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Alginate gene expression by Pseudomonas syringae pv. tomato DC3000 in host and non-host plants

Ronald C. Keith, Lisa M. W. Keith, Gustavo Hernández-Guzmán, Srinivasa R. Uppalapati, Carol L. Bender

  • 127 Noble Research Center, Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078, USA
  • Correspondence
    Carol L. Bender
    cbender{at}okstate.edu

    • Microbiology 2003; 149(5):1127–1138 · https://doi.org/10.1099/mic.0.26109-0

    View at publisher PubMed

    Abstract

    Pseudomonas syringae produces the exopolysaccharide alginate, a copolymer of mannuronic and guluronic acid. Although alginate has been isolated from plants infected by P. syringae, the signals and timing of alginate gene expression in planta have not been described. In this study, an algD : : uidA transcriptional fusion, designated pDCalgDP, was constructed and used to monitor alginate gene expression in host and non-host plants inoculated with P. syringae pv. tomato DC3000. When leaves of susceptible collard plants were spray-inoculated with DC3000(pDCalgDP), algD was activated within 72 h post-inoculation (p.i.) and was associated with the development of water-soaked lesions. In leaves of the susceptible tomato cv. Rio Grande-PtoS, algD activity was lower than in collard and was not associated with water-soaking. The expression of algD was also monitored in leaves of tomato cv. Rio Grande-PtoR, which is resistant to P. syringae pv. tomato DC3000. Within 12 h p.i., a microscopic hypersensitive response (micro-HR) was observed in Rio Grande-PtoR leaves spray-inoculated with P. syringae pv. tomato DC3000(pDCalgDP). As the HR progressed, histochemical staining indicated that individual bacterial cells on the surface of resistant tomato leaves were expressing algD. These results indicate that algD is expressed in both susceptible (e.g. collard, tomato) and resistant (Rio Grande-PtoR) host plants. The expression of algD in an incompatible host–pathogen interaction was further explored by monitoring transcriptional activity in leaves of tobacco, which is not a host for P. syringae pv. tomato. In tobacco inoculated with DC3000(pDCalgDP), an HR was evident within 12 h p.i., and algD expression was evident within 8-12 h p.i. However, when tobacco was inoculated with an hrcC mutant of DC3000, the HR did not occur and algD expression was substantially lower. These results suggest that signals that precede the HR may stimulate alginate gene expression in P. syringae. Histochemical staining with nitro blue tetrazolium indicated that the superoxide anion () is a signal for algD activation in planta. This study indicates that algD is expressed when P. syringae attempts to colonize both susceptible and resistant plant hosts.

    • ↵†Present address: Pacific Basin Tropical Plant Genetic Resource Management Unit, USDA-ARS, PO Box 4487, Hilo, HI 96720, USA.

    INTRODUCTION

    Pseudomonas syringae is a Gram-negative plant pathogen that elicits a wide variety of symptoms in plants, including blights (rapid death of tissue), leaf spots and galls. The species is divided into pathogenic variants (pathovars) that vary in host range. The infection of host plants by P. syringae involves epiphytic (surface) colonization, entry, establishment of infection sites in the intercellular spaces (apoplast), multiplication within host tissue and production of disease symptoms (Alfano & Collmer, 1996; Boch et al., 2002; Hirano & Upper, 2000). The genetic basis of pathogenicity in P. syringae is complex and includes global regulatory genes (Rich et al., 1994), virulence factors such as phytotoxins (Bender et al., 1999) and the hrp/hrc gene cluster. The hrp/hrc genes encode a type III secretion system (TTSS), which is functionally similar to the TTSS identified in Gram-negative animal pathogens (Galán & Collmer, 1999). In P. syringae, the TTSS is required for growth in susceptible host plants and the activation of plant defence in non-host plants; the latter is often associated with the hypersensitive response (HR) (Staskawicz et al., 2001). The HR is a complex form of programmed cell death that is generally characterized by the presence of brown, dead cells at the infection site (Heath, 2000).

    Both P. syringae and Pseudomonas aeruginosa produce the exopolysaccharide alginate, a copolymer of O-acetylated β-1,4 linked d-mannuronic acid and its C-5 epimer, l-guluronic acid. P. aeruginosa is an important human pathogen that causes opportunistic pulmonary infections in patients suffering from cystic fibrosis. Some strains of P. aeruginosa also cause disease in plants (Rahme et al., 1995), although the symptoms (soft rotting) and mechanistic aspects of entry into plant tissue are clearly different for P. aeruginosa and P. syringae (Plotnikova et al., 2000). A critical aspect of pulmonary infection by P. aeruginosa is the conversion of this bacterium to a mucoid, alginate-overproducing phenotype during the chronic-infection stage of cystic fibrosis (Lyczak et al., 2002). In cystic fibrosis patients, the alginate capsule has been reported to inhibit opsonic and non-opsonic phagocytosis and to enhance bacterial survival during the macrophage-mediated oxidative burst (Cabral et al., 1987; Simpson et al., 1988, 1989).

    Studies have shown that alginate functions in the virulence of some P. syringae strains (Osman et al., 1986; Gross & Rudolph, 1987; Yu et al., 1999). Alginate production by P. syringae has been associated with increased epiphytic fitness, resistance to desiccation and toxic molecules, and the induction of water-soaked lesions on infected leaves (Fett & Dunn, 1989; Rudolph et al., 1994; Yu et al., 1999). In plants inoculated with the alginate-defective mutant P. syringae pv. syringae 3525.L, symptoms were less severe and bacterial multiplication was reduced relative to the wild-type (Yu et al., 1999), suggesting that alginate facilitates the colonization and/or dissemination of this strain in planta. Strains of P. syringae, like P. aeruginosa, are normally non-mucoid; however, some of the signals for conversion to the mucoid phenotype differ in these species. For example, copper-based sprays are frequently applied to plants for the control of bacterial diseases and exposure to copper ions dramatically increases alginate production in certain strains of P. syringae but not in P. aeruginosa (Kidambi et al., 1995). As in P. aeruginosa, increased levels of NaCl and sorbitol activate the transcription of alginate promoters in P. syringae, indicating that elevated osmolarity is a signal for alginate production in both pseudomonads (Keith & Bender, 1999; Peñaloza-Vázquez et al., 1997). Another conserved signal for alginate gene expression in P. aeruginosa and P. syringae is exposure to reactive oxygen species (ROS) (Keith & Bender, 1999; Mathee et al., 1999).

    Although alginate was shown to be the major exopolysaccharide produced by P. syringae in water-soaked lesions (Fett & Dunn, 1989), until recently there was no evidence that alginate gene expression was induced in planta. Boch et al. (2002) used in vivo expression technology (IVET) to show that a variety of genes are specifically induced during the infection of Arabidopsis thaliana by P. syringae pv. tomato DC3000. One of the plant-inducible genes identified in the IVET screen was algA, which encodes a bifunctional enzyme in the alginate biosynthetic pathway (Shinabarger et al., 1991). Although the IVET study clearly demonstrated that alginate genes are expressed in A. thaliana, the temporal expression of the algA : : uidA promoter fusion was not investigated (Boch et al., 2002). The primary objective of our study was to construct a defined algD : : uidA transcriptional fusion and use this to monitor alginate gene expression in host and non-host plants infected with P. syringae pv. tomato DC3000, which is a also a pathogen of tomato and Brassica spp. (e.g. cauliflower, collard) (Moore et al., 1989; Zhao et al., 2000). algD encodes GDP-mannose dehydrogenase, which is the committed step in alginate biosynthesis and the first gene to be transcribed in the alginate structural gene cluster of P. syringae (Peñaloza-Vázquez et al., 1997). In the current study, algD expression was monitored in susceptible hosts (tomato and collard), a resistant host (tomato carrying the PtoR resistance gene) and a non-host plant (tobacco), which undergoes the HR in response to infection with P. syringae pv. tomato.

    METHODS

    Bacterial strains, plasmids and media.

    The bacterial strains and plasmids used in this study are described in Table 1⇓. P. syringae strains were grown at 28 °C on mannitol-glutamate medium (MG) (Keane et al., 1970). Escherichia coli strains were grown on Luria–Bertani (LB) medium (Miller, 1972) at 37 °C. Antibiotics were used at the following concentrations (μg ml−1): kanamycin (Km), 25; ampicillin (Ap), 100; chloramphenicol (Cm), 25; spectinomycin (Sp), 25; tetracycline (Tc), 12·5.

    Table 1.

    Bacterial strains and plasmids used in this study

    Molecular genetic techniques.

    Restriction enzyme digests, agarose gel electrophoresis, PCR and other routine molecular methods were performed using standard protocols (Sambrook et al., 1989). Plasmids were isolated from P. syringae as described by Kado & Liu (1981). Clones were mobilized into recipient strains by a triparental mating procedure using the mobilizer plasmid pRK2013 (Bender et al., 1991). Nucleotide sequencing reactions were performed by the dideoxynucleotide method using AmpliTaq DNA polymerase and the ABI PRISM Dye Primer Cycle Sequencing Kit (Perkin Elmer). The synthesis of oligonucleotide primers and automated DNA sequencing were provided by the Recombinant DNA/Protein Resource Facility at Oklahoma State University. Sequence data were aligned and homology searches were executed using the National Center for Biotechnology Information blast Network server.

    Construction of an algD : : uidA transcriptional fusion.

    A genomic library of P. syringae pv. tomato DC3000 was previously constructed in pRK7813 (Boch et al., 2002). In this study, a 3·9 kb KpnI fragment from pSKK3.9, which contains algD from P. syringae pv. syringae FF5 (Peñaloza-Vázquez et al., 1997), was used to screen the DC3000 genomic library for clones containing algD. A cosmid clone designated pRCK1 contained an 8 kb BamHI fragment that hybridized with the probe. This fragment was subcloned into pBluescript SK(+), resulting in pBRCK1, which was shown by sequence analysis to contain the algD gene and the predicted promoter region (Keith, 2002). The algD promoter in the related pathogen, P. syringae pv. syringae FF5, was previously defined by deletion analysis (Peñaloza-Vázquez et al., 1997). The corresponding region in P. syringae pv. tomato DC3000 was sequenced in pBRCK1 and showed 81·6 % nucleotide identity with the algD promoter of FF5 (Keith, 2002). Therefore, the strategy used for subcloning the DC3000 algD promoter was based on prior results with FF5, since these two strains were highly homologous in their algD upstream regions.

    pBBR.Gus, which contains a promoterless glucuronidase (GUS) gene (uidA) downstream of the polylinker in pBBR1MCS, was used to create an algD : : uidA transcriptional fusion. To obtain the algD promoter region in the transcriptionally active orientation, a 1 kb PCR product was cloned into the HindIII/PstI sites of pBBR.Gus. The promoter region was amplified from pBRCK1 by using the forward primer 5′-CGGAAAGCTTTAAACCAGTTCGATG-3′ (the HindIII site is underlined) and the reverse primer 5′-CCGCCTGCAGGGTAACAACTAGTTCAG-3′ (PstI site is underlined). The amplified region contained 956 nt upstream of the algD translational start site, including the recognition sequence for σ22, an alternate sigma factor that mediates transcription at the algD promoter (Keith & Bender, 1999; Wozniak & Ohman, 1994). After amplification of the 1 kb PCR product, cloning into pBBR.Gus as a HindIII/PstI fragment, and transformation into E. coli DH5α, plasmid pDCalgDP was recovered. Expression of the algD promoter was also monitored in P. syringae pv. tomato DC3000-hrcC, a Cmr strain. In these experiments, the algD promoter was subcloned from pDCalgDP as a 1 kb HindIII/PstI fragment into pBBR.Gus.Km (Table 1⇑), resulting in pDCalgDP.Km. The presence of the algD promoter region in both pDCalgDP and pDCalgDP.Km was confirmed by sequence analysis.

    Quantitative GUS assays.

    Liquid cultures of DC3000(pBBR.Gus) and DC3000(pDCalgDP) were incubated at 28 °C in MG broth supplemented with 25 μg chloramphenicol ml−1. Each strain carrying an individual construct was inoculated into triplicate aliquots of medium (100 ml MG broth) and incubated at 28 °C. At different time points (0, 12, 24 and 48 h), a 1 ml sample of bacterial cells was removed from each tube and assayed for GUS activity as described previously (Peñaloza-Vázquez & Bender, 1998). The protein concentration of cell lysates was determined using the Bio-Rad protein assay kit, following the manufacturer's protocol. GUS activity was expressed in units (U) (mg protein)−1, with 1 U equivalent to 1 nmol of methylumbelliferone formed min−1.

    Plant growth and inoculation procedures.

    Tomato (Lycopersicum esculentum cv. Rio Grande-PtoS and -PtoR) and collard (Brassica oleracea var. viridis L. cv. Vates) seedlings were maintained in a growth chamber at 24–25 °C in 30–40 % relative humidity, with a photoperiod of 12 h. Plants were maintained at ⩾90 % relative humidity for 48 h before inoculation. Derivatives of P. syringae pv. tomato DC3000 were grown at 28 °C for 48 h on MG agar supplemented with the appropriate antibiotics and cells were resuspended to an OD600 of 0·3 (∼5×108 c.f.u. ml−1) in sterile distilled water. Silwet L77 (Osi Specialties) was added to the bacterial inoculum at a concentration of 0·2 μg ml−1. Six-week-old plants were spray-inoculated with an airbrush (55·2 kPa) until leaf surfaces were uniformly wet. After inoculation, tomato and collard plants were incubated at 24 °C in 60 % relative humidity with a 12 h photoperiod for the duration of the experiment.

    Tobacco (Nicotiana tabacum cv. Petite Havana) leaves were infiltrated (OD600=0·1) with selected bacterial strains using established methods (Schaad, 1988).

    Histochemical detection of GUS activity.

    Tomato and collard leaves were sampled at 0, 24, 48, 72, 120 and 168 h post-inoculation (p.i.) and vacuum-infiltrated with a substrate surfactant solution [5-bromo-4-chloro-3-indoyl β-d-glucuronide (X-gluc), 0·5 mg ml−1, and L77 at 0·2 μl ml−1 in 50 mM sodium phosphate buffer, pH 7·0]. Vacuum-infiltrated leaves were incubated at 37 °C overnight and fixed and destained in 80 % ethanol at 37 °C (Hugouvieux et al., 1988). Samples from infiltrated tobacco leaves were excised at 2, 4, 6, 8, 10 and 12 h p.i. with a sterile cork borer (1 cm diam.) and assayed for GUS activity as described above.

    Determination of bacterial growth in planta.

    In experiments designed to follow the total population of the DC3000 strains in collard and tomato, random leaf samples were taken at the time points indicated above. Each leaf was weighed separately (three replicates per time point) and macerated in 2 ml (tomato) or 5 ml (collard) sterile distilled H2O. Bacterial populations in tobacco leaves were monitored by removing leaf disks (1 cm diameter) at 0, 6 and 12 h after infiltration with DC3000 strains. Bacterial counts were determined by plating dilutions of the leaf homogenate onto MG agar supplemented with 25 μg chloramphenicol ml−1. Colonies were counted after incubating the plates for 48–96 h and the experiment was then repeated twice. Bacterial population counts were similar on media with and without antibiotic selection, indicating that the vector was stably maintained in planta.

    Detection of
    Figure image not available in archive
    .

    Tobacco leaves were monitored for the superoxide anion (

    Figure image not available in archive
    ) by infiltration with nitro blue tetrazolium (NBT) using methods similar to those described by Doke (1983). Tobacco leaf disks were excised at 2, 4, 6, 8, 10 and 12 h after inoculation with DC3000, DC3000-hrcC or sterile distilled H2O. Leaf disks were then vacuum-infiltrated with 0·05 M sodium phosphate buffer (pH 7·5) containing 0·5 mM NBT. Leaf tissue was incubated at ambient temperature under cool fluorescent lighting for 20 min and the reaction was then stopped with 95 % ethanol. Chlorophyll was removed by repeated changes of 95 % ethanol.

    RESULTS

    Construction of the algD : : uidA fusion

    Our first experiment was designed to determine whether the algD : : uidA fusion in pDCalgDP was transcriptionally active. GUS activity of DC3000(pBBR.Gus) and DC3000(pDCalgDP) was measured at various intervals after inoculation into MG broth. At 48 h, GUS activity in DC3000(pDCalgDP) was 16-fold higher than DC3000(pBBR.Gus) (data not shown). GUS activity in DC3000(pBBR.Gus) was negligible throughout the sampling period. These results confirmed that the algD promoter was active in DC3000(pDCalgDP) and the level of expression in vitro was consistent with values previously obtained for algD : : uidA fusions (Fakhr et al., 1999; Peñaloza-Vázquez et al., 1997).

    Symptoms and algD expression in collard leaves

    Collard leaves inoculated with P. syringae pv. tomato were examined for disease symptoms and GUS activity to determine whether the manifestation of a particular symptom was correlated with alginate gene expression. In collard leaves inoculated with DC3000(pBBR.Gus) and DC3000(pDCalgDP), water-soaked lesions were first visible at 72 h p.i. (Fig. 1⇓i), and chlorosis and necrosis were evident beginning at 72–96 h p.i. (Fig. 1b⇓). Histochemical staining was used to study expression of the algD promoter in planta. When the algD promoter is activated, GUS is produced and leaves incubated with the chromogenic substrate X-gluc stain blue where the bacteria are located. At 72 h p.i., the water-soaked lesions on collard leaves inoculated with DC3000(pDCalgDP) (Fig. 1i⇓) stained blue when the tissue was infiltrated with X-gluc (Fig. 1j⇓). Collard leaves inoculated with DC3000(pBBR.Gus) did not stain when treated with X-gluc, regardless of the time following inoculation (Fig. 1l⇓). Both DC3000(pBBR.Gus) and DC3000(pDCalgDP) grew equally well in collard reaching a population of 5×109 c.f.u. g−1 in 120 h (Fig. 1m⇓), which indicates that the constructs pBBR.Gus and pDCalgDP did not have a negative impact on the growth of DC3000 in planta.

    Figure image not available in archive
    Fig. 1.

    Symptom development and histochemical localization of GUS activity in collard cv. Vates spray-inoculated with P. syringae pv. tomato DC3000(pDCalgDP) and DC3000(pBBR.Gus), which contain an algD : : uidA fusion and a promoterless uidA gene, respectively. (a–d) Symptom development in collard leaves spray-inoculated with DC3000(pDCalgDP) and photographed at 24, 72, 120 and 168 h p.i. (e–h) GUS activity in collard leaves inoculated with DC3000(pDCalgDP) at 24, 72, 120 and 168 h p.i. (i) Disease lesions on upper surface of a collard leaf inoculated with DC3000(pDCalgDP) and photographed 72 h p.i. (red arrows show water-soaked lesions). (j) GUS activity in lower surface of leaf shown in panel (i). (k) GUS activity in disease lesions (×5 magnification) and photographed at 96 h p.i.; and (l) absence of GUS activity in lesions induced by DC3000(pBBR.Gus) at 96 h p.i. (×5 magnification). This experiment was repeated and leaves were examined for symptom development and GUS activity; the results shown in this figure are typical of both experiments. (m) Bacterial population dynamics in collards inoculated with DC3000(pDCalgDP) (circles) and DC3000(pBBR.Gus) (squares). Bacteria were inoculated as described in Methods and random leaf samples were removed at 0, 24, 48, 72, 120 and 168 h p.i. Bacterial counts were determined as described in Methods and the experiment was repeated with similar results.

    algD expression in tomato cv. Rio Grande

    Since the onset of visible symptoms and the expression of virulence genes can vary with different host plants (Wang et al., 2002), we also monitored disease symptoms and GUS activity in leaves of tomato, another host infected by DC3000. When tomato cv. Rio Grande-PtoS was inoculated with DC3000(pBBR.Gus) and DC3000(pDCalgDP), necrotic lesions were visible to the unaided eye at 96 h p.i. (Fig. 2⇓c). Chlorotic lesions with necrotic centres were visible at 168 h p.i. (Fig. 2d⇓), which coincided with visible GUS staining (Fig. 2h⇓). It is important to note that water-soaked lesions were not visible on tomato and GUS activity in tomato was visibly lower than that observed in collard leaves. No GUS activity was evident when tomato leaves were inoculated with DC3000(pBBR.Gus), regardless of the sampling period (data not shown). Both DC3000(pBBR.Gus) and DC3000(pDCalgDP) grew equally well in Rio Grande-PtoS and multiplied to a population of 5×109 c.f.u. ml−1 in 72 h (Fig. 2i⇓), indicating that neither pBBR.Gus nor pDCalgDP had a negative impact on the growth of DC3000 in tomato. These data also indicate that algD expression is not simply correlated with bacterial growth. For example, DC3000 grew well in both collard (Fig. 1m⇑) and tomato (Fig. 2i⇓), but expression was reproducibly low in tomato.

    Figure image not available in archive
    Fig. 2.

    Symptoms and GUS activity in tomato cv. Rio Grande-PtoS spray-inoculated with P. syringae pv. tomato DC3000(pDCalgDP) and DC3000(pBBR.Gus), which contain an algD : : uidA fusion and a promoterless uidA gene, respectively. Tomato leaves were inoculated and stained for GUS activity as described in Methods. (a–d) Symptoms in tomato leaves spray-inoculated with DC3000(pDCalgDP). Red arrows indicate location of necrotic lesions at 96 h p.i. (c). (e–h) GUS activity in tomato leaves inoculated with DC3000(pDCalgDP) at 24, 48, 96 and 168 h p.i. Red arrows show necrotic lesions associated with GUS activity at 168 h p.i. (h). (i) Bacterial population dynamics in Rio Grande-PtoS inoculated with DC3000(pDCalgDP) (circles) and DC3000(pBBR.Gus) (squares). All experiments were repeated with similar results.

    The next experiment was designed to determine if algD was expressed in a tomato line that is resistant to P. syringae pv. tomato DC3000. When cv. Rio Grande containing the PtoR resistance gene was spray-inoculated with DC3000(pDCalgDP) and DC3000(pBBR.Gus), no macroscopic symptoms were apparent from 0 to 168 h p.i. (Fig. 3⇓a). However, upon removal of chlorophyll, leaf tissue became increasingly brown (Fig. 3b⇓); this suggested that cellular damage was occurring since the browning of tissue was absent in mock (buffer)-inoculated plants. Rio Grande-PtoR leaves were then examined using light microscopy to determine if individual cells were responding to spray-inoculated bacteria. When investigated at higher magnification (×500) at 12 h p.i., cells adjacent to individual stomata were necrotic (Fig. 3c, d⇓). At 24 h p.i., epiphytic bacteria associated with the guard cells stained blue, indicating that the algD promoter was active (Fig. 3e⇓). At 48 h p.i., the intercellular spaces adjacent to cells undergoing the HR stained intensively (Fig. 3f⇓). At 96 and 120 h p.i., the intensity of blue staining decreased, while cells undergoing the HR increased in number and could be visualized as small circular lesions at×10 magnification (data not shown). In tomato leaves infected with DC3000(pBBR.Gus), cells undergoing necrosis were evident, but did not stain blue due to the absence of an active uidA gene (data not shown). The rapid onset of necrosis and the desiccated appearance of necrotic cells in Rio Grande-PtoR are typical of the rapid cell death associated with the HR, a form of programmed cell death associated with incompatible host–pathogen interactions (Heath, 2000).

    Figure image not available in archive
    Fig. 3.

    Incompatible response of tomato cv. Rio Grande-PtoR inoculated with P. syringae pv. tomato DC3000(pDCalgDP). Tomato leaves were inoculated and stained for GUS activity as described in the legend to Fig. 1. (a) Leaves photographed at 0, 120 and 168 h p.i. (b) Histochemical staining of leaves shown in panel (a) for GUS activity. (c) Onset of cell death in Rio Grande-PtoR inoculated with P. syringae pv. tomato DC3000(pDCalgDP). Photo was taken at 12 h p.i. at ×500 magnification; the cell adjacent to the stomata is undergoing an HR (arrow). (d) Cells in the substomatal cavity undergoing an HR (arrows) at 24 h p.i. (e) Individual bacteria (red arrows) on guard cells expressing GUS at 24 h p.i. (×500 magnification). (f) The intercellular spaces adjacent to cells undergoing the HR stain intensively at 48 h p.i. The experiment was repeated and leaves were examined for symptom development and GUS activity; the results shown in this figure are typical of both experiments.

    algD expression in tobacco cv. Petite Havana

    Our results indicated that algD was expressed in both susceptible (PtoS) and resistant (PtoR) lines of tomato cv. Rio Grande, suggesting that signals generated in both compatible and incompatible interactions may stimulate alginate gene expression. algD was expressed in Rio Grande-PtoR very soon after the HR (Fig. 3c, d⇑), suggesting that alginate may be induced by factors associated with the HR. This hypothesis was investigated by analysing algD expression in tobacco, which undergoes a rapid HR in response to DC3000.

    When tobacco cv. Petite Havana was infiltrated with DC3000(pDCalgDP) (Fig. 4⇓a) and DC3000(pBBR.Gus) (not shown), an HR was visualized within 12 h p.i. An HR was also visualized in tobacco leaves inoculated with DC3000 containing pCPP2438, which contains the hrpA promoter fused to a promoterless GUS gene (Fig. 4a⇓). The hrpA gene encodes the major subunit of the TTSS appendage known as the Hrp pilus and is essential for the physical translocation of virulence effectors (Jin & He, 2001; Wei et al., 2000). After vacuum infiltration with X-gluc, incubation and destaining, a high level of GUS activity was observed in tobacco tissue inoculated with DC3000(pCPP2438) at 2–12 h p.i. (Fig. 4c⇓). Activation of the algD : : uidA fusion was delayed in comparison to the hrpA : : uidA fusion, with GUS activity first appearing 8 h p.i. (Fig. 4c⇓). No GUS expression was apparent in tobacco leaves inoculated with DC3000(pBBR.Gus), regardless of the sampling period (Fig. 4c⇓). When the bacterial population was monitored during this experiment, the DC3000(pDCalgDP) and DC3000(pBBR.Gus) populations showed a decline during the 6–12 h sampling period (Fig. 4d⇓). This is possibly due to the accumulation of toxic defence compounds that are produced during the HR.

    Figure image not available in archive
    Fig. 4.

    Tobacco cv. Petite Havana infiltrated with P. syringae pv. tomato DC3000 and DC3000-hrcC. Bacterial inoculum was infiltrated into tobacco leaves as described in Methods and photographed 24 h p.i. (a) DC3000 containing pDCalgDP (algD : : uidA) and pCPP2438 (hrpA : : uidA) elicit an HR in tobacco. (b) DC3000-hrcC containing pCPP2438, pDCalgDP and pBBR.Gus fail to elicit an HR due to the absence of a functional TTSS. (c) GUS activity in tobacco infiltrated with P. syringae pv. tomato DC3000 containing pCPP2438, pDCalgDP and pBBR.Gus (promoterless uidA gene). Tobacco tissue was infiltrated with X-gluc as described in the legend to Fig. 1 and photographed at 2, 4, 6, 8, 10 and 12 h p.i. (d) Population dynamics of DC3000(pDCalgDP) (triangles) and DC3000(pBBR.Gus) (circles) in tobacco tissue sampled at 0, 6 and 12 h p.i. (e) GUS activity in tobacco infiltrated with P. syringae pv. tomato DC3000-hrcC containing pCPP2438, pDCalgDP.Km and pBBR.Gus. (f) Population dynamics of DC3000-hrcC(pDCalgDP.Km) (triangles) and DC3000-hrcC(pBBR.Gus) (circles) in tobacco leaves. All experiments were repeated and the results shown are typical of all experiments.

    During pathogenesis, enhanced expression of the hrp genes can be detected beginning 1–2 h after infiltration into the tissue (Rahme et al., 1992; Xiao et al., 1992). However, it is not known what plant signals induce hrp expression during pathogenesis and whether the same signals stimulate alginate gene expression. The data presented above suggest that hrp expression precedes algD expression, possibly because a functional Hrp system can result in ‘signals' that stimulate algD activity. This was investigated by examining algD expression in tobacco leaves inoculated with DC3000-hrcC. The hrcC gene encodes an outer-membrane protein that is essential for type III protein secretion and has a primary role in protein translocation across the outer membrane (Charkowski et al., 1997). It is important to note that both DC3000 and DC3000-hrcC produce similar amounts of alginate in vitro [130–175 μg alginate (mg protein)−1]. Thus there is no evidence suggesting that the hrcC mutation has any effect on alginate production or algD expression in DC3000. When tobacco leaves were infiltrated with DC3000-hrcC containing pCPP2438 (hrpA : : uidA), pDCalgDP.Km (algD : : uidA) and pBBR.Gus (promoterless uidA), no HR was observed (Fig. 4b⇑), which is consistent with previous results (Deng et al., 1998). When tobacco tissue was harvested and monitored for GUS activity, a high level of gene expression was observed in tissue infiltrated with DC3000-hrcC(pCPP2438) (Fig. 4e⇑); this was not surprising since the hrpA gene is controlled by hrpL and its expression should be independent of hrcC (Hutcheson, 1999). However, GUS activity in tobacco leaves infiltrated with D3000-hrcC(pDCalgDP.Km) was negligible throughout the sampling period (Fig. 4e⇑) and virtually identical to DC3000-hrcC containing the promoterless uidA gene (Fig. 4e⇑).

    Production of
    Figure image not available in archive
    in tobacco inoculated with DC3000

    When P. syringae was exposed to compounds that are known to generate ROS, the expression of algT was activated in vitro (Keith & Bender, 1999). The algT gene encodes σ22, an alternative sigma factor that is required for expression of algD (Peñaloza-Vázquez et al., 1997; Wozniak & Ohman, 1994). Since alginate gene expression is known to be activated by ROS in vitro, we reasoned that these signals might also activate alginate production in the plant. For example, the algD : : uidA fusion was transcriptionally activated when tobacco was infiltrated with DC3000(pDCalgDP) (Fig. 4c⇑), but not with DC3000-hrcC(pDCalgDP) (Fig. 4e⇑). Since DC3000-hrcC does not induce an HR, these results suggest that the algD promoter is activated by signals generated prior to the HR, and these signals are absent or reduced when the HR is not elicited. To explore this hypothesis, pathogen-inoculated tobacco leaves were infiltrated with NBT, which facilitates the detection of

    Figure image not available in archive
    , one of the primary oxygen species produced during the HR (Baker & Orlandi, 1995). When NBT reacts with
    Figure image not available in archive
    , a dark blue insoluble formazan compound is produced (Beyer & Fridovich, 1987), which identifies the regions of superoxide formation in leaf tissue.
    Figure image not available in archive
    is thought to be the major oxidant species responsible for reducing NBT to formazan (Doke, 1983; Fryer et al., 2002). A transient production of
    Figure image not available in archive
    was observed in tobacco leaves inoculated with DC3000 and DC3000-hrcC at 4 h p.i. (Fig. 5⇓). However, in leaves inoculated with DC3000, a second burst of
    Figure image not available in archive
    production was detected at 8–10 h p.i. (Fig. 5⇓); this second phase of
    Figure image not available in archive
    accumulation was visibly reduced in tobacco leaves inoculated with DC3000-hrcC (Fig. 5⇓).

    Figure image not available in archive
    Fig. 5.

    Detection of

    Figure image not available in archive
    in tobacco tissue.
    Figure image not available in archive
    was monitored by NBT staining as described in Methods. Tobacco leaves were infiltrated with sterile distilled H2O, P. syringae pv. tomato DC3000, or DC3000-hrcC and stained with NBT at 2, 4, 6, 8, 10 and 12 h p.i. The arrow at 4 h p.i. indicates the initial, transient production of
    Figure image not available in archive
    (phase I) and the brackets indicate the more prolonged production of
    Figure image not available in archive
    (phase II); the latter occurs when tobacco is inoculated with DC3000, but not DC3000-hrcC. The experiment was repeated with similar results and the results shown are typical of both experiments.

    DISCUSSION

    In this study, algD expression was monitored in collard and tomato, which are hosts for P. syringae pv. tomato DC3000. The induction of algD in susceptible hosts is consistent with the role of alginate as a virulence factor; however, it is important to note that the disease lesion phenotype, the onset of algD expression and the visible level of algD expression differed with these two hosts. The expression of algD in susceptible tomatoes was visibly less than collard, was only visible in the late stages of infection (e.g. 168 h p.i.) and coincided with the production of necrotic lesions that lacked a water-soaked appearance (Fig. 2d⇑). However, the lesions developing on collard leaves initially had a water-soaked appearance (Fig. 1i⇑) and exhibited visible levels of GUS activity early in the infection process (e.g. 72 h p.i.). Alginate may be responsible for the water-soaked appearance of lesions since the polymer is highly hydrophilic and would help maintain moisture in the extracellular milieu.

    In many plant–pathogen interactions, protection from pathogen invasion is controlled by plant disease resistance (R) genes (Flor, 1971). R genes activate defence reactions by recognizing the presence of a corresponding avirulence (avr) gene of pathogen origin. The PtoR resistance gene in tomato encodes a serine-threonine protein kinase and confers resistance to isolates of P. syringae that express the avirulence gene avrPto, including strain DC3000 (Martin et al., 1993; van Dijk et al., 1999). When P. syringae pv. tomato DC3000 was spray-inoculated onto tomato Rio Grande-PtoR, an HR was visible when the tissue was examined by light microscopy (Fig. 3c, d⇑); however, necrotic cells were not visible without the aid of the microscope. This approach enabled us to monitor algD expression in internal and epiphytic populations of P. syringae pv. tomato DC3000. Interestingly, algD expression was observed in epiphytic populations of bacteria near plant cells undergoing an HR (Fig. 3e⇑) and in the apoplast near mesophyll cells that were responding hypersensitively (Fig. 3f⇑).

    As expected, P. syringae pv. tomato DC3000 elicited a prominent HR when infiltrated into leaves of tobacco, which is not a host for this pathogen (Fig. 4a⇑). Furthermore, algD was expressed in tobacco leaves undergoing the HR (Fig. 4c⇑), although algD expression was delayed and lower than that observed for the hrpA : : uidA fusion (Fig. 4c⇑). The lag period preceding algD expression in tobacco suggested that signals that are evolved during the onset of the HR might stimulate alginate gene expression. This was further investigated by analysing algD expression in DC3000-hrcC, which does not elicit an HR in tobacco leaves (Fig. 4b⇑). The hrpA : : uidA fusion in DC3000-hrcC was strongly induced in the absence of an HR (Fig. 4e⇑); this is consistent with regulation of hrpA transcription via the hrpL promoter, which should be functional in the hrcC mutant (Collmer et al., 2000). However, we found that algD expression was negligible in tobacco leaves inoculated with DC3000-hrcC (Fig. 4e⇑), which suggests that manifestation of the HR is a prerequisite for algD transcription in tobacco leaves inoculated with P. syringae pv. tomato DC3000. These results also suggest that the hrpA and algD promoters respond to different signals in the DC3000–tobacco interaction.

    The HR is often associated with an oxidative burst, which involves the production of potentially cytotoxic quantities of H2O2 and

    Figure image not available in archive
    (Wojtaszek, 1997). This phenomenon was first identified in mammalian phagocytes and was described as the respiratory burst (Segal & Abo, 1993). It is tempting to speculate that the ROS produced during the oxidative burst may function as signals for alginate gene induction in planta. The oxidative burst is generally described as a biphasic phenomenon in plant cells. During phase I, an immediate and transient production of ROS occurs and is non-specifically stimulated by compatible, incompatible and saprophytic bacteria (Baker & Orlandi, 1995). We speculate that phase I production of ROS occurred at approximately 4 h p.i., since NBT staining (Fig. 5⇑) indicated the presence of
    Figure image not available in archive
    in tobacco leaves infiltrated with both DC3000 (produces HR) and DC3000-hrcC (no HR). Furthermore, the detection of ROS at 4 h p.i. is consistent with the timing of phase I in previous studies (Wojtaszek, 1997). Phase II is characterized by a more prolonged production of ROS and is stimulated by incompatible bacteria that induce an HR (Baker & Orlandi, 1995; Levine et al., 1994). In our studies, the wild-type DC3000 is incompatible on tobacco, produces an HR and induces a second phase of ROS at 8–10 h p.i. (Fig. 5⇑). We hypothesize that this second burst (phase II) of
    Figure image not available in archive
    stimulates expression of the algD gene, either directly or indirectly. Since the DC3000-hrcC mutant does not elicit an HR in tobacco and does not induce phase II production of ROS, the signals for triggering algD expression may be absent or suboptimal in the DC3000-hrcC/tobacco interaction. In a highly relevant study, hrp mutants of P. syringae pv. tomato DC3000 failed to elicit significant levels of ROS in an incompatible interaction (Grant et al., 2000), which is consistent with our results.

    It is likely that ROS also function as signals for algD activation in tomato Rio Grande-PtoR inoculated with P. syringae pv. tomato DC3000. The interaction of AvrPto, which is present in DC3000, with the PtoR-encoded kinase initiates multiple defence pathways, including the production of ROS and elicitation of the HR (Jin & He, 2001; Sessa & Martin, 2000). Chandra et al. (1996) used tomato suspension cells to follow the oxidative burst in tomato lines challenged with P. syringae pv. tomato. Their results demonstrate that phase II of the oxidative burst is dependent on co-expression of PtoR in the tomato host and avrPto in the pathogen, an interaction that results in the HR (Chandra et al., 1996). In Rio Grande-PtoS, the second oxidative burst is either absent or occurs at sublethal levels, which do not have an impact on the multiplication of the bacteria.

    Although the plant signals that stimulate alginate production have not been identified, ROS are likely signals since they are known to stimulate alginate production in P. syringae and P. aeruginosa in vitro (Keith & Bender, 1999; Mathee et al., 1999). Furthermore, Venisse et al. (2001) recently demonstrated that a sustained production of ROS was required for initiation of necrotic lesion development by Erwinia amylovora in pear. Consequently, ROS may have a role in compatible interactions and could trigger alginate production in selected host–pathogen combinations. Further studies designed to identify the specific ROS that trigger algD expression are under way.

    Acknowledgments

    This work was supported by the Oklahoma Agricultural Experiment Station and grant AI 43311 (C. L. B) from the National Institutes of Health. We thank Aswathy Sreedharan for technical assistance, Alejandro Peñaloza-Vázquez and Chris Allen for helpful advice and comments, and Barbara Kunkel for providing the genomic library of P. syringae DC3000.

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