Molecular Characterization of Natural Erwinia pyrifoliae Strains Deficient in Hypersensitive Response

Abstract

From necrotic tissue of a Nashi pear tree, 24 Erwinia pyrifoliae strains, found to be identical by pulsed-field gel electrophoresis analysis, were isolated. Thirteen strains were not virulent on immature pears and did not induce a hypersensitive response in tobacco leaves. The defective gene hrpL was complemented with intact genes from E. pyrifoliae and Erwinia amylovora.

A bacterial disease similar to fire blight was observed on Nashi pear trees (Pyrus pyrifolia) in South Korea, caused by the novel species Erwinia pyrifoliae (16). The pathogen was consistently isolated from orchards in the area of Chuncheon from 1995 to 1998 and identified on semiselective agar plates and by PCR (15, 16). E. pyrifoliae is restricted mainly to pear and is highly related to pear-pathogenic Erwinia strains from Japan (14). It can be distinguished from the fire blight pathogen Erwinia amylovora by several microbiological and molecular features (13). Fire blight was first described for North America and has spread to New Zealand, Europe, and the Mediterranean region (6, 10). The disease has not been reported in countries of the Southern hemisphere other than New Zealand, except for a transient occurrence in 1997 in Australia (11). The pathogenicity of E. amylovora depends on both the synthesis of the acidic exopolysaccharide (EPS) amylovoran (2, 17) and a functional type III secretion system, involved in induction of a hypersensitive response (HR) (1, 5, 9, 17). The pear pathogen E. pyrifoliae shares substantial homologies to ams genes and also requires capsular EPS for pathogenicity (12).

Natural HR-deficient strains.

In 1997, necrotic tissue from a Nashi pear tree was obtained from Korea and extracted for isolation of the causative pathogen. White colonies on Luria-Bertani (LB) agar (with 50 μg of cycloheximide/ml to avoid fungal growth) were assayed for the mucoid, yellowish colony morphology typical of E. pyrifoliae on MM2Cu agar as described before (13, 15, 16). The isolates were further assayed on LB plates with 5% sucrose and on MM1Cu agar as described for identification of E. amylovora (3). A lack of levan synthesis and no growth on MM1Cu agar were additional properties expected from E. pyrifoliae. The synthesis of capsular polysaccharide of the isolates on MM2Cu agar excluded being deficient in a gene associated with EPS synthesis. The 24 isolates were confirmed to be E. pyrifoliae by PCR analysis (4) with primers derived from the rRNA genes and the cps region (15). The strains were then assayed on pear slices for formation of an exudate (ooze) to assess their virulence and infiltrated into tobacco leaves in order to determine their capacity to elicit an HR (Fig. 1, leaf sections 1 and 2). Fewer than one-half of these isolates produced exudate on pear slices and the typical necrotic symptoms at the infiltrations sites on tobacco leaves (Table 1). The ability to cause HR is a requirement for virulence of E. amylovora strains (1, 17). Harpin, encoded by hrpN, is transported to the surface of the bacteria and elicits HR in the plant tissue (20). The dsp locus, adjacent to the hrp gene cluster, encodes Avr-like proteins (5, 9) and is required for pathogenicity but does not affect HR. A deficiency of dsp-related genes of E. pyrifoliae was therefore not assumed for the nonpathogenic isolates.

FIG. 1.

HR assays on tobacco (cv. Samsun) with the HR-negative strain Ep2/97 (section 1), the coisolated positive strain Ep4/97 (section 2), and Ep2/97(pGEM-hrpL) with hrpL from strain Ep4/97 (section 3). The bacteria were grown in LB medium, incubated in inducing medium (7), suspended in water, and then infiltrated into tobacco leaves at 10⁸ CFU/ml.

TABLE 1.

Results of assays for identification and characterization of E. pyrifoliae

Strain(s) tested	Result for indicated assaya					Reference(s) or source
	HR	Vir	PCR	MM2Cu	KB
E. amylovora
CFBP1367 (Ea321), CFBP1430, Ea1/79	+	+	−	+/+	−	1, 8, 21
Other bacterium
SLR21	−	−	−	−	−	South Koreab
E. pyrifoliae
Ep8/95, Ep1/96, Ep16/96, Ep28/96, Ep31/96, Ep4/97, Ep5/97, Ep42/97, Ep44/97, Ep45/97, Ep53/97, Ep43/97, Ep55/97, Ep56/97, Ep58/97, Ep60/97	+	+	+	+/+	−	16, this studyc
Ep2/97, Ep3/97, Ep41/97, Ep46/97, Ep47/97, Ep48/97, Ep49/97, Ep50/97, Ep51/97, Ep52/97, Ep54/97, Ep57/97, Ep59/97	−	−	+	+/+	−	This studyc

^aHR, assay on tobacco; Vir, assay for virulence on pear slices; PCR, assays were done as described before (3) with primers for specific detection of E. pyrifoliae (15); MM2Cu, colony morphology on MM2Cu agar; KB, fluorescence on King's medium B (16); +, positive for E. pyrifoliae; −, negative for E. pyrifoliae; +/+, yellow to slightly yellow color and mucoid.

^bIsolated from necrotic Nashi pear tissue in 1996 in South Korea; this strain is not E. pyrifoliae.

^cStrains ending with 97 were isolated from the bark of a branch from a diseased Nashi pear tree (Pyrus pyrifolia) in 1997 obtained from South Korea.

Identity of PFGE patterns.

To confirm genomic similarity between the pathogenic, HR-positive strain Ep4/97 and the nonpathogenic, HR-negative strain Ep2/97, we applied pulsed-field gel electrophoresis (PFGE) analysis after digestion with restriction enzymes XbaI and SpeI. PFGE analysis of E. pyrifoliae strains can be used as a stringent method for identification of the pathogen. E. amylovora strains isolated at different times in various European and Mediterranean regions were closely related in their PFGE patterns after digestion with XbaI and SpeI, and a common origin for these strains was assumed (10, 21). For both E. pyrifoliae strains, the XbaI fragments obtained were identical, matching strains Ep8/95, Ep1/96, and Ep16/96 isolated in 1995 and 1996 in South Korea, but the DNA fragments were different for strain SLR21 (Fig. 2). This strain was isolated from necrotic Nashi pear tissue in 1996 and also differed from E. pyrifoliae by other molecular and microbiological properties (Table 1). Although the SpeI fragments of E. pyrifoliae strains have been more diverse than XbaI patterns (15), the digests of genomic DNA from strains Ep2/97 and Ep4/97 with SpeI also produced identical patterns in PFGE analysis (data not shown). Matching of the PFGE patterns supported the assumption that the HR-deficient mutant Ep2/97 was derived from Ep4/97 during development of the disease in the affected Nashi pear tree and excluded isolation of strains with microbiological and molecular properties that were merely similar to those of E. pyrifoliae. This coexistence of virulent and nonvirulent strains in the population is a rare example of a possible advantage for a nonpathogenic variant of a bacterial pathogen. The spontaneous hrp mutants seem to be well suited to support the HR-positive parent strain during colonization by the pathogen and may also enhance pathogen survival in necrotic plant tissue.

FIG. 2.

PFGE analysis to show the relatedness of E. pyrifoliae strains Ep2/97 and Ep4/97. Genomic DNA of agar-embedded cells was digested with restriction enzyme XbaI, and the PFGE was performed in a 1% agarose gel (Ultra Pure DNA grade agarose; Bio-Rad) with running buffer (3.5 mM HEPES, 3.5 mM sodium acetate, 0.35 mM EDTA [pH 8.3]) for 22 h at 14°C and a ramping time from 1 to 25 s. M, positions of marker DNA in kilobases. Multimeric λ genomes were run in a separate lane. Lanes: 1, E. pyrifoliae Ep8/95 (pattern identical with those of strains Ep1/96 and Ep16/96 in XbaI digests); 2, SLR21 (from Asian pear; not E. pyrifoliae); 3, Ep2/97; 4, Ep4/97; 5, E. amylovora Ea1/79.

Complementation.

The properties of the 13 isolated defective E. pyrifoliae strains showed a correlation of virulence on pears and HR on tobacco (Table 1). An EPS-deficient mutant of E. pyrifoliae had previously been complemented with ams genes of E. amylovora (12). In a similar approach, we attempted to complement the HR-deficient strain Ep2/97 with hrp genes of E. amylovora. Spontaneous mutants with streptomycin resistance were selected, and strain Ep2/97Sm was conjugated with Escherichia coli strain S17-1 carrying cosmids pPV130, pPV132, or pPV133 (1). The cosmids contain partially overlapping regions of the hrp cluster (Fig. 3). Ep2/97Sm(pPV130) and Ep2/97Sm(pPV133) gave a positive HR after incubation in inducing medium (7). To narrow down the area responsible for complementation, several smaller plasmids (9) covering DNA insertions of the positive fragment were introduced by electroporation: pMAB5, -31, -32, -38, -40, and -71 (Fig. 3). Only Ep2/97(pMAB71) induced a positive HR, indicating that the mutation affected either hrpL or hrpJ. To confirm the ability of pMAB71 for complementation of HR-deficient variants, this plasmid was also introduced into strains Ep41/97, Ep46/97, Ep49/97, Ep52/97, and Ep57/97 (Table 1), where HR was restored in each case. To dissect the involvement of hrpL or hrpJ, primers were designed from the nucleotide sequences of the E. amylovora genes (EMBL nucleotide sequence database accession numbers U36244 and L25828) as follows: for amplification of hrpL, HRPL1 (5′-GGCACAAGCCTTGCTAA) and HRPL2c (5′-CGGCAAGACAGGACACT), and for amplification of hrpJ, HRPJ1 (5′-TATGTCGCTGGCGACTT) and HRPJ2c (5′-CTGATGGCGAGGCGATT). In the case of the hrpJ primers, only PCR fragments from CFBP1430 and Ea321 could be cloned into pGEM-T (Promega). A faint PCR band but no cloning product was obtained with E. pyrifoliae strains as a template. Ep2/97 was not complemented with E. amylovora hrpJ clones, although a putative promoter region was part of the amplified DNA. In PCR amplifications of hrpL, the E. amylovora strains CFBP1430, Ea321, and Ea1/79 as well as the E. pyrifoliae strains Ep1/96, Ep16/96, Ep2/97, and Ep4/97 produced DNA fragments which were cloned into plasmid pGEM-T. The resulting plasmids were assayed with restriction enzyme PstI for insertion of the gene in the direction of the lac promoter (pGEM-hrpL). Strain Ep2/97 was successfully complemented for HR on tobacco with pGEM-hrpL carrying inserts from the HR-positive E. pyrifoliae strains Ep4/97 (Fig. 1, leaf section 3), Ep1/96, and Ep16/96 and also from E. amylovora strains CFBP1430, Ea321, and Ea1/79. With the cloned hrpL fragment from the nonpathogenic strain Ep2/97 in pGEM-T, HR on tobacco leaves and virulence on pear tissue were not restored for Ep2/97, confirming the nonfunctional gene in this strain. On the other hand, Ep2/97 with pGEM-hrpL of virulent E. amylovora and E. pyrifoliae strains caused necrotic symptoms on pear seedlings, which were slightly retarded compared to those of the wild-type strain Ep4/97, possibly due to the instability of the complementing plasmid. Attempts to find spontaneous HR-negative mutants by passaging Ep4/97 on nutrient agar plates or by reisolation of cells from slime formed on pear slices were not successful. Mutation to HR deficiency is therefore not frequent for E. pyrifoliae wild-type strains.

FIG. 3.

Restriction map of cosmids and subclones used for complementation of the HR-deficient E. pyrifoliae strain Ep2/97. The dark central line indicates the dsp-hrp region of E. amylovora CFBP1430 (1). The plasmids named by numbers are insertions in vector pUC18, except for pMAB40 with an insertion in pRK767. Restriction sites are indicated for EcoRI (long lines pointing down, E), BamHI (short lines down, B), HindIII (short lines up, H), and SalI (long lines up, S). The hrp cluster and the dsp region (left) are indicated by broken lines.

Change of single base pair.

The insertions obtained from strains Ep2/97 and Ep4/97 were sequenced with labeled SP6 and T7 primers in both strands by using an ALF sequencer, and the nucleotide sequences were independently confirmed by commercial sequencing. A comparison of the nucleotide sequences of the HR-positive and the HR-negative strains revealed a change of nucleotide C into T at position 277 (Fig. 4), converting the codon TCA (for serine) into TTA (for leucine), thereby changing a polar amino acid into an unpolar amino acid. In the amino acid sequences of HrpL of E. amylovora (SWISS-PROT accession number Q46616) and of Pseudomonas syringae (P37929), this amino acid is also serine, which may be needed for the function of the protein. The amino acid sequences for HrpL of E. pyrifoliae and E. amylovora were 95% identical to each other and differed at eight positions, which were located mainly at both ends of the protein (Fig. 5). The nucleotide sequence of hrpL from Ep16/96 corresponded with the nucleotide sequence from Ep4/97 with C at position 277. To reconfirm the hrpL sequences, DNA from strains Ep2/97, Ep4/97, and Ep16/96 was also amplified with a proofreading DNA polymerase (AccuTaq; Sigma). The same sequence as in Fig. 4 was obtained for the three wild-type strains, and the change from C to T at position 277 for strain Ep2/97 was observed.

FIG. 4.

Nucleotide sequence of the hrpL gene of E. pyrifoliae strain Ep4/97. The nucleotide change in Ep2/97 (from C to T) is indicated with dark shading. The PCR primers used for amplification are in italics and underlined at both ends of the nucleotide sequence, and a putative ribosome binding site is printed in bold. The triplets indicate the range of the structural gene.

FIG. 5.

Alignment of the HrpL proteins from E. amylovora strain Ea1/79 and E. pyrifoliae strains Ep4/97 and Ep2/97. Identical amino acids are indicated by dots, and the amino acid change (to L) in Ep2/97 is shaded in black.

Growth properties.

HrpL of E. amylovora has been associated with the function of a σ-factor, controlling expression of the hrp gene cluster (19). The mutation in the investigated population of E. pyrifoliae from the necrotic tissue of Nashi pears can be explained by a spontaneous base change. The mutants lacking recognition by plant defense mechanisms could derive an advantage in some stages of the pathogen's life cycle. We coinoculated the HR-deficient strain Ep2/97Sm and wild-type Ep4/97 mixed at different ratios (10:1, 1:1, and 1:10) into slices of immature pears. In most cases, approximately equal amounts of cells from each strain were recovered in the exudate developed after 1 week. These data indicate a helper function of the HR-positive strain for efficient multiplication of the HR-negative variant in host plant tissue. After inoculation of E. amylovora hrp mutants, which had been complemented with cosmids carrying parts of the hrp cluster, a large proportion of the recovered cells had lost the cosmid, indicating feeding by the other bacteria (1), similar to in vivo complementation between E. amylovora ams and dsp mutants (2). Although Ep2/97 was not virulent due to its inability to damage plant cells, the wild-type strain relieved the restriction, allowing both strains to grow. In the presence of wild-type cells, a spontaneous HR-deficient variant may be advantageous in pear tissue, because it does not challenge the plant defense mechanism (18). In addition, the variant could also have a better chance than the HR-positive parent strain for survival during decline of the population in a late stage of plant colonization.

Nucleotide sequence accession number.

The nucleotide sequence of hrpL of E. pyrifoliae strain Ep4/97 has been deposited in the EMBL nucleotide sequence database with the accession number AJ438881.