Plant Cell, Vol. 10, 917-924, June 1998, Copyright © 1998, American Society of Plant Physiologists
Inactivation of the sapA to sapF Locus of Er winia chr ysanthemi Reveals Common Features in Plant and Animal Bacterial Pathogenesis
Emilia López-Solanillaa,
Francisco García-Olmedoa, and
Pablo Rodríguez-Palenzuelaa
a Departamento de Biotecnología, Universidad Politécnica de Madrid, E.T.S. Ingenieros Agrónomos, Ciudad Universitaria s/n, E-28040 Madrid, Spain
Correspondence to:
Pablo Rodríguez-Palenzuela, pablo{at}bit.etsia.upm.es (E-mail), 341-3365757 (fax).
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ABSTRACT |
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We investigated the role in pathogenesis of bacterial resistance to plant antimicrobial peptides. The sapA to sapF (for sensitive to antimicrobial peptides) operon from the pathogenic bacterium Erwinia chrysanthemi has been characterized. It has five open reading frames that are closely related (71% overall amino acid identity) and are in the same order as those of the sapA to sapF operon from Salmonella typhimurium. An E. chrysanthemi sap mutant strain was constructed by marker exchange. This mutant was more sensitive than was the wild type to wheat 
-thionin and to snakin-1, which is the most abundant antimicrobial peptide from potato tubers. This mutant was also less virulent than was the wild-type strain in potato tubers: lesion area was 37% that of the control, and growth rate was two orders of magnitude lower. These results indicate that the interaction of antimicrobial peptides from the host with the sapA to sapF operon from the pathogen plays a similar role in animal and in plant bacterial pathogenesis.
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INTRODUCTION |
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Antimicrobial peptides are essential effectors of innate immunity, a nonspecific defense mechanism that seems to be shared by animals and plants (reviewed in Garcia-Olmedo et al. 1992 
, Garcia-Olmedo et al. 1995 ; Gabay 1994 ; Boman 1995 ; Hoffman 1995 ; Broekaert et al. 1997 ; Shewry and Lucas 1997 ). Peptide families identified in animals include both linear and disulfide-folded types (Gabay 1994 ; Ganz and Lehrer 1994 ; Boman 1995 ; Hoffman 1995 ), whereas only disulfide-containing antimicrobial peptides have been found in plants (Garcia-Olmedo et al. 1992 , Garcia-Olmedo et al. 1995 ; Broekaert et al. 1997 ; Shewry and Lucas 1997 ). Among the cysteine-rich plant peptides that have been shown to be active in vitro against pathogens are thionins, hevein- and knottin-like peptides, lipid transfer proteins (LTPs), and defensins (reviewed in Broekaert et al. 1997 ; Shewry and Lucas 1997 ) as well as snakins (Moreno 1995 ; A. Segura, M. Moreno, F. Madueño, A. Molina, and F. García-Olmedo, manuscript in preparation). The expression patterns of these peptides have been found to be compatible with a defense role, and enhanced tolerance to different pathogens has been observed through transgenic overexpression of genes encoding some of them, such as thionins (Carmona et al. 1993 ; Epple et al. 1997 ), defensins (Terras et al. 1995 ), or LTPs (Molina and Garcia-Olmedo 1997 ).
The role of antimicrobial peptides in animal innate immunity has been highlighted by the observation of increased susceptibility to infection in Drosophila mutants affected in their synthesis (Lemaitre et al. 1996 ) and by the fact that certain human disorders characterized by recurrent infections are associated with a lack of a particular class of antimicrobial peptides (defensins) in blood phagocytes (Ganz et al. 1988 ), but no such evidence is available with respect to plant peptides because appropriate plant mutants with decreased peptide levels have not yet been obtained. An alternative line of evidence about the role of antimicrobial peptides is based in the production of peptide-sensitive mutants of a pathogen. The hypothesis that peptides are involved in defense would be supported by a decrease of virulence in this type of mutant. Indeed, in the animal pathogen Salmonella typhimurium, both rough lipopolysaccharide (LPS) mutants and sapA to sapF (sap stands for sensitive to antimicrobial peptides) mutants, which show increased sensitivity to antimicrobial peptides, have reduced virulence, suggesting that resistance to host peptides has a direct role in Salmonella pathogenesis (Macias et al. 1990 ; Groisman et al. 1992 ; Parra-Lopez et al. 1993 ). Similarly, we found previously that thionin- and LTP-sensitive mutants of Ralstonia (Pseudomonas) solanacearum were altered in their LPS structure and were avirulent in tobacco (Titarenko et al. 1997 ).
Analysis of sap mutants from S. typhimurium has led to the discovery of genes sapA to sapF. These are required for peptide resistance and for virulence (Parra-Lopez et al. 1993 ). These genes are organized in a single operon and exhibit sequence similarity with ABC transporters described in prokaryotes and eukaryotes. The proposed mechanism of action for the Sap system includes binding of the periplasmic component SapA to the antimicrobial peptide, followed by peptide transport to the cytoplasm, where peptide degradation and/or activation of resistance determinants occurs.
We have now investigated the sap operon in Erwinia chrysanthemi, which is an economically important phytopathogenic bacterium that causes soft rot diseases in a wide range of crops (Perombelon and Kelman 1980 ). Active virulence mechanisms are known to contribute to the pathogenesis of this bacterium. The most important are the secretion of hydrolytic enzymes that attack the pectic fraction of the plant cell wall (Collmer and Keen 1986 ) and the induction of plant necrosis elicited by hrp (for hypersenstive response and pathogenicity) gene products (Bauer et al.. 1994 ). However, little is known about the passive virulence mechanisms that enable E. chrysanthemi to resist the action of antimicrobial agents from the plant host. Although S. typhimurium and E. chrysanthemi have very different pathogenic behavior, the fact that they are phylogenetically related and that antimicrobial peptides occur in their respective animal and plant hosts led us to investigate the possible role of the Sap system in plant–pathogen interactions. We report here that sap genes are structurally conserved in E. chrysanthemi with respect to S. typhimurium and that their inactivation by insertion mutagenesis makes the bacterium more sensitive to certain plant antimicrobial peptides and less virulent to plants. These data suggest that the SapA to SapF resistance mechanism plays a similar role in plant and animal pathogenesis.
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RESULTS |
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Cloning and Analysis of the sap Locus of E. chrysanthemi
To investigate the presence of a sap operon homolog in E. chrysanthemi, we analyzed genomic DNA from the AC4150 strain of this bacterium by DNA gel blotting. A fragment from plasmid pEG6179 was used as a probe (Parra-Lopez et al. 1993 ). pEG6179 contains most of the S. typhimurium sap operon. Two fragments that hybridized with this probe were cloned in the 
ZAPII vector and subcloned in pBluescript SK- (pB103, 6.5-kb fragment; pB104, 1.7-kb fragment). To obtain a clone containing the complete sapA to sapF operon, we made an E. chrysanthemi genomic library in the vector FIX II and screened it with probes obtained from inserts of plasmids pB103 and pB104. A positive phage was isolated, and the EcoRI-EcoRI 8.2-kb fragment, containing the whole operon, was subcloned in pBluescript SK- (Figure 1, pB105).
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Figure 1.
Genetic and Physical Map of the sap Region from E. chrysanthemi.
The deletion in sapA and insertion of the  interposon used for the construction of mutant BT105 are represented by bars. Binary alignments of the corresponding E. chrysanthemi– and S. typhimurium–deduced proteins were performed, and the identity percentage is indicated. E, EcoRI; P, PstI; S, SacII; Smr, streptomycin resistance; Spr, spectinomycin resistance.
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The nucleotide sequences of the inserts in plasmids pB103 and pB104 were determined (EMBL accession number AJ222649) and found to contain five open reading frames that were homologs of the five sap genes described for S. typhimurium by Parra-Lopez et al. 1993 . The deduced amino acid sequences aligned with the corresponding ones from S. typhimurium products, showing that the sap operon has the same structure in the two species. The average identity of the amino acid sequences of the homologous genes from the two species was 71%, which justified the designation of the E. chrysanthemi locus as sap and the corresponding open reading frames as sapA to sapF. Figure 1 shows a genetic and physical map of the sap region from E. chrysanthemi. Parra-Lopez et al. 1993 noted the occurrence of homologous sap sequences in E. herbicola. As shown in Figure 2, we have found sequences homologous to sap in seven out of eight species of Erwinia tested by DNA gel blot hybridization, using a 964-bp PstI fragment internal to sapA from pB103 as a probe.
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Figure 2.
Distribution of sap Sequences among Erwinia spp.
Gel blot hybridization analysis was performed using total chromosomal DNA digested with EcoRI and probed with the labeled 964-bp PstI fragment internal to the sap operon (Figure 1), as described in Methods. Lane 1, E. chrysanthemi AC4150; lane 2, E. carotovora subsp carotovora NCPPB312; lane 3, E. stewartii 2994; lane 4, E. c. subsp carotovora ATCC15713; lane 5, E. rhapontici 1025; lane 6, E. lupinicola 3299; lane 7, E. nigrifluens 3026; and lane 8, E. rubrifaciens 3032. Length standards are given at left in kilobases.
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Insertional Inactivation of the sap Locus in E. chrysanthemi
To ascertain the involvement of the sapA to sapF operon from E. chrysanthemi in resistance to antibiotic peptides, we mutated this locus. A region containing part of the sapA sequence was replaced by the interposon (Prentki and Krisch 1984 ) and then marker exchanged into the E. chrysanthemi chromosome (see Figure 1). Out of several recombinants (data not shown), one mutant strain, named BT105, was selected for further analysis. To investigate the effect of the sap mutation on outer membrane permeability, the susceptibility of BT105 to lysozyme, erythromycin, and rifampicin was assayed, and no difference with respect to the wild type was found (data not shown). Furthermore, no difference in the mutant with respect to the wild type was found for the following characteristics: growth rate, colony size and morphology, cell size and appearance, and production of pectic enzymes (data not shown).
Sensitivity of the BT105 mutant to antimicrobial peptides was compared with that of the wild type. Inhibition tests were performed in vitro with two peptides from potato tubers, namely, defensin-Pth1 and snakin-1, -thionin from wheat endosperm, and salmon protamine. As shown in Figure 3, the mutant had an increased sensitivity for snakin-1 and -thionin but not for defensin-Pth1 or protamine.
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Figure 3.
Susceptibility of E. chrysanthemi AC4150 and Mutant BT105 to Different Antimicrobial Proteins.
Bacteria and peptides were incubated at 30°C for 6 hr, diluted, and plated in King's B agar plates (King et al. 1954 ). Bacterial survival was measured by counting colony-forming units (cfu). The results are for a typical experiment from three independent trials, and the magnitude of standard errors was smaller than are the symbols in all cases. Closed circles, E. chrysanthemi AC4150; open circles, mutant BT105.
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Mutant BT105 was transformed with pB105, which contains the whole sapA to sapF operon, and the complemented strain recovered the resistance to snakin-1 and -thionin to wild-type levels.
Reduced Virulence of the sap Mutant
To investigate the possible effect on virulence of decreased resistance in vitro to antimicrobial peptides, potato tubers were chosen because typically they are affected by this pathogen and contain up to 0.3 mmol/kg fresh weight of snakin-1 (Moreno 1995 ; A. Segura, M. Moreno, F. Madueño, A. Molina, and F. García-Olmedo, manuscript in preparation). The sap mutant showed increased sensitivity in vitro to snakin-1. In each of three independent experiments, potato tubers were pair inoculated with a suspension containing the wild-type or the mutant strain. Necrotic areas of the developed lesions were measured in all of the tubers after 48 hr. The data shown in Table 1 were subjected to statistical analysis. The average necrotic area of lesions produced by the mutant was 37% that of the wild type, and this difference was statistically significant. A typical result is shown in Figure 4A and Figure 4D.
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Figure 4.
Soft-Rot Symptoms Produced by E. chrysanthemi AC4150 and Mutant BT105 on Potato Tubers.
Each tuber was pair inoculated (AC4150 at left and BT105 at right) three times with 50 µL of a suspension containing 5 x 10 5 cells plus the peptide.
(A) and (D) Control.
(B) Thionin (100 µM).
(C) Thionin (200 µM).
(E) Snakin (70 µM).
(F) Snakin (140 µM).
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The effect of added peptide on lesion size was investigated by addition of purified snakin-1 (0, 70, and 140 µM) or -thionin (0, 100, and 200 µM) to the inoculum (Figure 4). The intermediate concentration abolished the symptoms in the mutant but not in the wild type (Figure 4B and Figure 4E). These concentrations were lower than those required for in vitro inhibition of the mutant (Figure 3). The highest concentration abolished symptoms in the mutant and in the wild type (Figure 4C and Figure 4F). These results are consistent with an additive effect of the incorporated peptide and those originally present in the plant tissue.
Growth rates in the plant of the wild type and the mutant were also determined by inoculation in potato tuber discs, as shown in Figure 5A. The population of the BT105 mutant strain was two orders of magnitude below that of the wild type along all of the time intervals. Figure 5B shows that browning of the inoculated discs correlated well with the estimated sizes of the bacterial populations.
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Figure 5.
Growth of E. chrysanthemi AC4150 and Mutant BT105 on Potato Tuber Discs.
After inoculation of the discs with 5 x 104 cells, bacterial populations were estimated at different times by grinding the tissue and plating appropriate dilutions in King's B agar plates (King et al. 1954 ).
(A) Bacterial population of E. chrysanthemi AC4150 (closed circles) and mutant BT105 (open circles) on potato discs at different times. Bars represent SE.
(B) Browning of potato discs at different times, inoculated as in (A).
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DISCUSSION |
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Mutation of the sap Operon Supports the Involvement of Plant Antimicrobial Peptides in Defense
In animal pathogenesis, it has been shown that a pathogen must overcome inhibitors from the host to be able to proliferate in it. Thus, LPS and sap mutants of the bacterium S. typhimurium, which are more sensitive to antimicrobial peptides than is the wild type, have decreased or no virulence (Macias et al. 1990 ; Groisman et al. 1992 ; Parra-Lopez et al. 1993 ). More recently, a correlation between in vitro peptide sensitivity and virulence has been found in the analysis of mutants and complemented strains of the r faF gene, which encodes a heptose transferase involved in LPS biosynthesis in the plant pathogen R. solanacearum (Titarenko et al. 1997 ). However, indirect effects of the mutation in the r faF gene could not be completely excluded as responsible for the changes in virulence. This type of effect is less likely to occur in the case of the sap mutation reported here, because we have failed to find any additional effects of this mutation other than differential peptide sensitivity. Our results are consistent with a significant role of the Sap system in E. chrysanthemi virulence, which is similar to what has been observed in animal pathogenesis. This finding further supports the hypothesis that antimicrobial peptides are part of a primitive defense system that predates the evolutionary separation of plants and animals.
The ability of the Sap system to discriminate among different types of plant antimicrobial peptides raises the possibility that the specificity of the interaction between plant and pathogen might depend not only on the recognition of avirulence factors by the products of plant resistance genes but also on the recognition of predominant plant antimicrobial peptides by the pathogen resistance system. Indeed, the significant differences in virulence observed in the assays with potato tubers are consistent with the snakin-1 peptide being a key determinant of the interaction. In a screening of antibacterial peptides of potato tubers, snakin-1 was the peptide found at the highest concentration (0.3 mmol/kg fresh weight) (Moreno 1995 ; A. Segura, M. Moreno, F. Madueño, A. Molina, and F. García-Olmedo, manuscript in preparation). This in vivo concentration is within the range of peptide concentrations in which differential inhibition of mutant BT105 was found in the in vitro assays. In contrast, the concentration of defensin Pth-1, the second most abundant antimicrobial peptide in potato tuber, was 100-fold lower (Moreno et al. 1994 ). Intraspecific and interspecific variations in peptide composition in plants could contribute significantly to the determination of host specificity and range in combination with observed natural differences in peptide sensitivity among strains of a given pathogen (Molina and Garcia-Olmedo 1993 ). Furthermore, at least part of the reported differences of virulence among different strains of E. chrysanthemi when confronted with different hosts (Dickey 1978 ; Boccara et al. 1991 ) could be related to variation in plant peptide composition and/or changes of peptide specificity of the sap operon among the different pathogen strains.
The quantitative importance of the sapA to sapF operon contribution to virulence can be ascertained by comparing the effects of its mutation with those of pelE and hrp genes. Thus, in the standard potato tuber virulence assay of E. chrysanthemi, the sap mutant produced lesions that were ~37% the size of those of the wild type, whereas a mutant lacking PelE, the most important pectolytic enzyme for maceration, has been reported to retain 50% of wild-type virulence (Payne et al. 1987 ).
There is ample evidence that Erwinia spp elicit the full array of defense reactions in the plant (Davis and Ausubel 1989 ; Castresana et al. 1990 ; Palva et al. 1992 ; Yang et al. 1992 ; Bauer et al. 1994 ). Nevertheless, E. chrysanthemi causes disease in a large number of different hosts, which suggests that this bacterium has evolved efficient mechanisms to withstand the host's defense mechanisms and that these mechanisms are important for pathogenicity. The Sap system is one of them.
Several recent reports have shown that animal and bacterial pathogens share common pathogenic strategies, namely, that some of the above-mentioned hrp genes from phytopathogenic bacteria are homologs to certain components of the type III secretion system found in Yersinia, Shigella, and Salmonella spp (Van Gijsegem et al.. 1993 ). The hrp genes play a key role in pathogenicity through their ability to deliver virulence proteins into the animal or plant host cells (Alfano and Collmer 1996 ). According to the results reported here, the Sap system is also a common feature in animal and plant bacterial pathogenesis.
Conservation and Specificity of the sapA to sapF Operon
The conservation of gene order and the sequence similarity of the sapA to sapF operon from E. chrysanthemi with respect to that from S. typhimurium are consistent with the evolutionary relatedness of the two bacterial species. However, functional divergence has probably occurred, as shown by the different behavior of the two versions of the operon with respect to protamine: mutation of the operon in the animal pathogen determined differential sensitivity to protamine and melittin but not to other peptides, such as magainin-2 and defensin NP-1 (Groisman et al. 1992 ; Parra-Lopez et al. 1993 ), whereas that reported here for the plant pathogen affects sensitivity to snakin-1 and -thionin but not to protamine and defensin-Pth1. This suggests that coevolution of the defense mechanism with the predominant pathogen(s) of the host may have occurred.
Our data show that the sapA to sapF operon is responsible for specific resistance to given antimicrobial peptides and not to a general one. The model for sap operon function in S. typhimurium proposes that the first step of the defense mechanism consists of the binding of the periplasmic component SapA to the antimicrobial peptide (Parra-Lopez et al. 1993 ). This binding could provide the observed specificity with respect to the different peptides. However, specificity could also reside on any of the other proposed steps of the Sap mechanism or even depend on other genetic factors outside of the sap operon.
In contrast to the mechanism of resistance to antimicrobial peptides associated with the LPS (Macias et al. 1990 ; Groisman 1994 ; Titarenko et al. 1997 ), the Sap mechanism is not associated with changes in outer membrane permeability because susceptibility to lisozyme, erythromycin, and rifampicin was unchanged in the mutant. This mechanism seems to have little or no involvement in other cellular processes, as determined by the lack of pleiotropic effects of the mutation on properties such as growth rate in liquid medium, colony size and morphology, cell size and appearance, and production of pectic enzymes. Our data show that the Sap system confers specific resistance to certain antimicrobial peptides and that this feature makes an important contribution to bacterial virulence. Determining the Sap mechanism of resistance should help us to understand the basis for its specificity and its importance in different plant hosts.
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METHODS |
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Microbiological Methods, and DNA Manipulation and Sequencing
Bacterial strains and plasmids used in this work are described in Table 2. Strains of Escherichia coli were cultivated at 37 °C in Luria-Bertani medium. Strains of Erwinia chrysanthemi were cultivated at 30°C in nutrient broth (Difco) or King's B medium (King et al. 1954 ). Antibiotics were added to the media at the following concentrations: ampicillin, 100 µg/mL; spectinomycin, 25 µg/mL; and streptomycin, 125 µg/mL (for multicopy plasmid resistance) or 10 µg/mL (for chromosomal resistance). Marker exchange in E. chrysanthemi was performed as described by Roeder and Collmer 1985 . A genomic library of E. chrysanthemi was constructed in ZAP II and in FIX II (Stratagene, La Jolla, CA). Phagemid pBluescript SK- (Stratagene) was used for subcloning. Standard molecular cloning techniques employed in this study (small- and large-scale plasmid and genomic DNA purification, restriction enzyme digestion [Pharmacia], agarose gel electrophoresis, DNA subcloning [Stratagene], blot and hybridization and colony screening by hybridization [Amersham]) were performed as described by Sambrook et al. 1989 and following the manufacturers' indications. DNA sequencing of both strands was done by the chain termination method on double-stranded DNA templates, using an Abiprism Dye Terminator cycle sequencing kit (Perkin-Elmer) in a 377 DNA Sequencer (Perkin-Elmer). Sequence alignments were performed at the National Center for Biotechnology Information (http:// www.ncbi.nlm.nih.gov/), using the Blast network service (Atschul et al. 1990 ).
Antimicrobial Peptide Purification
Thionin was purified from wheat flour, as described by Ponz et al. 1982 . Defensin-Pth1 and snakin-1 were purified from potato tuber as follows: 20 g of frozen material was ground to powder in liquid nitrogen, using a mortar and pestle, and extracted once with 80 mL of buffer (0.1 M Tris-HCl, 10 mM EDTA, pH 7.5) and twice with 80 mL of H2O. The resulting pellet was then extracted with 50 mL of 1.5 M LiCl at 4°C for 1 hr, and the extract was dialyzed against 5 liters of water using a Spectra/Por 6 (molecular weight cutoff, 3000; Spectrum, Laguna Hills, CA) membrane and freeze dried. The extract was subjected to reverse-phase HPLC, as described by Molina et al. 1993 . Collected peaks were analyzed by SDS-PAGE in preformed gradient gels (4 to 20%; Bio-Rad). Protamines were purchased from Sigma.
Susceptibility and Virulence Assays
Susceptibility to antimicrobial peptides was assayed as follows: Log phase cells grown in nutrient broth were diluted to 10 5 colony-forming units per mL in 0.3 x nutrient broth, and 10 µL of the diluted material was placed in an Eppendorf tube and added to the appropiate amount of peptide dissolved in the same solution to reach the desired concentration. Cells and peptides were incubated for 6 hr at 30°C with shaking, and then a portion of each sample was diluted and plated on nutrient broth agar plates to assess bacterial viability.
Virulence of E. chrysanthemi AC4150 wild type and BT105 mutant was assayed by inoculating 50 µL of a suspension containing 5 x 10 5 bacteria in a potato tuber, cv Jaerla, purchased in a local supermarket. The bacteria were contained in a plastic tip, which was inserted at a constant depth of 1.5 cm. Two inoculations (wild-type and mutant strains) were made in each tuber. Potatoes were left at 30°C with 100% relative humidity for 48 hr. After this time, tubers were sliced at the inoculation point, and the damage was estimated by measuring the affected area. Differences between the wild type and the mutant were assessed statistically using a paired Student's t test. To monitor bacterial growth in potato tuber, 50 µL of a bacterial suspension containing 10 4 bacteria was inoculated in 1-cm in diameter potato discs. Discs were incubated at 30°C and high humidity, and were recovered at different times and ground with a tissue homogenizer in 500 µL of 10 mM MgCl2. Bacterial colony-forming units in the homogenate were determined by dilution plating.
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ACKNOWLEDGMENTS |
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We gratefully acknowledge Dr. Eduardo Groisman for the generous gift of plasmid pEG6179 and Drs. Javier Paz-Ares, Josefa Muñoz Alamillo, and José Manuel Palacios for critical reading of the manuscript. We also acknowledge Joaquín García, Carlos Rojas, Angeles Rubio, and Dolores Lamoneda for technical assistance. This work was financed by the Comisión Interministerial de Ciencia y Tecnología (Spain), No. DGICYT P92-0325. E.L.-S. was a recipient of a fellowship from the Ministerio de Educación y Ciencia (Spain).
Received December 29, 1997; accepted April 17, 1998.
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ABSTRACT
INTRODUCTION
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