Transgenic Expression ofaFungalendo-Polygalacturonase ... · Transgenic Expression ofaFungalendo-Polygalacturonase Increases Plant Resistance to Pathogens and Reduces Auxin Sensitivity1[W]

Transgenic Expression of a Fungal endo-PolygalacturonaseIncreases Plant Resistance to Pathogens and ReducesAuxin Sensitivity1[W]

Simone Ferrari2, Roberta Galletti2, Daniela Pontiggia2, Cinzia Manfredini, Vincenzo Lionetti,Daniela Bellincampi, Felice Cervone, and Giulia De Lorenzo*

Dipartimento di Biologia Vegetale, Universita degli Studi di Roma La Sapienza, 00185 Rome, Italy

Polygalacturonases (PGs), enzymes that hydrolyze the homogalacturonan of the plant cell wall, are virulence factors of severalphytopathogenic fungi and bacteria. On the other hand, PGs may activate defense responses by releasing oligogalacturonides(OGs) perceived by the plant cell as host-associated molecular patterns. Tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsisthaliana) plants expressing a fungal PG (PG plants) have a reduced content of homogalacturonan. Here, we show that PG plantsare more resistant to microbial pathogens and have constitutively activated defense responses. Interestingly, either in tobacco PGor wild-type plants treated with OGs, resistance to fungal infection is suppressed by exogenous auxin, whereas sensitivity toauxin of PG plants is reduced in different bioassays. The altered plant defense responses and auxin sensitivity in PG plants mayreflect an increased accumulation of OGs and subsequent antagonism of auxin action. Alternatively, it may be a consequence ofperturbations of cellular physiology and elevated defense status as a result of altered cell wall architecture.

The plant cell wall possesses mechanical featuresdetermining strength and plasticity of a tissue andsignaling properties affecting expansion, growth, anddevelopment (Carpita and McCann, 2000). The cellwall also represents a barrier against invading micro-organisms. During early stages of infection, pathogensproduce enzymes that degrade the various compo-nents of the wall, thus releasing compounds that areused as carbon sources. The major components ofprimary cell walls of higher plants are complex poly-saccharides; increasing evidence indicates that thesemolecules contribute to disease resistance not just asmechanical barriers but also as sensors for incominginfections (Vorwerk et al., 2004). For example, partialdegradation of homogalacturonan (HGA) by fungal endo-polygalacturonases (PGs) releases oligogalacturonides(OGs) with a degree of polymerization between 10 and15 that show elicitor activity (Cervone et al., 1987a, 1987b,1989). Treatment with OGs causes accumulation of reac-tive oxygen species, biosynthesis of phytoalexins (Hahnet al., 1981), and expression of pathogenesis-related (PR)

proteins (Davis and Hahlbrock, 1987; Broekaert andPneumas, 1988) in several plant species. In Arabidopsis(Arabidopsis thaliana), OGs induce the expression of sev-eral defense genes and proteins (Ferrari et al., 2003b, 2007;Casasoli et al., 2008), including AtPGIP1 and PAD3,which encode, respectively, an inhibitor of fungal PGsand the Cyt P450 CYP71B15 that catalyzes the last step ofcamalexin biosynthesis. Exogenous OGs protect Arabi-dopsis and grapevine leaves against the necrotrophicpathogen Botrytis cinerea (Aziz et al., 2004; Ferrari et al.,2007). In analogy with the role of hyaluronan fragmentsin animal cells, OGs may be regarded as host-associatedmolecular patterns involved in the innate immunity(Stern et al., 2006; Taylor and Gallo, 2006).

Besides inducing defense responses, OGs can alsoaffect several aspects of plant growth and develop-ment. In particular, a number of reports from ourlaboratory indicate that exogenously added OGs areable to antagonize the action of auxin, as in the case ofpea (Pisum sativum) stem elongation (Branca et al.,1988), tobacco (Nicotiana tabacum) adventitious rootformation (Bellincampi et al., 1993), and pericycle celldifferentiation (Altamura et al., 1998). OGs preventrhizogenesis in tobacco leaf explants expressing theAgrobacterium tumefaciens rolB gene by inhibiting theauxin-induced expression of the transgene (Bellincampiet al., 1996). Furthermore, in tobacco, OGs inhibit the in-duction of the late auxin-responsive genes Nt114, rolB,and rolD (Mauro et al., 2002). In cucumber (Cucumissativus) seedlings, OGs allow for a more rapid recoveryof root growth in auxin-treated roots (Spiro et al., 2002).To date, the mechanism underlying the antagonisticeffect of OGs on auxin-induced responses is not known.

We have previously generated tobacco and Arabidop-sis transgenic lines (hereafter referred to as PG plants)

1 This work was supported by the Ministero dell’Universita e dellaRicerca (Fondo per gli Investimenti della Ricerca di Base RBNE01KZE7and PRIN 2005052297), by the Giovanni Armenise-Harvard Founda-tion, and by the Institute Pasteur-Fondazione Cenci Bolognetti.

2 These authors contributed equally to the article.* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Giulia De Lorenzo ([email protected]).

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.107.109686

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expressing an attenuated version of PGII of Aspergillusniger (Capodicasa et al., 2004). PG plants of both speciesaccumulate the enzyme in their tissues and displaymorphological alterations, such as dwarfism andslightly curled leaves, but no other severe aberrations(Capodicasa et al., 2004). These phenotypes are depen-dent on the enzyme activity because expression of aninactive form of A. niger PGII has no obvious impact onArabidopsis development. Moreover, crossing tobaccoPG plants with a line expressing high levels of the bean(Phaseolus vulgaris) PG-inhibiting protein PvPGIP2,an inhibitor of A. niger PGII, completely blocked PGactivity and reverted the dwarf phenotype. The ob-served morphological alterations are associated to areduced content of HGA in both tobacco and Arabi-dopsis, suggesting that the phenotype of PG plants isdue to an enhanced degradation of HGA (Capodicasaet al., 2004). Interestingly, tobacco PG plants show noalterations of ion-mediated increase in xylem hydraulicconductivity (Nardini et al., 2007) and have a photo-synthesis rate and stomatal conductance similar tothose of wild-type plants (R. Galletti and D. Pontiggia,unpublished data). Therefore, PG plants, despite theirgrowth defects, do not show alterations that are typicalof plants suffering abiotic stresses, such as water stress(Flexas and Medrano, 2002).

Because the signaling potential of pectin and pectin-derived fragments may be critical for the outcome of aplant-pathogen interaction (Vorwerk et al., 2004), wehave investigated whether the modifications causedby the expression of PG affect responses of plants tomicrobial pathogens. Here, we show that PG plantsexhibit enhanced resistance to the necrotrophic fungalpathogen B. cinerea and to the virulent bacterial path-ogen Pseudomonas syringae, and constitutive expres-sion of defense responses. Notably, tobacco PG plantsshow a susceptibility to B. cinerea similar to wild-typeplants when treated with auxin, as well as a reducedsensitivity to auxin in different bioassays. Further-more, we show that auxin inhibits OG-induced resis-tance in wild-type tobacco plants. The relationshipamong pectin degradation, auxin responses, and de-fense against pathogens is discussed.

RESULTS

PG Plants Are Less Susceptible to Pathogen Infection

Tobacco PG plants were inoculated with B. cinerea, andtheir susceptibility to the fungus was compared to that ofwild-type plants and plants expressing the bean PvPGIP2,which inhibits several fungal PGs, including B. cinereaPG (PGIP2 plants; Leckie et al., 1999; Capodicasa et al.,2004). Upon infection, wild-type plants exhibited typicalsoft rot symptoms with rapidly expanding water-soakedlesions, whereas PG plants displayed lesions with areduced size (Fig. 1A). Furthermore, inoculation of PGplants resulted in dry lesions unable to develop beyondthe inoculation site in a significant number of cases (Fig.

1B). PGIP2 plants also displayed reduced lesions that,unlike the lesions observed in PG plants, were typicallywater soaked (Fig. 1, A and B). Leaves from plantsobtained by crossing line PG16 with the PGIP2 line(PG163PGIP2 plants), expressing PGIP2 in great excesswith respect to PG and showing no detectable PGactivity in their tissues (Capodicasa et al., 2004), exhib-ited symptoms comparable to those developed on PGIP2plants (Fig. 1, A and B).

Arabidopsis Wassilewskija (Ws) PG plants also dis-played a significant reduction of lesion size afterfungal inoculation (Fig. 1C). Line PG1, expressing thehighest levels of PG (Capodicasa et al., 2004), devel-oped smaller lesions (Fig. 1C) and showed a significantreduction of the number of spreading lesions com-pared to the other lines (Fig. 1D). Transcript levels ofthe B. cinerea actin gene Bcact were dramatically re-duced in inoculated PG1 plants (Supplemental Fig.S1), confirming that the reduced lesion developmentmirrored a reduced fungal growth. On the other hand,leaves of a transgenic line (PG201), expressing aninactive form of A. niger PGII (van Santen et al., 1999;Federici et al., 2001), showed symptoms similar tothose of wild-type plants (Fig. 1, C and D), indicatingthat enzymatic activity is required to confer enhancedresistance.

To determine whether the enhanced resistance ob-served in PG plants is specific for B. cinerea or is effectiveagainst other pathogens, leaves of tobacco wild-typeand PG plants were inoculated with a virulent strain ofthe bacterial pathogen P. syringae pv tabaci. Infiltrationwith a bacterial suspension produced the collapse ofthe inoculated area in wild-type plants but no visiblesymptoms in line PG16 and only very mild symptomsin lines PG5 and 7 (Fig. 2). PGIP2 and PG163PGIP2plants showed symptoms similar to those observed inwild-type plants (Fig. 2). This indicates that PvPGIP2has no effect on resistance to P. syringae and, moreimportantly, that inhibition of enzymatic activity of PGby PvPGIP2 completely blocks PG-mediated resis-tance. An active and free (uncomplexed) PG is thereforerequired to confer resistance to plants against organ-isms as different as the fungal pathogen B. cinerea andthe bacterial pathogen P. syringae.

PG Plants Have Enhanced Defense Responses

PGs may act not only as virulence factors of phyto-pathogenic microorganisms but may also induce de-fense responses, by either releasing endogenous OGelicitors or affecting cell wall integrity (Hahn et al.,1981; Davis et al., 1986; Cervone et al., 1987a). Stainingof leaves with 3-3#-diaminobenzidine (DAB) revealedaccumulation of hydrogen peroxide (H2O2) in tobaccoPG16 and PG7 and, to a lesser extent, PG5 leaves(Fig. 3A). No significant staining was observed inwild-type, PGIP2, and PG163PGIP2 plants, indicatingthat accumulation of H2O2 correlates with the level ofactive PG in the tissues. Similarly, constitutive accu-mulation of H2O2 was observed in leaves of Arabidopsis

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PG1 and, to a lesser extent, PG5 plants, whereas accu-mulation of H2O2 in Arabidopsis wild-type and PG201plants was restricted to the severed petioles (Fig. 3B).Leaves of tobacco and Arabidopsis PG plants exhibiteda bright fluorescence when illuminated with UV light,possibly due to the accumulation of secondary metab-olites (Supplemental Fig. S2).

We also determined total peroxidase and 1,3-b-glucanase enzymatic activities, which are known tobe regulated by biotic stress (Van Loon and van Strien,1999). Total peroxidase activity was significantly higherin leaves of tobacco plants PG16 and PG7 as comparedto control plants, whereas in PG5 as well as in PG163PGIP2 plants it was only slightly, but still significantly,higher than in wild-type plants or plants expressingPvPGIP2 alone (Fig. 4A). Analysis of peroxidase activ-ity in the intercellular washing fluids (IWFs) preparedfrom control and transgenic tobacco plants indicatedthat the increase of peroxidase activity of PG plants wasmainly due to extracellular isoforms (Fig. 4B). Peroxi-dase activity was also significantly increased in leavesof Arabidopsis plants belonging to line PG1, comparedto the other genotypes (Fig. 4C). Basal 1,3-b-glucanaseactivity was significantly higher only in tobacco andArabidopsis lines expressing high levels of PG (Fig. 5, Aand B).

Finally, we examined the expression of genes poten-tially involved in pathogen responses. Expression of twotobacco genes, EAS1/2, encoding a 5-epi-aristolochenesynthase required for the biosynthesis of the phyto-alexin capsidiol (Facchini and Chappell, 1992), andPOX, a gene encoding an apoplastic anionic peroxidase(Diaz-De-Leon et al., 1993), was clearly induced by B.cinerea infection in both wild-type and PG16 plants (Fig.6A). However, expression of POX was detectable also inuninfected PG16 plants and increased earlier and to agreater extent in response to fungal infection (Fig. 6A).EAS1/2 mRNA levels were not constitutively expressedin PG16 plants but increased earlier and to a greaterextent in response to fungal infection (Fig. 6A). InArabidopsis wild-type plants, expression of AtPGIP1,PR-1, and the defensin gene PDF1.2 steadily increasedup to 2 d after infection with B. cinerea (Fig. 6, B–D). The

Figure 1. Resistance of PG plants to fungal infection. Development ofsymptoms in tobacco (A and B) and Arabidopsis (C and D) untrans-formed (WT) and PG plants inoculated with B. cinerea is shown. Fullyexpanded leaves from untransformed tobacco plants (WT) from threeindependent transgenic lines expressing PG (PG5, PG7, and PG16)from plants overexpressing bean PvPGIP2 (PGIP2) or generated by

crossing line PG16 with the line expressing PvPGIP2 (PG163PGIP2)were inoculated with B. cinerea. Lesion area (A) and percentage ofexpanding lesions (B) were measured after 6 d. Bars in A indicateaverage lesion area 6 SE (SE; n . 36); this experiment was repeated fourtimes with similar results. Bars in B represent the average percentage ofspreading lesions 6 SE of five experiments (n . 36 in each experiments).Adult rosette leaves from untransformed Arabidopsis plants (WT) andfrom transgenic plants expressing PG (PG1 and PG5) or an inactiveversion of the A. niger PGII (PG201) were inoculated with B. cinerea,and lesion size was determined after 2 d (C), whereas the number ofexpanding lesions was determined after 3 d (D). Bars in C indicate theaverage lesion area 6 SE (n . 12); this experiment was repeated threetimes with similar results. Bars in D represent the average percentage ofspreading lesions 6 SE of three experiments (n . 12 in each experi-ment). Different letters represent data sets significantly different,according to ANOVA analysis followed by Tukey’s test (P , 0.01).

Fungal Polygalacturonase Decreases Susceptibility to Pathogens

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basal mRNA levels of all three genes were higher inPG1 plants than in wild-type plants (Fig. 6, B–D).However, their expression differed after inoculationwith B. cinerea. AtPGIP1 expression increased at 1 dpostinfection (dpi) but returned to basal levels at 2 dpi(Fig. 6B). In contrast, transcripts of PR-1 and PDF1.2showed only a mild increase after fungal infection ofPG plants (Fig. 6, C and D). These findings are consis-tent with previous data correlating the expression levelsof both genes to fungal lesion development rather thanto basal resistance (Ferrari et al., 2003a).

A possible explanation for the enhanced expressionof defense genes observed in healthy PG plants is theaccumulation of OG elicitors released from the cellwall by the action of the fungal PG. We therefore de-termined whether the expression of these genes is up-regulated by exogenous OGs. Both POX and EAS1/2mRNA levels increased in tobacco leaf explants treatedfor 4 h with OGs (Fig. 7, A and B). Similarly, AtPGIP1and PR-1 transcripts accumulated in response to OGs inArabidopsis wild-type seedlings (Fig. 8, A and B), whileno significant increase of PDF1.2 mRNA levels wasobserved (Fig. 8C). Taken together, these data supportthe hypothesis that the constitutive expression of atleast some of the defense responses observed in PGplants is mediated by OGs released by the fungalenzyme expressed in the transgenic plants.

Increased Resistance of Tobacco PG Plants Is Abolishedby Auxin

Because OGs have auxin-antagonistic activity andtreatment with OGs leads to a decreased sensitivity toauxin in tobacco plants (Branca et al., 1988; Bellincampiet al., 1993, 1996; Altamura et al., 1998; Mauro et al.,2002), a support for the possible involvement of OGsin the phenotype of PG plants might be provided bythe ability of auxin to revert some of the phenotypicfeatures as well as by a decreased sensitivity to auxin ofthe PG plants.

We therefore investigated whether the increasedresistance against B. cinerea of the tobacco PG plantscould be reverted by exogenous auxin. Leaf discs fromwild-type and PG16 tobacco plants were treated with3-indoleacetic acid (IAA) and then inoculated withB. cinerea. Notably, auxin pretreatment of PG16 leafdiscs restored their susceptibility to a level comparableto that of wild-type plants, whereas it did not signif-icantly increase the susceptibility of wild-type leafdiscs (Fig. 9A). Interestingly, whereas pretreatment ofwild-type leaf discs with exogenous OGs reducedlesion development after B. cinerea inoculation, co-treatment with OGs and IAA did not produce anyeffect, and the susceptibility was comparable to that ofuntreated tissues (Fig. 9B).

We also evaluated the sensitivity to auxin of tobaccoPG plants by analyzing the auxin-induced root forma-tion in leaf explants. While a concentration of 0.57 mM

IAA was sufficient to induce rhizogenesis in wild-typeexplants, no roots were observed in PG explants

treated with up to 1.7 mM IAA; however, at higherconcentrations, IAA induced root formation in PGexplants in a dose-dependent fashion (Fig. 10A). Theability of PG plants to respond to auxin was also testedusing a root growth inhibition assay. Wild-type plant-lets showed a significant reduction of primary rootgrowth in the presence of 1027

M IAA, whereas con-centrations of IAA up to 1026

M were not effective inPG16 plants (Fig. 10B). However, when auxin concen-tration was increased to 1025

M, a reduction of rootlength was observed in PG16 plants, consistent withthe hypothesis that they are not completely resistantbut only less sensitive to auxin. Taken together, theseresults indicate that auxin-induced developmentaland growth responses are impaired in PG plants.This is not due to increased catabolism of IAA becauseno significant IAA oxidase activity could be detectedin extracts from PG leaves (data not shown). We

Figure 2. Resistance of tobacco PG plants to bacterial infection.Disease symptoms of (from top to bottom and from left to right)tobacco untransformed plants (WT) and transgenic PG5, PG7, PG16,PG163PGIP2, and PGIP2 plants injected with a virulent strain of P.syringae pv tabaci. Inoculated leaves of the indicated genotypes werephotographed 5 d after inoculation. This experiment was repeatedtwice with similar results.

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therefore conclude that auxin signaling is partly com-promised in PG plants.

Because high concentrations of IAA induce ethyleneproduction in several plant species (Abeles, 1966) andethylene-controlled responses play a role in both re-sistance against B. cinerea (Thomma et al., 1999; Diazet al., 2002; Chague et al., 2006) and inhibition of rootdevelopment (Ortega-Martinez et al., 2007; Stepanovaet al., 2007), we investigated whether PG plants showincreased ability to produce ethylene. Ethylene pro-duced by wild-type and PG16 leaf explants was mea-sured after water or IAA treatment. Water-treatedwild-type or PG16 leaf explants did not release anydetectable accumulation of ethylene; in contrast,1-aminocyclopropane-1-carboxylic acid (ACC) inducedhigh levels of ethylene in both genotypes, indicatingthat they have a similar ability of producing this hor-mone (Fig. 11). Furthermore, treatment with 100 mM

IAA resulted in the accumulation of lower, but signif-icant and comparable, levels of ethylene in wild-typeand PG16 explants (Fig. 11). OGs had no significantimpact on ethylene production in water- or IAA-treatedwild-type tobacco explants (data not shown). A role ofethylene in the basal resistance, in the IAA-mediatedreversion of the resistant phenotype, and in the reducedability to form adventitious roots in response to IAA oftobacco PG plants is therefore unlikely.

DISCUSSION

In this article, we have shown that the expressionof a fungal PG in Arabidopsis and tobacco increasesplant resistance to microbial pathogens. The resistantphenotype is not exhibited by transgenic tobaccoplants expressing both PG and its inhibitor PvPGIP2or by Arabidopsis plants expressing a mutagenizedand inactive AnPGII (PG201), indicating that resis-tance is dependent on the enzymatic activity of the

expressed PG and is likely a consequence of the deg-radation of the host pectin accomplished by the en-zyme. Interestingly, PG plants appear to have reducedsensitivity to exogenous auxin, and auxin treatmentsrevert their resistant phenotype. The data presentedhere allow us to consider the reasons that might ex-plain why heterologous expression of a fungal PGincreases plant resistance, providing a link betweenHGA degradation, auxin perception, and activation ofdefense responses.

PG plants have an altered pectin composition with areduced GalUA and HGA content (Capodicasa et al.,2004). A direct negative effect of the lack of consum-able GalUA on pathogen growth appears unlikely,because we did not observe any significant reductionof growth when B. cinerea was cultivated in the pres-ence of cell walls from PG plants as the sole carbonsource (data not shown). On the other hand, PG plantsconstitutively express a number of defense responses,such as the accumulation of UV-fluorescent metabo-lites, H2O2, b-1,3-glucanase, and peroxidase, and ex-pression of defense-related genes that are normallyinduced only in the presence of pathogens. High levelsof H2O2 in PG plants are concomitant with an in-creased peroxidase activity, which, at least in tobacco,corresponds to an anionic and apoplastic enzyme pos-sibly encoded by the constitutively expressed POXgene (see Fig. 6A). In the presence of H2O2, peroxidasesmay generate free radicals that have direct antimicro-bial activity (Bolwell et al., 2002) or may modify theplant cell wall (Passardi et al., 2004). On the other hand,H2O2 itself may be responsible for the induction ofPR proteins (Apostol et al., 1989; Chen et al., 1993).Whether the high peroxidase activity and elevatedH2O2 levels in PG plants directly contribute to theirincreased resistance to pathogens still needs to be de-termined. Besides causing the constitutive activationof defense responses, PG expression makes plantsmore prone to respond to infection. Following B. cinerea

Figure 3. Accumulation of H2O2 in PG plants. A,DAB staining of detached leaves from (from left toright) tobacco untransformed (WT) and transgenicPG5, PG7, PG16, PG3PGIP2, and PGIP2 plants. B,DAB staining of detached leaves from (from left toright) Arabidopsis untransformed (WT) and trans-genic PG201, PG5, and PG1 plants.





inoculation, tobacco PG plants accumulate more POXand EAS1/2 transcripts than wild-type plants, and Arabi-dopsis plants show enhanced expression of AtPGIP1.

The enhanced expression of diverse defense re-sponses is likely the ultimate reason for their increasedresistance to pathogens. We could not observe a per-fect correlation between the levels of a specific defenseresponse and the degree of resistance observed in

independent PG lines, suggesting that multiple defenseresponses, activated to different extents in different PGlines, contribute to the final resistant phenotype. Theobservation that tobacco PG163PGIP2 plants or Arabi-dopsis PG201 plants, which have no PG activity, dis-play neither altered expression of defense responsesnor increased resistance indicates that these pheno-types are mediated by HGA degradation in PG plantsrather than by the recognition per se of the heterologousprotein by plant cells. Enhanced expression of basaland inducible defense responses and increased resis-tance to pathogens were previously observed in plantsexpressing pectin-degrading enzymes. Basal levels ofpolyphenoloxidase activity and induction of Phe am-monia lyase upon wounding are enhanced in trans-genic potato tubers expressing a bacterial pectate lyase(Wegener, 2002), and accumulation of b-1,3-glucanaseand H2O2 is induced in tobacco leaves by the transientexpression of a PG from Colletotrichum lindemuthianum(Boudart et al., 2003). Moreover, H2O2 accumulatesin tomato plants that have increased PG activity uponantisense expression of the regulatory subunit of a

Figure 5. Glucanase activity in PG plants. A, Levels of b-1,3-glucanaseactivity, expressed as enzymatic units per milligram of total proteins, inleaves of tobacco untransformed (WT) and transgenic PG5, PG7, PG16,PG3PGIP2, and PGIP2 plants. Bars indicate average activity of threeindependent samples 6 SE. B, Levels of b-1,3-glucanase activity,expressed as enzymatic units per milligram of total proteins, in leavesof Arabidopsis untransformed (WT) and transgenic PG201, PG5, andPG1 plants. Bars indicate the average activity of three independentsamples 6 SE. Different letters represent data sets significantly different,according to ANOVA analysis followed by Tukey’s test (P , 0.01).

Figure 4. Peroxidase activity in PG plants. A, Peroxidase activity intotal protein extracts from leaves of tobacco untransformed (WT) andtransgenic PG5, PG7, PG16, PGIP2, and PG3PGIP2 plants. B, Perox-idase activity in IWFs (black bars) and intracellular proteins extractedafter recovery of IWFs (white bars) from the same tobacco lines shownin A. C, Peroxidase activity in total protein extracts from leaves ofArabidopsis untransformed (WT) and transgenic PG201, PG5, and PG1plants. Bars indicate the average activity, expressed as enzymatic unitsper milligram of total proteins, of three samples 6 SE. Different lettersrepresent data sets significantly different, according to ANOVA analysisfollowed by Tukey’s test (P , 0.01).

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wound-inducible endogenous PG (PGbS); these plantsexhibit constitutive expression of wound-induciblegenes and enhanced resistance to insect attack (Orozco-Cardenas and Ryan, 2003).

One explanation for the observed phenotypes in thePG plants is that altered cell wall integrity may beperceived as a signal of pathogen attack or mechanicaldamage, leading to the induction of defense mecha-nisms (Humphrey et al., 2007). Links between pectin andcytoplasm, mediated by specific cell wall-associatedtransmembrane proteins, may allow the host cell tosense alterations caused by microbial pathogens andregulate the activation of defense responses. The obser-vation that the Arabidopsis wall-associated kinaseWAK1 (Decreux and Messiaen, 2005), which is able tobind polygalacturonic acid, is induced by bacterial in-fection and that this induction is required for survivalupon treatments with chemical inducers of resistancesupports this hypothesis (He et al., 1998). Apoplasticproteins that have domains interacting with pectin alsoinclude a peroxidase (Carpin et al., 2001) and PGIP(Spadoni et al., 2006); these proteins may participateto perceive alterations of pectin structure and transmitthis information across the plasma membrane withmech-anisms not yet investigated.

Indeed, increased disease resistance is observed inplants with alterations of cell wall structural compo-nents other than pectin. For example, mutations inan Arabidopsis cellulose synthase (CESA3) cause notonly decreased cellulose content but also constitutiveexpression of defense responses (Ellis et al., 2002;Cano-Delgado et al., 2003). More recently, it has beenreported that mutations in Arabidopsis cellulosesynthase genes confer resistance to different patho-gens through a mechanism that is independent ofsalicylic acid (SA), ethylene, and jasmonic acid signal-ing (Hernandez-Blanco et al., 2007). Defects in thecuticle cause complete resistance to B. cinerea, and thisresistance is also independent of SA, ethylene, andjasmonic acid (Chassot et al., 2007). It is therefore likelythat plants have evolved mechanisms to perceive thepresence of a potential pathogen by monitoring theintegrity of different components of their own cellwall. However, a direct mechanistic link betweenmodifications of these cell wall components and acti-vation of defense responses is still missing.

Another possible explanation for the enhanced re-sistance of plants with altered cell wall components isthat plant cells perceive molecules released in theapoplast as a consequence of the hydrolysis of specificstructural polymers and these molecules act as elicitors

Figure 6. Expression of defense genes in PG plants after infection withB. cinerea. A, Fully expanded leaves from tobacco untransformed (WT)and transgenic PG16 (PG) plants were inoculated with B. cinerea andtotal RNA was extracted at the indicated time points (dpi). The RNA gelblot was hybridized with the indicated probes. Equal loading wasverified by methylene blue staining of ribosomal RNA. B to D, Fullyexpanded leaves from Arabidopsis untransformed (WT, white bars) or

transgenic PG1 (PG, black bars) plants were inoculated with B. cinerea,and total RNA was extracted at the indicated time points (dpi). Theexpression of AtPGIP1 (B), PR-1 (C), and PDF1.2 (D) was analyzed byreal-time RT-PCR and normalized using the expression of UBQ5 ineach sample. The insets in C and D show gene expression in untreatedleaves (n.d., not detectable). Bars represent the average expression 6 SD

of two replicates.





of defense responses. In particular, OGs generatedupon degradation of HGA by fungal PGs act as elicitorsof defense responses in several plant systems (forreview, see Ridley et al., 2001). It is possible that inplanta expression of PG confers resistance to pathogensbecause of the release of OGs or other wall-derivedelicitors. Circumstantial evidence supporting this hy-pothesis is presented here. First, PG plants show en-hanced expression of defense responses similar to thoseobserved in wild-type plants after addition of exoge-nous OGs. Reactive oxygen species accumulation is ahallmark of OG-mediated responses (Ridley et al., 2001;Aziz et al., 2004), and we detected a dramatic increaseof H2O2 levels in both tobacco and Arabidopsis PGplants. Furthermore, we could demonstrate that ex-pression of EAS1/2 and POX is induced by both OGsand B. cinerea in tobacco and that their expression isincreased in PG plants. In Arabidopsis PG plants, highbasal levels of three defense-related genes, AtPGIP1,PR-1, and PDF1.2, are also enhanced compared to thewild type. Expression of AtPGIP1 was previously dem-onstrated to be responsive to OGs (Ferrari et al., 2003b),and PR-1, a marker of SA-mediated defense responses(Delaney et al., 1994), is also up-regulated by OGs in the

Ws ecotype, though at later time points than AtPGIP1.PDF1.2, which is not significantly regulated by OGs butis responsive to oxidative stress (Penninckx et al., 1996),may be high in PG plants because of the high levels ofH2O2 in their tissues. The reduced mRNA levels of bothPR-1 and PDF1.2 in inoculated PG plants reflect thefact that their expression correlates to fungal growth(reduced in PG plants) rather than to plant resistance(Ferrari et al., 2003a; Chassot et al., 2007). The observa-tion that PG plants accumulate higher levels of mRNAcorresponding to genes that are responsive to OGssuggests that HGA alterations cause the activation ofa defense-related pathway that is also activated by

Figure 8. Regulation of defense gene expression by OGs in Arabidopsisplants. A to C, Real-time RT-PCR analysis of the expression of AtPGIP1(A), PR-1 (B), and PDF1.2 (C) in untransformed Arabidopsis seedlingstreated for the indicated times with OGs. The expression of each genewas normalized using the expression of UBQ5 in each sample. Barsrepresent the average gene expression 6 SD of two replicates.

Figure 7. Regulation of defense gene expression by OGs in tobaccoplants. A and B, Real-time RT-PCR analysis of the expression of EAS1/2(A) and POX (B) in untransformed tobacco leaf explants treated for theindicated times with water (control, white bars) or OGs (OG, blackbars). The expression of each gene was normalized using the expressionof the actin gene Tob66 in each sample. Bars represent the averagegene expression 6 SD of two replicates.

Ferrari et al.




elicitors; this activation may be either directly mediatedby pectin fragments or by other mechanisms. It shouldbe noted that the levels of defense-related transcriptsaccumulating upon infection are dramatically higherthan those observed in either OG-treated wild-typeplants or in untreated PG plants. This may be due tothe concomitant activation of multiple defense-relatedpathways during pathogen infection and/or the mas-sive release of different elicitors in infected tissues.

A second line of evidence suggesting that pecticfragments with elicitor activity accumulate in PG plantsis that auxin reverts the susceptibility to B. cinerea oftobacco PG plants to wild-type levels and also sup-presses OG-induced protection against this fungus inwild-type plants. Furthermore, auxin-dependent re-sponses are impaired in tobacco PG plants, and ourprevious work has shown that OGs suppress differentresponses regulated by auxin in tobacco (Bellincampi

et al., 1993, 1996; Mauro et al., 2002). In particular,tobacco PG plants show reduced sensitivity to IAA inexperiments of rhizogenesis in leaf explants and inhi-bition of primary root growth. This reduced sensitivitydoes not appear to depend on IAA degradation, assuggested by the absence of IAA oxidase activity in PGplants.

We also show that tobacco PG plants do not differfrom wild-type plants in their ability to produceethylene, and, on the other hand, OGs do not inducesignificant production of ethylene in wild-type plants.This suggests that a role of this hormone in the basalresistance, as well as in the IAA-mediated reversion ofthe resistant phenotype and in the reduced ability toform adventitious roots in response to IAA, is unlikely.

Figure 9. Susceptibility to B. cinerea in tobacco PG plants treated withauxin. A, Leaf discs from tobacco untransformed (WT, white bars) ortransgenic PG16 (PG, black bars) plants were incubated for 3 h in liquidmedium containing water (control) or 100 mM IAA (IAA) and inoculatedwith B. cinerea. Lesion area was measured after 24 h. Bars indicateaverage area 6 SE of at least 10 lesions. Asterisks indicate statisticallysignificant difference between lesions in wild-type and transgenicplants (***, P , 0.01). This experiment was repeated three times withsimilar results. B, Leaf discs from untransformed tobacco plants wereincubated for 3 h in liquid medium containing water (control), 100 mM

IAA (IAA), 200 mg mL21 OGs (OG), or both (OG 1 IAA) and inoculatedwith B. cinerea. Lesion area was measured after 24 h. Bars indicate theaverage area 6 SE of at least 10 lesions. Different letters indicate datasets significantly different, according to ANOVA followed by Tukey’stest (P , 0.05). This experiment was repeated twice with similar results.

Figure 10. Auxin sensitivity of tobacco PG plants. A, Leaf explants fromtobacco untransformed (WT, white squares) and transgenic PG16 (PG,black squares) plants were treated with the indicated concentrations ofIAA for 15 d and the number of explants forming roots was measured.Each data point represents the average percentage of explants formingroots 6 SD calculated in three independent experiments (n . 8 in eachexperiment). B, Length of primary roots of tobacco untransformed (WT)and transgenic PG16 (PG) plants grown for 12 d on solid mediumcontaining the indicated IAA concentrations. Bars represent the aver-age length of at least eight plants 6 SE. Asterisks indicate statisticallysignificant difference between treated and control samples, accordingto the Student’s t test (P , 0.01). This experiment was repeated twicewith similar results.





Other mechanisms, such as an alteration of the bio-mechanical properties of the cell wall, may be respon-sible for the observed responses of the PG plants.However, no obvious correlation between auxin-induced wall loosening and auxin-mediated reversionof the resistant phenotype of PG explants could beobserved. Indeed, IAA did not increase susceptibilityin wild-type explants, which showed curling in re-sponse to auxin, whereas it did so in PG explants thatdid not show any curling (data not shown). Therefore,it may be argued that the loosening of the wall thatleads to curling in wild-type explants has no apparenteffect on the susceptibility to B. cinerea, while theincreased susceptibility of PG explants in the presenceof auxin occurs in the absence of significant wallloosening/curling. Furthermore, because expansionof discs from PG plants in water is similar or evenhigher than that of discs from wild-type plants(R. Galletti and D. Pontiggia, unpublished data), analteration of biomechanical properties (for example,more rigid cell walls) that can be compensated bytreatment with auxin is probably not the reason for theresistant phenotype and the reduced ability to formadventitious roots. On the other hand, the ability ofauxin to revert the altered responses of PG plants alsosuggests that irreversible wall modifications such aslignification or cross-links are unlikely to be involved.

Auxin and elicitors can have opposite effects ondefense gene expression. For instance, IAA inhibitsinduction of PR proteins in tobacco protoplasts treatedwith elicitors from the cell wall of the oomycetePhytophthora megasperma f. sp. glycinea (Jouanneauet al., 1991) and inhibits the expression of the wound-inducible pinII gene (Kernan and Thornburg, 1989).IAA secreted by Pseudomonas savastanoi has the abilityto suppress the hypersensitive response (Robinetteand Matthysse, 1990), whereas the expression of thepathogen-induced gene CEV-1 correlates with a defect

of perception of auxin (Mayda et al., 2000). Importantly,it has been reported that the flagellin-derived peptideflg22 represses auxin signaling through the induction ofa miRNA directed against the auxin receptors TIR1,AFB2, and AFB3, and this leads to an increased resis-tance to bacterial infection (Navarro et al., 2006). It ispossible that release of the auxin-mediated inhibition ofdefense gene expression is a crucial step in the trans-duction pathway activated by general elicitors, includ-ing OGs. According to this model, OGs, by negativelyregulating auxin signaling, allow for increased expres-sion of defense responses. However, our observationsimply that, if the phenotype of the PG plants is not dueto accumulation of OGs but to the perception of adefective wall structure, this second mechanism alsoinvolves a cross-talk with auxin. Perception of cell wall-derived fragments and of alterations of cell wall bio-mechanical properties may all converge in a commonsignaling pathway (Humphrey et al., 2007) that featuresa negative cross-talk with the auxin response pathway.

In conclusion, we have shown that the expression ofa fungal PG and the subsequent degradation of HGAincreases resistance of plants to microbial pathogens,likely through a pre-activation of plant defense re-sponses, and that auxin reverts the enhanced resis-tance to fungal infection. Our data also indicate that inmuro pectin degradation by PG leads to decreasedauxin sensitivity. This is reminiscent of the effects ob-served in untransformed plants treated with OGs,suggesting a possible role of these pectic fragments inthe resistant phenotypes of PG plants. Alternatively,an altered cell wall architecture may be directly per-ceived by the plant cell as a signal of the presence of apathogen, leading to an increased activation of defenseresponses. The molecular mechanisms linking pectindegradation, auxin signaling, and activation of de-fense responses still remain to be investigated. Webelieve that this line of research will provide newinsights in the regulation of plant defense responsesduring plant-pathogen interactions.

MATERIALS AND METHODS

Transgenic Lines, Plant Growth, and Treatments

Generation of transgenic Arabidopsis (Arabidopsis thaliana) Ws and tobacco

(Nicotiana tabacum) Petit Havana-SR1 plants expressing PG or PvPGIP2 and

their cross were described previously (Capodicasa et al., 2004). The PG

expressed in these plants had a point mutation (N178D) that has an estimated

activity approximately 20-fold lower than the native Aspergillus niger PGII

(data not shown). The expression of a PG with reduced enzymatic activity

allowed us to generate viable plants. Arabidopsis line PG201 expresses an

inactive version of A. niger PGII with a point mutation in the catalytic site

(D201N) that causes complete loss of enzymatic activity (van Santen et al.,

1999; Federici et al., 2001). These plants express a level of the inactive enzyme

comparable to that observed in line PG5, as estimated by immunoblot analysis

(data not shown), and show no obvious developmental defects (Capodicasa

et al., 2004). Arabidopsis plants were grown in growth chambers at 22�C and

70% relative humidity, with a 12-h photoperiod (100 mmol m22 s21 of fluo-

rescent light). Tobacco plants were grown in a greenhouse at 23�C and 60%

relative humidity, with a 16-h photoperiod (130 mmol m22 s21).

For elicitor treatments on Arabidopsis seedlings, seeds were surface-

sterilized and germinated in multiwell plates (approximately 10 seeds per

Figure 11. Ethylene production in tobacco PG leaf explants. Leafexplants from tobacco untransformed (WT, white bars) and transgenicPG16 (PG, black bars) plants were incubated with water, 250 mM ACC,or 100 mM IAA (IAA) in sealed flasks. Ethylene accumulating in the flaskwas determined by gas chromatography. Bars indicate ethylene con-centration after 24 h of treatment. No detectable accumulation ofethylene could be observed in control samples. This experiment wasrepeated twice with similar results.

Ferrari et al.




well) containing 1 mL per well of 13 Murashige and Skoog (MS) medium

(Sigma; Murashige and Skoog, 1962) supplemented with 0.5% Suc. Plates were

incubated at 22�C with a 16-h photoperiod and a light intensity of 100 mmol

m22 s21. After 8 d, the medium was replaced, and after two additional days

treatments with 100 mg L21 OGs or water were performed (Ferrari et al.,

2003b). Tobacco leaf explants (5 3 10 mm) were obtained from apical leaves of

4-week-old SR1 plants, washed six times for 30 min with 0.253 MS medium

containing 2% Suc, and incubated in the same medium supplemented with

10 mg mL21 OGs. The OGs used in this work were a pool with average degree

of polymerization of 10 to 15, prepared as described previously (Bellincampi

et al., 2000).

Ethylene Measurements

Approximately 20 to 30 tobacco leaf explants (approximately 500 mg fresh

weight) were prepared from 7-week-old tobacco leaves, avoiding the midrib,

and extensively washed with sterile distilled water. The explants were placed

in sealed 500-mL flasks containing 100 mL of sterile water alone or supple-

mented with 250 mM ACC, 100 mM IAA sodium salt, or 200 mg L21 OGs.

Gas samples were withdrawn from the flasks after 24 h and analyzed with a

gas chromatographer equipped with a flame ionization detection system

(Carlo Erba). Chromatographic separations were carried out on a Porapak Q

80-100 mesh column (4-mm i.d., 1.5 mL). The flow rate of the carrier gas (N2;

0.80 kg cm22) was 40 mL min21. Column, injector, and detector temperatures

were 30�C, 50�C, and 120�C, respectively. Heating rate was 18�C min21 to

170�C. Air flow was 1.60 kg cm22 and hydrogen flow was 0.70 kg cm22.

Ethylene concentration in each sample was normalized using dry weight of

the leaf explants.

Pathogen Growth and Infection

Botrytis cinerea (a kind gift of J. Plotnikova, Massachusetts General Hos-

pital) was grown for 7 to 10 d at 22�C under constant light on 20 g L21 malt

extract, 10 g L21 mycological peptone, and 15 g L21 agar until sporulation.

Conidia were collected with 10 mL of sterile water containing 0.05% Tween 20,

filtered with sterile glass wool, and centrifuged for 5 min at 5,000g. Before

plant inoculation, fresh spores were resuspended in 24 g L21 potato dextrose

broth and incubated for 3 h at room temperature to allow uniform germina-

tion. Inoculation of detached Arabidopsis leaves was performed as described

previously (Ferrari et al., 2003b). Fully developed leaves of 8-week-old tobacco

plants were detached and placed in large petri dishes, with the petioles

embedded in 0.5% agar, and inoculated with a spore suspension in 24 g L21

potato dextrose broth (106 spores mL21). Six droplets of spore suspension

(10 mL each) were placed on the adaxial surface of each leaf. Plates containing

the inoculated leaves were wrapped with a transparent plastic film and

incubated in a growth chamber at 22�C under fluorescent light for a 16-h

photoperiod. Disease progress was scored as described by ten Have et al.

(1998). Results were analyzed by one-way ANOVA followed by Tukey’s

Student range test. For leaf disc infections, discs (10-mm diameter) were cut

from surface-sterilized fully expanded tobacco leaves and placed floating in

12-well plates containing MS medium supplemented with 0.5% Suc and

water, 100 mM IAA, or 200 mg L21 OGs (1 mL per well; final ethanol con-

centration in all samples was 0.01%). After 3 h, the discs were drop inoculated

with a B. cinerea spore suspension (105 spores mL21) and incubated in a growth

chamber as above indicated. Lesion area was scored 24 h after inoculation.

Ethanol alone had no significant effect on lesion size (data not shown).

Pseudomonas syringae pv tabaci DC3000 was cultured in King’s B broth at

28�C for 2 d, and a bacterial suspension was prepared in 10 mM MgCl2 (5 3 104

colony-forming units cm22). Challenge inoculation was performed by infil-

tration of the bacterial suspension using a 1-mL syringe without a needle.

Determination of H2O2 Content; Glucanase, Peroxidase,

and IAA Oxidase Activity; and Visualization ofUV-Fluorescent Compounds

For H2O2 visualization, leaves were cut from adult plants using a razor

blade and dipped for 12 h in a solution containing 1 mg mL21 DAB, pH 5.0.

Chlorophyll was extracted for 10 min with boiling ethanol and for 2 h with

ethanol at room temperature prior to photography (Orozco-Cardenas and

Ryan, 1999).

For total protein extraction, frozen leaves or stems were homogenized in

1 M NaCl, 20 mM sodium acetate, pH 4.7, incubated under gentle shaking for

1 h, and centrifuged for 20 min at 10,000g. IWFs were prepared from tobacco

stems using 0.3 M NaCl and 20 mM sodium acetate, pH 4.7, buffer, as described

previously (Terry and Bonner, 1980). After IWF extraction, stem sections were

homogenized with 1 M NaCl, 20 mM sodium acetate, pH 4.7, as described

above for total protein extraction, to obtain intracellular proteins. Total protein

concentration was determined by the Bradford method (Bradford, 1976).

Activity of b-1,3-glucanase was determined by incubating 50 mg and

150 mg of tobacco or Arabidopsis total proteins, respectively, at 37�C with 2 mg

mL21 laminarin (Sigma) in 50 mM sodium acetate, pH 5.2. The produced

reducing sugars were determined colorimetrically with dinitrosalicylic acid

(Sigma; Miller, 1959). Peroxidase activity was measured using a modified

version of the method described in Smith and Barker, 1988. Enzymatic activity

was determined by the change in A515 due to the oxidation of aminoantipyrine

and 3,5-dicloro-2-idroxibenzensulphonic acid. IAA oxidase activity was de-

termined as described in Pressey, 1990.

The presence of UV-fluorescent compounds was visualized by photo-

graphing detached leaves under UV light (l 5 320 nm) using the ‘‘Image-

Master’’ VDS (Pharmacia Biotech).

RNA Gel-Blot Analysis

Tobacco leaf discs of 5-mm radius around the site of inoculation were

frozen in liquid nitrogen, homogenized, and total RNA was extracted with

Tri-reagent (Sigma). RNA was separated on agarose-formaldehyde gel and

transferred to a nylon membrane as described previously (Ferrari et al., 2003b).

Equal RNA loading and transfer were verified by staining the membrane with a

solution containing 0.03% methylene blue (w/v) and 0.3 M sodium acetate, pH

4.7. Prehybridization was performed for 6 h at 42�C in 50% formamide, 63

sodium chloride/sodium phosphate/EDTA, 13 Denhardt’s solution, 0.1%

SDS, and 250 mg mL21 denatured herring sperm DNA. Filters were hybridized

overnight at 42�C using the following solution: 50% formamide, 63 sodium

chloride/sodium phosphate/EDTA, 13 Denhardt’s solution, 0.1% SDS, 5%

dextran sulfate, 100 mg mL21 denatured herring sperm DNA, and 100 ng of

denatured probe. 32P-labeled probes were prepared by PCR; as templates for

the tobacco probes, two DNA fragments, corresponding respectively to a region

of a tobacco POX gene (GeneBank accession no. L02124; Diaz-De-Leon et al.,

1993) conserved also in different anionic peroxidase genes, and to the EAS1 and

EAS2 genes (Facchini and Chappell, 1992), were amplified from cDNA of

infected tobacco leaves, using the following primers: 5#-TAATGTAGGTGG-

CAGGAGG-3# (EAS1/2 forward), 5#-CTAGGAATATCACTATTAGC-3# (EAS1/

2 reverse), 5#-ACATCGTATTTGGGCATG-3# (POX forward), and 5#-GTTGC-

AATTTGTCCCCT-3# (POX reverse). Probes were purified using ProbeQuant

G-50 microcolumns (Amersham Pharmacia Biotech). Hybridized blots were

washed once with 23 SSC, 0.1% SDS at room temperature and twice with 0.13

SSC, 0.1% SDS at 65�C. Images were taken after overnight exposure using a

phosphor imager (Typhoon 9200; Amersham).

Real-Time Reverse Transcription-PCR Analysis

Total RNA was extracted with Tri-reagent (Sigma) and treated with Turbo-

DNase I (Ambion). First-strand cDNA was synthesized using ImProm-II

Reverse Transcriptase (Promega). Real-time PCR analysis was performed

using an Iq-Cycler (Bio-Rad) according to the manufacturer’s guide. A total of

2 mL of cDNA (corresponding to 120 ng of total RNA) was amplified in 30 mL

of reaction mix containing IQ SYBR Green Supermix (Bio-Rad) and 0.4 mM

each primer. Primer sequences for POX and EAS1/2 are described above. The

tobacco actin gene (Tob66, accession no. U60491) was amplified using the

following primers: 5#-CTGCCATGTATGTTGCTATT-3# and 5#-AGTCTCC-

AACTCTTGCTCAT-3#. The primers for PDF1.2, PR-1, AtPGIP1, and UBQ5

were described previously (Penninckx et al., 1996; Rogers and Ausubel, 1997;

Ferrari et al., 2006). The primers utilized for the amplification of the B. cinerea

Bcact actin gene (accession no. AJ000335) were the following: 5#-AAGTG-

TGATGTTGATGTCC-3# and 5#-CTGTTGGAAAGTAGACAAAG-3#. Relative

expression of the reverse transcription (RT)-PCR products was determined

using a modified version of the Pfaffl method (Pfaffl, 2001; Ferrari et al., 2006).

Auxin Responses in Tobacco Leaf Explants and Seedlings

For rhizogenesis of leaf explants, the second apical leaves from tobacco

plants grown for 4 weeks on soil were harvested and surface sterilized. Ten

explants of about 0.4 3 0.8 cm were excised in correspondence with the midrib

vein and placed, abaxial side down, in petri dishes containing 10 mL of sterile





MS medium, pH 5.7, supplemented with 2% Suc, 0.8% plant agar, and IAA at

the indicated concentrations. Leaf explants were incubated at 25�C under low

intensity light (60 mE m22 s22) for 15 d. For seedling assays, seeds were

sterilized and germinated in 0.53 MS liquid medium supplemented with 2%

Suc, pH 5.8. After 1 week, seedlings were transferred to full-strength MS

medium, pH 5.7, supplemented with 2% Suc, 1.5% plant agar, and IAA.

Primary root length was measured after 12 d of growth at 22�C with a

photoperiod of 16 h.

Sequence data from this article can be found in the GenBank/EMBL data

libraries under accession number L02124.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Expression of the B. cinerea Bcact gene in PG

plants after infection.

Supplemental Figure S2. Accumulation of UV-fluorescent compounds in

PG plants.

ACKNOWLEDGMENTS

We thank Gianni Salvi for the preparation of OGs and Lucia Tufano and

Lorenzo Mariotti for assistance. We are grateful to Francesco Loreto and

Francesco Brilli (Istituto di Biologia Agro-ambientale e Forestale, Consiglio

Nazionale delle Ricerche) for their help in the analysis of physiological

parameters and in the gas chromatography experiments.

Received September 24, 2007; accepted November 28, 2007; published De-

cember 7, 2007.

LITERATURE CITED

Abeles FB (1966) Auxin stimulation of ethylene evolution. Plant Physiol 41:

585–588

Altamura MM, Zaghi D, Salvi G, De Lorenzo G, Bellincampi D (1998)

Oligogalacturonides stimulate pericycle cell wall thickening and cell

divisions leading to stoma formation in tobacco leaf explants. Planta

204: 429–436

Apostol I, Heinstein PF, Low PS (1989) Rapid stimulation of an oxidative

burst during elicitation of cultured plant cells. Plant Physiol 90: 109–116

Aziz A, Heyraud A, Lambert B (2004) Oligogalacturonide signal trans-

duction, induction of defense-related responses and protection of

grapevine against Botrytis cinerea. Planta 218: 767–774

Bellincampi D, Cardarelli M, Zaghi D, Serino G, Salvi G, Gatz C,

Cervone F, Altamura MM, Costantino P, De Lorenzo G (1996) Oligo-

galacturonides prevent rhizogenesis in rolB-transformed tobacco ex-

plants by inhibiting auxin-induced expression of the rolB gene. Plant

Cell 8: 477–487

Bellincampi D, Dipierro N, Salvi G, Cervone F, De Lorenzo G (2000)

Extracellular H2O2 induced by oligogalacturonides is not involved in

the inhibition of the auxin-regulated rolB gene expression in tobacco leaf

explants. Plant Physiol 122: 1379–1385

Bellincampi D, Salvi G, De Lorenzo G, Cervone F, Marfa V, Eberhard S,

Darvill A, Albersheim P (1993) Oligogalacturonides inhibit the forma-

tion of roots on tobacco explants. Plant J 4: 207–213

Bolwell GP, Bindschedler LV, Blee KA, Butt VS, Davies DR, Gardner SL,

Gerrish C, Minibayeva F (2002) The apoplastic oxidative burst in

response to biotic stress in plants: a three-component system. J Exp Bot

53: 1367–1376

Boudart G, Charpentier M, Lafitte C, Martinez Y, Jauneau A, Gaulin E,

Esquerre-Tugaye MT, Dumas B (2003) Elicitor activity of a fungal

endopolygalacturonase in tobacco requires a functional catalytic site

and cell wall localization. Plant Physiol 131: 93–101

Bradford MM (1976) A rapid and sensitive method for quantification of

microgram quantities of protein utilizing the principle of protein-dye

binding. Anal Biochem 72: 248–254

Branca C, De Lorenzo G, Cervone F (1988) Competitive inhibition of the

auxin-induced elongation by a-D-oligogalacturonides in pea stem seg-

ments. Physiol Plant 72: 499–504

Broekaert WF, Pneumas WJ (1988) Pectic polysaccharides elicit chitinase

accumulation in tobacco. Physiol Plant 74: 740–744

Cano-Delgado A, Penfield S, Smith C, Catley M, Bevan M (2003) Reduced

cellulose synthesis invokes lignification and defense responses in

Arabidopsis thaliana. Plant J 34: 351–362

Capodicasa C, Vairo D, Zabotina O, McCartney L, Caprari C, Mattei B,

Manfredini C, Aracri B, Benen J, Knox JP, et al (2004) Targeted

modification of homogalacturonan by transgenic expression of a fungal

polygalacturonase alters plant growth. Plant Physiol 135: 1294–1304

Carpin S, Crevecoeur M, de Meyer M, Simon P, Greppin H, Penel C (2001)

Identification of a Ca(21)-pectate binding site on an apoplastic perox-

idase. Plant Cell 13: 511–520

Carpita NC, McCann MC (2000) The cell wall. In BB Buchanan, W

Gruissem, R Jones, eds, Biochemistry and Molecular Biology of Plants.

American Society Plant Physiologists, Rockville, MD, pp 52–108

Casasoli M, Spadoni S, Lilley KS, Cervone F, De Lorenzo G, Mattei B

(2008) Identification by 2D-DIGE of apoplastic proteins regulated by

oligogalacturonides in Arabidopsis thaliana. Proteomics (in press)

Cervone F, De Lorenzo G, Degra L, Salvi G (1987a) Elicitation of necrosis

in Vigna unguiculata Walp. by homogeneous Aspergillus niger endo-

polygalacturonase and by a-D-galacturonate oligomers. Plant Physiol

85: 626–630

Cervone F, De Lorenzo G, Degra L, Salvi G, Bergami M (1987b) Purifi-

cation and characterization of a polygalacturonase-inhibiting protein

from Phaseolus vulgaris L. Plant Physiol 85: 631–637

Cervone F, Hahn MG, De Lorenzo G, Darvill A, Albersheim P (1989)

Host-pathogen interactions XXXIII. A plant protein converts a fungal

pathogenesis factor into an elicitor of plant defence responses. Plant

Physiol 90: 542–548

Chague V, Danit LV, Siewers V, Schulze-Gronover C, Tudzynski P,

Tudzynski B, Sharon A (2006) Ethylene sensing and gene activation

in Botrytis cinerea: a missing link in ethylene regulation of fungus-plant

interactions? Mol Plant Microbe Interact 19: 33–42

Chassot C, Nawrath C, Metraux JP (2007) Cuticular defects lead to full

immunity to a major plant pathogen. Plant J 49: 972–980

Chen Z, Silva H, Klessig DF (1993) Active oxygen species in the induc-

tion of plant systemic acquired resistance by salicylic acid. Science 262:

1883–1886

Davis KR, Darvill A, Albersheim P, Dell A (1986) Host-pathogen inter-

actions XXIX. Oligogalacturonides released from sodium polypectate

by endopolygalacturonic acid lyase are elicitors of phytoalexins in

soybean. Plant Physiol 80: 568–577

Davis KR, Hahlbrock K (1987) Induction of defense responses in cultured

parsley cells by plant cell wall fragments. Plant Physiol 85: 1286–1290

Decreux A, Messiaen J (2005) Wall-associated kinase WAK1 interacts with

cell wall pectins in a calcium-induced conformation. Plant Cell Physiol

46: 268–278

Delaney TP, Uknes S, Vernooij B, Friederich L, Weymann K, Negrotto D,

Gaffney T, Gut-Rella M, Kessmann H, Ward E, et al (1994) A central

role of salicylic acid in plant disease resistance. Science 266: 1247–1250

Diaz J, ten Have A, van Kan JA (2002) The role of ethylene and wound

signaling in resistance of tomato to Botrytis cinerea. Plant Physiol 129:

1341–1351

Diaz-De-Leon F, Klotz KL, Lagrimini LM (1993) Nucleotide sequence of

the tobacco (Nicotiana tabacum) anionic peroxidase gene. Plant Physiol

101: 1117–1118

Ellis C, Karafyllidis I, Wasternack C, Turner JG (2002) The Arabidopsis

mutant cev1 links cell wall signaling to jasmonate and ethylene re-

sponses. Plant Cell 14: 1557–1566

Facchini PJ, Chappell J (1992) Gene family for an elicitor-induced sesqui-

terpene cyclase in tobacco. Proc Natl Acad Sci USA 89: 11088–11092

Federici L, Caprari C, Mattei B, Savino C, Di Matteo A, De Lorenzo G,

Cervone F, Tsernoglou D (2001) Structural requirements of endopoly-

galacturonase for the interaction with PGIP (polygalacturonase-inhibiting

protein). Proc Natl Acad Sci USA 98: 13425–13430

Ferrari S, Galletti R, Vairo D, Cervone F, De Lorenzo G (2006) Antisense

expression of the Arabidopsis thaliana AtPGIP1 gene reduces Polygalac-

turonase-Inhibiting Protein accumulation and enhances susceptibility

to Botrytis cinerea. Mol Plant Microbe Interact 19: 931–936

Ferrari S, Galletti R, Denoux C, De Lorenzo G, Ausubel FM, Dewdney J

(2007) Resistance to Botrytis cinerea induced in Arabidopsis by elicitors

is independent of salicylic acid, ethylene or jasmonate signaling but

requires PAD3. Plant Physiol 144: 367–379

Ferrari et al.




Ferrari S, Plotnikova JM, De Lorenzo G, Ausubel FM (2003a) Arabidopsis

local resistance to Botrytis cinerea involves salicylic acid and camalexin

and requires EDS4 and PAD2, but not SID2, EDS5 or PAD4. Plant J 35:

193–205

Ferrari S, Vairo D, Ausubel FM, Cervone F, De Lorenzo G (2003b)

Tandemly duplicated Arabidopsis genes that encode polygalacturonase-

inhibiting proteins are regulated coordinately by different signal transduc-

tion pathways in response to fungal infection. Plant Cell 15: 93–106

Flexas J, Medrano H (2002) Drought-inhibition of photosynthesis in C3

plants: stomatal and non-stomatal limitations revisited. Ann Bot (Lond)

89: 183–189

Hahn MG, Darvill AG, Albersheim P (1981) Host-pathogen interactions.

XIX. The endogenous elicitor, a fragment of plant cell wall polysaccha-

ride that elicits phytoalexin accumulation in soy beans. Plant Physiol 68:

1161–1169

He ZH, He D, Kohorn BD (1998) Requirement for the induced expression

of a cell wall associated receptor kinase for survival during the pathogen

response. Plant J 14: 55–63

Hernandez-Blanco C, Feng DX, Hu J, Sanchez-Vallet A, Deslandes L,

Llorente F, Berrocal-Lobo M, Keller H, Barlet X, Sanchez-Rodriguez C,

et al (2007) Impairment of cellulose synthases required for Arabidopsis

secondary cell wall formation enhances disease resistance. Plant Cell 19:

890–903

Humphrey TV, Bonetta DT, Goring DR (2007) Sentinels at the wall: cell

wall receptors and sensors. New Phytol 176: 7–21

Jouanneau JP, Lapous D, Guern J (1991) In plant protoplasts, the sponta-

neous expression of defense reactions and the responsiveness to exog-

enous elicitors are under auxin control. Plant Physiol 96: 459–466

Kernan A, Thornburg RW (1989) Auxin levels regulate the expression of a

wound-inducible proteinase inhibitor II-chloramphenicol acetyl trans-

ferase gene fusion in vitro and in vivo. Plant Physiol 91: 73–78

Leckie F, Mattei B, Capodicasa C, Hemmings A, Nuss L, Aracri B, De

Lorenzo G, Cervone F (1999) The specificity of polygalacturonase-

inhibiting protein (PGIP): a single amino acid substitution in the solvent-

exposed beta-strand/beta-turn region of the leucine-rich repeats (LRRs)

confers a new recognition capability. EMBO J 18: 2352–2363

Mauro ML, De Lorenzo G, Costantino P, Bellincampi D (2002) Oligo-

galacturonides inhibit the induction of late but not of early auxin-

responsive genes in tobacco. Planta 215: 494–501

Mayda E, Marques C, Conejero V, Vera P (2000) Expression of a pathogen-

induced gene can be mimicked by auxin insensitivity. Mol Plant

Microbe Interact 13: 23–31

Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of

reducing sugar. Anal Chem 31: 426–428

Murashige T, Skoog F (1962) Revised medium for rapid growth and

bioassays with tobacco cultures. Physiol Plant 15: 437–479

Nardini A, Gasco A, Cervone F, Salleo S (2007) Reduced content of

homogalacturonan does not alter the ion-mediated increase in xylem

hydraulic conductivity in tobacco. Plant Physiol 143: 1975–1981

Navarro L, Dunoyer P, Jay F, Arnold B, Dharmasiri N, Estelle M, Voinnet

O, Jones JD (2006) A plant miRNA contributes to antibacterial resis-

tance by repressing auxin signaling. Science 312: 436–439

Orozco-Cardenas M, Ryan CA (1999) Hydrogen peroxide is generated

systemically in plant leaves by wounding and systemin via the octa-

decanoid pathway. Proc Natl Acad Sci USA 96: 6553–6557

Orozco-Cardenas ML, Ryan CA (2003) Polygalacturonase beta-subunit

antisense gene expression in tomato plants leads to a progressive

enhanced wound response and necrosis in leaves and abscission of

developing flowers. Plant Physiol 133: 693–701

Ortega-Martinez O, Pernas M, Carol RJ, Dolan L (2007) Ethylene modu-

lates stem cell division in the Arabidopsis thaliana root. Science 317:

507–510

Passardi F, Penel C, Dunand C (2004) Performing the paradoxical: how

plant peroxidases modify the cell wall. Trends Plant Sci 9: 534–540

Penninckx IA, Eggermont K, Terras FR, Thomma BP, De Samblanx GW,

Buchala A, Metraux JP, Manners JM, Broekaert WF (1996) Pathogen-

induced systemic activation of a plant defensin gene in Arabidopsis

follows a salicylic acid-independent pathway. Plant Cell 8: 2309–2323

Pfaffl MW (2001) A new mathematical model for relative quantification in

real-time RT-PCR. Nucleic Acids Res 29: e45

Pressey R (1990) Anions activate the oxidation of indoleacetic acid by

peroxidases from tomato and other sources. Plant Physiol 93: 798–804

Ridley BL, O’Neill MA, Mohnen D (2001) Pectins: structure, biosynthesis,

and oligogalacturonide-related signaling. Phytochemistry 57: 929–967

Robinette D, Matthysse AG (1990) Inhibition by Agrobacterium tumefa-

ciens and Pseudomonas savastanoi of development of the hypersensi-

tive response elicited by Pseudomonas syringae pv. phaseolicola.

J Bacteriol 172: 5742–5749

Rogers EE, Ausubel FM (1997) Arabidopsis enhanced disease susceptibil-

ity mutants exhibit enhanced susceptibility to several bacterial patho-

gens and alterations in PR-1 gene expression. Genetics 146: 381–392

Smith TA, Barker JH (1988) The di- and polyamine oxidases of plants. Adv

Exp Med Biol 250: 573–587

Spadoni S, Zabotina O, Di Matteo A, Mikkelsen JD, Cervone F, De Lorenzo

G, Mattei B, Bellincampi D (2006) Polygalacturonase-inhibiting protein

interacts with pectin through a binding site formed by four clustered

residues of arginine and lysine. Plant Physiol 141: 557–564

Spiro MD, Bowers JF, Cosgrove DJ (2002) A comparison of oligogalacturonide-

and auxin-induced extracellular alkalinization and growth responses in

roots of intact cucumber seedlings. Plant Physiol 130: 895–903

Stepanova AN, Yun J, Likhacheva AV, Alonso JM (2007) Multilevel

interactions between ethylene and auxin in Arabidopsis roots. Plant

Cell 19: 2169–2185

Stern R, Asari AA, Sugahara KN (2006) Hyaluronan fragments: an infor-

mation-rich system. Eur J Cell Biol 85: 699–715

Taylor KR, Gallo RL (2006) Glycosaminoglycans and their proteoglycans:

host-associated molecular patterns for initiation and modulation of

inflammation. FASEB J 20: 9–22

ten Have A, Mulder W, Visser J, van Kan JA (1998) The endopolygalactu-

ronase gene Bcpg1 is required for full virulence of Botrytis cinerea. Mol

Plant Microbe Interact 11: 1009–1016

Terry ME, Bonner BA (1980) An examination of centrifugation as a method

of extracting an extracellular solution from peas, and its use for the

study of indoleacetic acid-induced growth. Plant Physiol 66: 321–325

Thomma BP, Eggermont K, Tierens KF, Broekaert WF (1999) Requirement

of functional ethylene-insensitive 2 gene for efficient resistance of

Arabidopsis to infection by Botrytis cinerea. Plant Physiol 121: 1093–1102

Van Loon LC, van Strien EA (1999) The families of pathogenesis-related

proteins, their activities, and comparative analysis of PR-1 type pro-

teins. Physiol Mol Plant Pathol 55: 85–97

van Santen Y, Benen JA, Schroter KH, Kalk KH, Armand S, Visser J,

Dijkstra BW (1999) 1.68-A crystal structure of endopolygalacturonase II

from Aspergillus niger and identification of active site residues by site-

directed mutagenesis. J Biol Chem 274: 30474–30480

Vorwerk S, Somerville S, Somerville C (2004) The role of plant cell wall

polysaccharide composition in disease resistance. Trends Plant Sci 9:

203–209

Wegener GB (2002) Induction of defence responses against Erwinia soft rot

by an endogenous pectate lyase in potatoes. Physiol Mol Plant Pathol 60:

91–100





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