplastic LTP and proposed it tointeract with a lipid-derived molecule to promote long-distancesignaling involved in SAR. The dir1-1 mutant exhibited wild typeresistance to P. syringae, so a direct effect of the knock-out of the LTPgene on plant resistance was not shown. Concerning their possiblerole in defense, one report describes the endogenous over-expression of three LTP-like genes (At4g12470, At4g12480,At4g12490) in A. thaliana resulting in enhanced tolerance topathogens, more specifically to B. cinerea [20]. However, these threeclustered genes, possibly involved in the resistance to B. cinerea,and the formerly described DIR1 gene, were not classified byArondel et al. [5] (Fig. 4).

6. Conclusion

In conclusion, PR proteins consist of a large variety of familieswith members that differ in occurrence, expression and biologicalactivities. In this review on PR peptides in the model plant A.thaliana, this variety among characterized and putative members ofthe PR-6, PR-12, PR-13 and PR-14 families was discussed. The studywas based on literature describing single-gene studies and onlineavailable micro-array data for the discussion of putative unchar-acterized members.

It is clear that PR peptides probably have varied physiologicalfunctions in the plant, not restricting them to proposed roles in

Pseud

omon

assy

ringa

e

Botryti

s cinere

aSA MeJ

AACC

Roots

Leav

esFlo

werSee

dStem

8.5

7.5

4.2

8.5

5.2

4.9

9.4

7.7

7.7B

9.1C

9.1C999

9.7

9.4A999

11.4B

9.3A

Cluster Core pI

d PR-14 encoding genes of Arabidopsis thaliana and calculated pI of the correspondingch gene is indicated as specified by Arondel et al. [5].


defense against phytopathogens. Since only some members offamilies of PR peptides have suppressive effects on specific patho-gens, but not on others, their direct role in restricting pathogengrowth appears to be limited and case-specific, as postulated byVan Loon et al. [120]. However, PR peptides appear to be part ofa larger set of SA-, ET-, JA-dependent defense responses in whicheach component may contribute to resistance against the attacker.As an example, the ein2 mutant of A. thaliana, which is impaired inET/JA signaling and shows increased susceptibility to necrotrophicpathogens like B. cinerea [109], has a whole set of PR genes forwhich diminished expression was observed, including genes fromPR families PR-12, PR-3 and PR-4 [110]. In analogy, the npr-1 mutant,which is impaired in SA signaling and displays an increased sus-ceptibility to most biotrophic pathogens like P. syringae, showeddecreased expression of genes from PR families of PR-1, PR-2 andPR-5 whereas PR-12 expression remained unaffected [110]. Theseexamples show the complexity of the disease signaling network inA. thaliana and the difficulty in elucidating the role of a specificfamily of PR peptides herein.

To determine a possible function for these PR peptides in vivo,their behavior in the plant of origin needs to be studied. The fact thatPR peptides are represented as multi-gene families (e.g. [5,111,120])makes it more difficult to determine a specific function for a par-ticular member, as redundancy could logically be a problem. Forexample, with respect to the PR family PR-12, it was mentionedearlier that in A. thaliana 3 different genes (AtPDF1.2a, AtPDF1.2b andAtPDF1.2c) encode the same mature peptide AtPDF1.2 [111]. In ad-dition, using gene specific primers we could demonstrate by qRT-PCR that these genes also exhibit very similar expression profilesupon treatment with pathogens or defense-related plant hormonessuch as SA, ET and JA (Sels, unpublished results). Moreover, a similarexpression profile was found for a fourth plant defensin gene,AtPDF1.3, which is 91% identical to the marker gene AtPDF1.2a (Sels,unpublished results). In view of this redundancy, it is very unlikelythat modulation of expression of a single gene can result in a clearphenotypical effect. Further, it is important to emphasize that forplant peptides in general, not only the total number but also thedifferent types of peptides have been heavily underestimated[93,94]. At the most recent annotation of the Arabidopsis genome by‘The Institute for Genomic Research’ [46], only proteins with a mo-lecular size exceeding 110 amino acid residues (330 bp) wereretained in order to avoid false positive predictions. Peptides areconsequently underestimated in the most updated Arabidopsis geneannotation and, as a consequence missing on commercially avail-able microarrays such as the CATMA arrays [1] or the AffymetrixATH1 chips [90] which are typically gene annotation-based. Inagreement, Silverstein et al. [93,94] recently described a great un-der-prediction of cysteine-rich peptides in plants, as numerousnewly un-annotated PDF-like, thionin-like and LTP-like sequenceswere found in A. thaliana (as well as in other plant species). Theimplementation of tiling arrays, which are gene annotation in-dependent, could be an alternative to study on a genome-widebasis, the expression profile of peptide genes [44], including thoseencoding PR peptides, following a specific treatment, inoculation orphysiological condition. Such a genome-wide gene expressionprofiling could provide a first basis to further unravel the still largelyunknown biological role of PR peptides.

Acknowledgements

This research was supported by predoctoral grants (to J.S.) andpostdoctoral grants (to J.M. and B.D.C.) from the ‘‘Institute for thePromotion of Innovation through Science and Technology in Flan-ders (IWT-Vlaanderen)’’, by the Flemish Fonds voor Weten-schappelijk Onderzoek (FWO Vlaanderen) (research projects

G.0288.04, G.0405.05) and by the University Research Foundation(GOA/08/011).

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Plant pathogenesis-related (PR) proteins: A focus on PR peptides