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First published online December 7, 2007; 10.1104/pp.107.109686 Plant Physiology 146:669-681 (2008) © 2008 American Society of Plant Biologists Transgenic Expression of a Fungal endo-Polygalacturonase Increases Plant Resistance to Pathogens and Reduces Auxin Sensitivity1,[W]Dipartimento di Biologia Vegetale, Università 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 several phytopathogenic 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 (Arabidopsis thaliana) plants expressing a fungal PG (PG plants) have a reduced content of homogalacturonan. Here, we show that PG plants are more resistant to microbial pathogens and have constitutively activated defense responses. Interestingly, either in tobacco PG or wild-type plants treated with OGs, resistance to fungal infection is suppressed by exogenous auxin, whereas sensitivity to auxin of PG plants is reduced in different bioassays. The altered plant defense responses and auxin sensitivity in PG plants may reflect an increased accumulation of OGs and subsequent antagonism of auxin action. Alternatively, it may be a consequence of perturbations of cellular physiology and elevated defense status as a result of altered cell wall architecture.
The plant cell wall possesses mechanical features determining strength and plasticity of a tissue and signaling properties affecting expansion, growth, and development (Carpita and McCann, 2000  ). The cell wall also represents a barrier against invading microorganisms. During early stages of infection, pathogens produce enzymes that degrade the various components of the wall, thus releasing compounds that are used as carbon sources. The major components of primary cell walls of higher plants are complex polysaccharides; increasing evidence indicates that these molecules contribute to disease resistance not just as mechanical barriers but also as sensors for incoming infections (Vorwerk et al., 2004). For example, partial degradation of homogalacturonan (HGA) by fungal endo-polygalacturonases (PGs) releases oligogalacturonides (OGs) with a degree of polymerization between 10 and 15 that show elicitor activity (Cervone et al., 1987a, 1987b, 1989). Treatment with OGs causes accumulation of reactive oxygen species, biosynthesis of phytoalexins (Hahn et al., 1981), and expression of pathogenesis-related (PR) proteins (Davis and Hahlbrock, 1987; Broekaert and Pneumas, 1988) in several plant species. In Arabidopsis (Arabidopsis thaliana), OGs induce the expression of several defense genes and proteins (Ferrari et al., 2003b, 2007; Casasoli et al., 2008), including AtPGIP1 and PAD3, which encode, respectively, an inhibitor of fungal PGs and the Cyt P450 CYP71B15 that catalyzes the last step of camalexin biosynthesis. Exogenous OGs protect Arabidopsis and grapevine leaves against the necrotrophic pathogen Botrytis cinerea (Aziz et al., 2004; Ferrari et al., 2007). In analogy with the role of hyaluronan fragments in animal cells, OGs may be regarded as host-associated molecular patterns involved in the innate immunity (Stern et al., 2006; Taylor and Gallo, 2006).
Besides inducing defense responses, OGs can also affect several aspects of plant growth and development. In particular, a number of reports from our laboratory indicate that exogenously added OGs are able to antagonize the action of auxin, as in the case of pea (Pisum sativum) stem elongation (Branca et al., 1988
We have previously generated tobacco and Arabidopsis transgenic lines (hereafter referred to as PG plants) expressing an attenuated version of PGII of Aspergillus niger (Capodicasa et al., 2004
Because the signaling potential of pectin and pectin-derived fragments may be critical for the outcome of a plant-pathogen interaction (Vorwerk et al., 2004
PG Plants Are Less Susceptible to Pathogen Infection
Tobacco PG plants were inoculated with B. cinerea, and their susceptibility to the fungus was compared to that of wild-type plants and plants expressing the bean PvPGIP2, which inhibits several fungal PGs, including B. cinerea PG (PGIP2 plants; Leckie et al., 1999
Arabidopsis Wassilewskija (Ws) PG plants also displayed a significant reduction of lesion size after fungal inoculation (Fig. 1C). Line PG1, expressing the highest levels of PG (Capodicasa et al., 2004 ), developed smaller lesions (Fig. 1C) and showed a significant reduction of the number of spreading lesions compared to the other lines (Fig. 1D). Transcript levels of the B. cinerea actin gene Bcact were dramatically reduced in inoculated PG1 plants (Supplemental Fig. S1), confirming that the reduced lesion development mirrored a reduced fungal growth. On the other hand, leaves of a transgenic line (PG201), expressing an inactive form of A. niger PGII (van Santen et al., 1999; Federici et al., 2001), showed symptoms similar to those of wild-type plants (Fig. 1, C and D), indicating that enzymatic activity is required to confer enhanced resistance. To determine whether the enhanced resistance observed in PG plants is specific for B. cinerea or is effective against other pathogens, leaves of tobacco wild-type and PG plants were inoculated with a virulent strain of the bacterial pathogen P. syringae pv tabaci. Infiltration with a bacterial suspension produced the collapse of the inoculated area in wild-type plants but no visible symptoms in line PG16 and only very mild symptoms in lines PG5 and 7 (Fig. 2 ). PGIP2 and PG16xPGIP2 plants showed symptoms similar to those observed in wild-type plants (Fig. 2). This indicates that PvPGIP2 has no effect on resistance to P. syringae and, more importantly, that inhibition of enzymatic activity of PG by PvPGIP2 completely blocks PG-mediated resistance. An active and free (uncomplexed) PG is therefore required to confer resistance to plants against organisms as different as the fungal pathogen B. cinerea and the bacterial pathogen P. syringae.
PG Plants Have Enhanced Defense Responses
PGs may act not only as virulence factors of phytopathogenic microorganisms but may also induce defense responses, by either releasing endogenous OG elicitors or affecting cell wall integrity (Hahn et al., 1981
We also determined total peroxidase and 1,3-β-glucanase enzymatic activities, which are known to be regulated by biotic stress (Van Loon and van Strien, 1999 ). Total peroxidase activity was significantly higher in leaves of tobacco plants PG16 and PG7 as compared to control plants, whereas in PG5 as well as in PG16xPGIP2 plants it was only slightly, but still significantly, higher than in wild-type plants or plants expressing PvPGIP2 alone (Fig. 4A
). Analysis of peroxidase activity in the intercellular washing fluids (IWFs) prepared from control and transgenic tobacco plants indicated that the increase of peroxidase activity of PG plants was mainly due to extracellular isoforms (Fig. 4B). Peroxidase activity was also significantly increased in leaves of Arabidopsis plants belonging to line PG1, compared to the other genotypes (Fig. 4C). Basal 1,3-β-glucanase activity was significantly higher only in tobacco and Arabidopsis lines expressing high levels of PG (Fig. 5, A and B
).
Finally, we examined the expression of genes potentially involved in pathogen responses. Expression of two tobacco genes, EAS1/2, encoding a 5-epi-aristolochene synthase required for the biosynthesis of the phytoalexin capsidiol (Facchini and Chappell, 1992 ), and POX, 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 in uninfected PG16 plants and increased earlier and to a greater extent in response to fungal infection (Fig. 6A). EAS1/2 mRNA levels were not constitutively expressed in PG16 plants but increased earlier and to a greater extent in response to fungal infection (Fig. 6A). In Arabidopsis wild-type plants, expression of AtPGIP1, PR-1, and the defensin gene PDF1.2 steadily increased up to 2 d after infection with B. cinerea (Fig. 6, B–D). The basal mRNA levels of all three genes were higher in PG1 plants than in wild-type plants (Fig. 6, B–D). However, their expression differed after inoculation with B. cinerea. AtPGIP1 expression increased at 1 d postinfection (dpi) but returned to basal levels at 2 dpi (Fig. 6B). In contrast, transcripts of PR-1 and PDF1.2 showed only a mild increase after fungal infection of PG plants (Fig. 6, C and D). These findings are consistent with previous data correlating the expression levels of both genes to fungal lesion development rather than to basal resistance (Ferrari et al., 2003a).
A possible explanation for the enhanced expression of defense genes observed in healthy PG plants is the accumulation of OG elicitors released from the cell wall by the action of the fungal PG. We therefore determined whether the expression of these genes is up-regulated by exogenous OGs. Both POX and EAS1/2 mRNA levels increased in tobacco leaf explants treated for 4 h with OGs (Fig. 7, A and B ). Similarly, AtPGIP1 and PR-1 transcripts accumulated in response to OGs in Arabidopsis wild-type seedlings (Fig. 8, A and B ), while no significant increase of PDF1.2 mRNA levels was observed (Fig. 8C). Taken together, these data support the hypothesis that the constitutive expression of at least some of the defense responses observed in PG plants is mediated by OGs released by the fungal enzyme expressed in the transgenic plants.
Increased Resistance of Tobacco PG Plants Is Abolished by Auxin
Because OGs have auxin-antagonistic activity and treatment with OGs leads to a decreased sensitivity to auxin in tobacco plants (Branca et al., 1988 We therefore investigated whether the increased resistance against B. cinerea of the tobacco PG plants could be reverted by exogenous auxin. Leaf discs from wild-type and PG16 tobacco plants were treated with 3-indoleacetic acid (IAA) and then inoculated with B. cinerea. Notably, auxin pretreatment of PG16 leaf discs restored their susceptibility to a level comparable to that of wild-type plants, whereas it did not significantly increase the susceptibility of wild-type leaf discs (Fig. 9A ). Interestingly, whereas pretreatment of wild-type leaf discs with exogenous OGs reduced lesion development after B. cinerea inoculation, cotreatment with OGs and IAA did not produce any effect, and the susceptibility was comparable to that of untreated tissues (Fig. 9B).
We also evaluated the sensitivity to auxin of tobacco PG plants by analyzing the auxin-induced root formation in leaf explants. While a concentration of 0.57 µM IAA was sufficient to induce rhizogenesis in wild-type explants, no roots were observed in PG explants treated with up to 1.7 µM IAA; however, at higher concentrations, IAA induced root formation in PG explants in a dose-dependent fashion (Fig. 10A ). The ability of PG plants to respond to auxin was also tested using a root growth inhibition assay. Wild-type plantlets showed a significant reduction of primary root growth in the presence of 10–7 M IAA, whereas concentrations of IAA up to 10–6 M were not effective in PG16 plants (Fig. 10B). However, when auxin concentration was increased to 10–5 M, a reduction of root length was observed in PG16 plants, consistent with the hypothesis that they are not completely resistant but only less sensitive to auxin. Taken together, these results indicate that auxin-induced developmental and growth responses are impaired in PG plants. This is not due to increased catabolism of IAA because no significant IAA oxidase activity could be detected in extracts from PG leaves (data not shown). We therefore conclude that auxin signaling is partly compromised in PG plants.
Because high concentrations of IAA induce ethylene production in several plant species (Abeles, 1966 ) and ethylene-controlled responses play a role in both resistance against B. cinerea (Thomma et al., 1999; Diaz et al., 2002; Chague et al., 2006) and inhibition of root development (Ortega-Martinez et al., 2007; Stepanova et al., 2007), we investigated whether PG plants show increased ability to produce ethylene. Ethylene produced by wild-type and PG16 leaf explants was measured after water or IAA treatment. Water-treated wild-type or PG16 leaf explants did not release any detectable accumulation of ethylene; in contrast, 1-aminocyclopropane-1-carboxylic acid (ACC) induced high levels of ethylene in both genotypes, indicating that they have a similar ability of producing this hormone (Fig. 11
). Furthermore, treatment with 100 µM IAA resulted in the accumulation of lower, but significant and comparable, levels of ethylene in wild-type and PG16 explants (Fig. 11). OGs had no significant impact on ethylene production in water- or IAA-treated wild-type tobacco explants (data not shown). A role of ethylene in the basal resistance, in the IAA-mediated reversion of the resistant phenotype, and in the reduced ability to form adventitious roots in response to IAA of tobacco PG plants is therefore unlikely.
In this article, we have shown that the expression of a fungal PG in Arabidopsis and tobacco increases plant resistance to microbial pathogens. The resistant phenotype is not exhibited by transgenic tobacco plants expressing both PG and its inhibitor PvPGIP2 or by Arabidopsis plants expressing a mutagenized and inactive AnPGII (PG201), indicating that resistance is dependent on the enzymatic activity of the expressed PG and is likely a consequence of the degradation of the host pectin accomplished by the enzyme. Interestingly, PG plants appear to have reduced sensitivity to exogenous auxin, and auxin treatments revert their resistant phenotype. The data presented here allow us to consider the reasons that might explain why heterologous expression of a fungal PG increases plant resistance, providing a link between HGA degradation, auxin perception, and activation of defense responses.
PG plants have an altered pectin composition with a reduced GalUA and HGA content (Capodicasa et al., 2004
The enhanced expression of diverse defense responses is likely the ultimate reason for their increased resistance to pathogens. We could not observe a perfect correlation between the levels of a specific defense response and the degree of resistance observed in independent PG lines, suggesting that multiple defense responses, activated to different extents in different PG lines, contribute to the final resistant phenotype. The observation that tobacco PG16xPGIP2 plants or Arabidopsis PG201 plants, which have no PG activity, display neither altered expression of defense responses nor increased resistance indicates that these phenotypes are mediated by HGA degradation in PG plants rather than by the recognition per se of the heterologous protein by plant cells. Enhanced expression of basal and inducible defense responses and increased resistance to pathogens were previously observed in plants expressing pectin-degrading enzymes. Basal levels of polyphenoloxidase activity and induction of Phe ammonia lyase upon wounding are enhanced in transgenic potato tubers expressing a bacterial pectate lyase (Wegener, 2002
One explanation for the observed phenotypes in the PG plants is that altered cell wall integrity may be perceived as a signal of pathogen attack or mechanical damage, leading to the induction of defense mechanisms (Humphrey et al., 2007
Indeed, increased disease resistance is observed in plants with alterations of cell wall structural components other than pectin. For example, mutations in an Arabidopsis cellulose synthase (CESA3) cause not only decreased cellulose content but also constitutive expression of defense responses (Ellis et al., 2002
Another possible explanation for the enhanced resistance of plants with altered cell wall components is that plant cells perceive molecules released in the apoplast as a consequence of the hydrolysis of specific structural polymers and these molecules act as elicitors of defense responses. In particular, OGs generated upon degradation of HGA by fungal PGs act as elicitors of defense responses in several plant systems (for review, see Ridley et al., 2001
A second line of evidence suggesting that pectic fragments with elicitor activity accumulate in PG plants is that auxin reverts the susceptibility to B. cinerea of tobacco PG plants to wild-type levels and also suppresses OG-induced protection against this fungus in wild-type plants. Furthermore, auxin-dependent responses are impaired in tobacco PG plants, and our previous work has shown that OGs suppress different responses regulated by auxin in tobacco (Bellincampi et al., 1993 We also show that tobacco PG plants do not differ from wild-type plants in their ability to produce ethylene, and, on the other hand, OGs do not induce significant production of ethylene in wild-type plants. This suggests that a role of this hormone in the basal resistance, as well as in the IAA-mediated reversion of the resistant phenotype and in the reduced ability to form adventitious roots in response to IAA, is unlikely. Other mechanisms, such as an alteration of the biomechanical properties of the cell wall, may be responsible for the observed responses of the PG plants. However, no obvious correlation between auxin-induced wall loosening and auxin-mediated reversion of the resistant phenotype of PG explants could be observed. Indeed, IAA did not increase susceptibility in wild-type explants, which showed curling in response to auxin, whereas it did so in PG explants that did not show any curling (data not shown). Therefore, it may be argued that the loosening of the wall that leads to curling in wild-type explants has no apparent effect on the susceptibility to B. cinerea, while the increased susceptibility of PG explants in the presence of auxin occurs in the absence of significant wall loosening/curling. Furthermore, because expansion of discs from PG plants in water is similar or even higher than that of discs from wild-type plants (R. Galletti and D. Pontiggia, unpublished data), an alteration of biomechanical properties (for example, more rigid cell walls) that can be compensated by treatment with auxin is probably not the reason for the resistant phenotype and the reduced ability to form adventitious roots. On the other hand, the ability of auxin to revert the altered responses of PG plants also suggests that irreversible wall modifications such as lignification or cross-links are unlikely to be involved.
Auxin and elicitors can have opposite effects on defense gene expression. For instance, IAA inhibits induction of PR proteins in tobacco protoplasts treated with elicitors from the cell wall of the oomycete Phytophthora megasperma f. sp. glycinea (Jouanneau et al., 1991 In conclusion, we have shown that the expression of a fungal PG and the subsequent degradation of HGA increases resistance of plants to microbial pathogens, likely through a pre-activation of plant defense responses, and that auxin reverts the enhanced resistance to fungal infection. Our data also indicate that in muro pectin degradation by PG leads to decreased auxin sensitivity. This is reminiscent of the effects observed in untransformed plants treated with OGs, suggesting a possible role of these pectic fragments in the resistant phenotypes of PG plants. Alternatively, an altered cell wall architecture may be directly perceived by the plant cell as a signal of the presence of a pathogen, leading to an increased activation of defense responses. The molecular mechanisms linking pectin degradation, auxin signaling, and activation of defense responses still remain to be investigated. We believe that this line of research will provide new insights in the regulation of plant defense responses during plant-pathogen interactions.
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
For elicitor treatments on Arabidopsis seedlings, seeds were surface-sterilized and germinated in multiwell plates (approximately 10 seeds per well) containing 1 mL per well of 1x Murashige and Skoog (MS) medium (Sigma; Murashige and Skoog, 1962
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 supplemented with 250 µM ACC, 100 µM IAA sodium salt, or 200 mg L–1 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 cm–2) was 40 mL min–1. Column, injector, and detector temperatures were 30°C, 50°C, and 120°C, respectively. Heating rate was 18°C min–1 to 170°C. Air flow was 1.60 kg cm–2 and hydrogen flow was 0.70 kg cm–2. Ethylene concentration in each sample was normalized using dry weight of the leaf explants.
Botrytis cinerea (a kind gift of J. Plotnikova, Massachusetts General Hospital) was grown for 7 to 10 d at 22°C under constant light on 20 g L–1 malt extract, 10 g L–1 mycological peptone, and 15 g L–1 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 L–1 potato dextrose broth and incubated for 3 h at room temperature to allow uniform germination. Inoculation of detached Arabidopsis leaves was performed as described previously (Ferrari et al., 2003b 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 x 104 colony-forming units cm–2). Challenge inoculation was performed by infiltration of the bacterial suspension using a 1-mL syringe without a needle.
For H2O2 visualization, leaves were cut from adult plants using a razor blade and dipped for 12 h in a solution containing 1 mg mL–1 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
Activity of β-1,3-glucanase was determined by incubating 50 µg and 150 µg of tobacco or Arabidopsis total proteins, respectively, at 37°C with 2 mg mL–1 laminarin (Sigma) in 50 mM sodium acetate, pH 5.2. The produced reducing sugars were determined colorimetrically with dinitrosalicylic acid (Sigma; Miller, 1959
The presence of UV-fluorescent compounds was visualized by photographing detached leaves under UV light (
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
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 µL of cDNA (corresponding to 120 ng of total RNA) was amplified in 30 µL 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'-AGTCTCCAACTCTTGCTCAT-3'. The primers for PDF1.2, PR-1, AtPGIP1, and UBQ5 were described previously (Penninckx et al., 1996
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 x 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 µE m–2 s–2) for 15 d. For seedling assays, seeds were sterilized and germinated in 0.5x 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.
The following materials are available in the online version of this article.
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 December 7, 2007.
1 This work was supported by the Ministero dell'Università e della Ricerca (Fondo per gli Investimenti della Ricerca di Base RBNE01KZE7 and PRIN 2005052297), by the Giovanni Armenise-Harvard Foundation, and by the Institute Pasteur-Fondazione Cenci Bolognetti. 
2 These authors contributed equally to the article. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Giulia De Lorenzo (giulia.delorenzo{at}uniroma1.it).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.107.109686 * Corresponding author; e-mail giulia.delorenzo{at}uniroma1.it.
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= 320 nm) using the "ImageMaster" VDS (Pharmacia Biotech). 
-D-oligogalacturonides in pea stem segments. Physiol Plant 72: 499–504
