Lehrstuhl für Genetik, Fakultät für Biologie, Universität Bielefeld, Postfach 100131, D-33501 Bielefeld, Germany1
Author for correspondence: Alfred Pühler. Tel: +49 521 106 5607. Fax: +49 521 106 5626. e-mail: alfred.puehler{at}genetik.uni-bielefeld.de
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ABSTRACT |
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Keywords: Xanthomonas campestris, Capsicum annuum, tonB, exbB, exbD
Abbreviations: HR, hypersensitive response
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INTRODUCTION |
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The hrp genes (hypersensitive response and pathogenicity) are essential for virulence on host plants and for induction of a HR on nonhost plants. hrp genes have been identified in Pseudomonas syringae spp., Ralstonia solanacearum (formerly Pseudomonas solanacearum), Erwinia amylovora and X. campestris pathovars (Bonas, 1994 ; Willis et al., 1991
). Several hrp gene products show sequence homology to proteins mediating the type III secretion of virulence factors from animal bacterial pathogens and are therefore predicted to be involved in the secretion of molecules essential for the interaction with the plant (Fenselau et al., 1992
; Fenselau & Bonas, 1995
; Wengelnik et al., 1996
). Hrp-dependent secretion has been demonstrated for the harpin proteins of E. amylovora, Erwinia chrysanthemi and P. syringae pv. syringae and the PopA protein of R. solanacearum (Arlat et al., 1994
; He et al., 1993
; Wei et al., 1992
).
Over 30 avirulence (avr) genes have been identified as essential for the HR in host and nonhost plants. In a gene-for-gene relationship, avirulence genes confer cultivar-specific interactions on resistant hosts as well as on nonhost plants (Dangl, 1994 ; Ritter & Dangl, 1995
). Members of the avirulence/pathogenicity gene family were shown to encode functional nuclear localization signals (Yang & Gabriel, 1995
). These proteins are therefore proposed to be translocated into the plant nucleus. Recently, the AvrBs3 protein from X. campestris pv. vesicatoria was shown to be recognized inside the plant cell and to require functional nuclear localization signals (Van den Ackerveken et al., 1996
).
Moreover, iron is considered to play a crucial role in plantphytopathogen interactions (Expert et al., 1996 ; Loper & Buyer, 1991
). Iron is one of the factors that limits bacterial growth in planta because micromolar concentrations of iron are necessary to support growth and multiplication. The common strategy of bacteria to overcome this problem is the utilization of siderophores with high affinity for ferric iron (Raymond et al., 1989
). Ferric iron is complexed by these chelators, which are transported into the cell via an energy-coupled process mediated by the TonB system (reviewed by Braun, 1995
). Outer-membrane receptors and the cytoplasmic membrane proteins TonB and ExbBD are involved in this process. In current models, the TonB protein induces a conformational change of the receptor proteins, which causes the release of bound ligands into the periplasmic space. Since the translocation into the periplasm is energy-dependent, TonB is considered to transduce the cytoplasmic membrane proton force to the receptor proteins (Bradbeer, 1993
). Two auxiliary proteins, ExbB and ExbD, forming a complex with TonB, are necessary for TonB-dependent uptake. In addition to import processes across the outer membrane, the TonB system mediates signal transduction from the cell surface to the cytoplasm. This was shown for the induction of transcription of ferric citrate and pseudobactin-transport genes of Escherichia coli and Pseudomonas putida, respectively (Härle et al., 1995
; Koster et al., 1994
).
Recently, we identified the tonB, exbB and exbD1 genes of X. campestris pv. campestris, which are essential for ferric iron uptake. In contrast to all bacteria investigated for TonB systems so far, a second exbD gene was identified in X. campestris pv. campestris (Wiggerich et al., 1997 ). The deduced amino acid sequences of the exbD1 and exbD2 genes showed 38·2% identity and 71·3% similarity to each other. Interestingly, we were able to show that exbD2 mutants are not impaired in ferric iron uptake. Moreover, the ExbD2 protein cannot substitute the ExbD1 protein in ferric-ironsiderophore uptake (Wiggerich et al., 1997
). All four genes, tonB, exbB, exbD1 and exbD2, were found to be clustered in X. campestris pv. campestris. Due to low coding probability and the wide spread of translational termination codons in all three reading phases, no other open reading frames were identified downstream of exbD2. Each gene was predicted to have its individual promoter as could be deduced from the analysis of several strains with insertional mutations (Wiggerich et al., 1997
). In the current paper, we report that the tonB, exbB, exbD1 and exbD2 genes are necessary for a HR on the nonhost plant pepper.
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METHODS |
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DNA methods, hybridization, transformation and electroporation.
Plasmid DNA was isolated using a rapid boiling lysis technique (Arnold & Pühler, 1988 ). Total DNA was isolated by lysozyme/sarcosyl/proteinase K lysis, followed by phenol/chloroform extraction and 2-propanol precipitation (Meade et al., 1982
). Restriction enzymes and other commercially available enzymes were obtained from Pharmacia or BRL and used in accordance with the manufacturers recommendations. Restriction-endonuclease-generated fragments of total DNA were separated by gel electrophoresis and subsequently vacuum blotted on nylon filters as described by Simon et al. (1991)
. Labelling of the probe (vector and DNA region) and hybridization were done as described elsewhere (Simon et al., 1991
). Transformation of E. coli cells and electroporation of X. campestris pv. campestris with plasmid DNA were performed as described by Morrison (1977)
and Wiggerich et al. (1997)
, respectively. Agarose gel electrophoresis was carried out as described by Maniatis et al. (1982)
.
Construction of plasmids.
Plasmids pHGW100 and pHGW31 were described previously (Wiggerich et al., 1997 ). Plasmids pHGW241, pHGW242 and pHGW243 harboured the tonB, exbB and exbD1 gene of X. campestris pv. campestris, respectively (Wiggerich et al., 1997
). A 1·3 kb BamHIXbaI subfragment of pHGW100 carrying the exbD2 gene of X. campestris pv. campestris resulted from nested deletions with exonuclease III and S1 nuclease (nested deletion kit from Pharmacia). This 1·3 kb BamHIXbaI DNA fragment was cloned into pHGW31 and the resulting plasmid was designated pHGW244 (Fig. 1
).
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Pathogenicity test.
Brassica oleracea cultivar Braunschweiger (cauliflower) was used for inoculation of mature host plants. The plants were maintained under growth room conditions described for the HR test. The plants were inoculated by injecting approximately 10 µl bacterial culture (1x109 cells ml-1) suspended in sterile water into the leaf margin. Symptom development was scored for 14 d after infection. Reisolation of bacteria from cauliflower leaf tissue was carried out as described for pepper.
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RESULTS AND DISCUSSION |
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The HR-inducing abilities of the mutant strains were also tested on the pepper cultivars Yolo Wonder and Pusztagold. The types of reaction were identical to those with cultivar California Wonder (data not shown).
To confirm that all effects observed were due to the insertions located in the genes investigated, we tested the tonB, exbB, exbD1 and exbD2 mutants complemented with a copy of the particular wild-type gene for their ability to induce an HR. To complement the exbD2 mutant B100-11.03, the suicide vector pHGW244 (exbD2), carrying the corresponding subfragment of the 3·6 kb HindIIISmaI wild-type DNA, was constructed and integrated into the genome by a single-crossover event. The correct integration was confirmed by Southern hybridization (data not shown). Mutants B100-5.05::pHGW241 (tonB), B100-7.03::pHGW242 (exbB) and B100-9.01::pHGW243 (exbD1) were described previously (Wiggerich et al., 1997 ). Each merodiploid mutant regained the ability to induce wild-type HR symptoms when infiltrated into pepper leaves. However, symptom formation was delayed by 2 d.
To rule out the possibility that the inability of the exbD1 mutant B100-9.01 to induce an HR was due to the lack of both ExbD1 and ExbD2 proteins, we tested strain B100-9.21 carrying an aacC1 gene in the exbD1 gene bringing the exbD2 gene under the control of the constitutively active Pout promoter of the aacC1 gene (Wiggerich et al., 1997 ). Like strain B100-9.01 mutant strain B100-9.21 induced only slight chlorosis in the infected leaf tissue (data not shown).
The X. campestris pv. campestris tonB, exbB and exbD1 mutants died rapidly, but the exbD2 mutant grew even better than the wild-type strain in pepper leaf tissue
As has been shown previously, the TonB system of X. campestris pv. campestris is involved in ferric iron uptake ex planta (Wiggerich et al., 1997 ). Therefore, the investigated mutant strains could have failed to acquire iron in planta, resulting in a lack of growth. To analyse if this was the reason why the tonB/exbBD1D2 mutant strains failed to induce an HR on pepper we scored the viable bacteria by reisolation from infiltrated pepper leaf tissue for the first 2 d after infection (Fig. 3
). Surprisingly, the mutants behaved differently. During the first 4 d after infiltration the bacterial titre of the wild-type strain B100 increased from 1x106 bacteria initially infiltrated to 7x107 bacteria per cm2 tissue. As expected, within the next 4 d, the bacterial titre decreased rapidly to a value of 5x106 cells per cm2. In contrast, the viable bacterial titre of the mutant strains B100-5.05 (tonB), B100-7.03 (exbB) and B100-9.01 (exbD1) decreased immediately after infiltration from a value of 1x106 bacteria per cm2 tissue to values two to three orders of magnitude lower. Not only were these mutants unable to grow in the pepper leaf tissue, they even died rapidly. Since these mutant strains were also found to grow only on medium supplemented with ferrous iron (Wiggerich et al., 1997
), they were considered to be unable to acquire iron in planta, thus explaining the absence of growth. The decreasing number of bacteria seems to be the reason that pepper did not establish an HR. An unexpected result was obtained with the exbD2 mutant B100-11.03. The bacterial titre of this mutant strain increased during the first 4 d after infiltration, as did the titre of the wild-type strain. However, in contrast to the viable bacterial titre of the wild-type strain B100, the bacterial titre of the exbD2 mutant B100-11.03 did not decrease at all. Instead, the bacteria multiplied to a titre of 9x107 cells per cm2 during the following 6 d. Similarly to the ex planta state (Wiggerich et al., 1997
), this mutant was probably able to acquire iron in planta. Interestingly, despite its higher titre, the exbD2 mutant strain did not induce HR symptoms on pepper, indicating the disturbance of the interaction between this mutant and the nonhost plant pepper. The bacterial titre of the intergenic mutant B100-6.01 increased during the first 5 d to 5x107 bacteria per cm2 tissue, then decreased to a value of 2x107 bacteria per cm2 tissue. The intergenic mutant B100-6.01, which is not affected in iron import (Wiggerich et al., 1997
), induced an HR with strongly reduced symptom formation. In accordance we found that this strain could multiply for a longer period of time in infected pepper leaf tissue than the wild-type strain. However, even this reduced HR led to a decreasing number of viable bacteria. Although the insertional mutation in strain B100-6.01 is intergenic, this mutation affected the interaction between this mutant strain and pepper. A reason for this effect can be the disturbance of a common regulation of the tonB/exbBD1D2 genes. Since the complemented mutant strains were in a merodiploid state, this regulation may also be affected in the complemented strains, resulting in a delay of symptom formation of 2 d. We conclude that the recognition between X. campestris pv. campestris and its nonhost plant pepper is disturbed in mutant strains with an ineffective TonB system independently of their capacity to import iron.
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The production of ferric-siderophores has been suggested as an elicitor of plant defence reactions (Expert et al., 1996 ). Chrysobactin, a siderophore produced by E. chrysanthemi, has been identified as a virulence factor in plant disease (Enard et al., 1988
). Since the X. campestris pv. campestris tonB, exbB and exbD1 mutant strains exhibited an increased siderophore production (Wiggerich et al., 1997
), the siderophore of X. campestris pv. campestris cannot be the elicitor of HR induction in pepper. Additional work is necessary to determine whether signal molecules are recognized or imported via the TonB system in X. campestris pv. campestris.
The X. campestris pv. campestris exbD2 mutant, but not the tonB, exbB and exbD1 mutants, induced typical black rot symptoms on the host plant cauliflower
Since the exbD2 mutant strain B100-11.03 was able to grow in pepper, we wanted to know whether this strain was also affected in the interaction with host plants. Therefore, the tonB, exbB, exbD1 and exbD2 mutant strains and the wild-type strain B100 were injected into leaf margins of B. oleracea (cauliflower). Symptoms caused by the wild-type strain were blackening of the leaf veins followed by sectorial drying and death of the leaf tissue (Fig. 4a). The iron-uptake genes tonB, exbB and exbD1 are therefore not only necessary for the interaction with nonhost plants but also with the host plant cauliflower, because strains carrying a mutation in one of these genes no longer induced the typical black rot symptoms. Since the sectorial drying of the leaf tissue is missing, the blockage of veins, which is commonly considered to be the reason for death of leaf tissue, did not occur. The symptoms caused by the exbD2 mutant strain were identical to those induced by the wild-type strain (Fig. 4b
). Only a slight delay of symptom formation during the first 2 d after injection could be detected (data not shown).
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The exbD2 gene of X. campestris pv. campestris increases the known scope of the TonB system function
The TonB system of Gram-negative bacteria is not only involved in ferric-ironsiderophore uptake but also serves as the central structure of the import pathways of several macromolecules (Postle, 1990 ; Braun, 1995
). Moreover, the TonB system and related components normally used for iron uptake were shown to mediate the induction of transcription of ferric citrate and pseudobactin transport genes from the cell surface in E. coli and P. putida, respectively (Härle et al., 1995
; Koster et al., 1994
). In X. campestris pv. campestris, a second ExbD protein (ExbD2) is present, which cannot substitute for the ExbD1 protein in ferric iron uptake (Wiggerich et al., 1997
). We have demonstrated here that this ExbD2 protein was found to be involved in the induction of an HR in pepper. To our knowledge, the existence of a second ExbD protein has not been reported for other Gram-negative bacteria so far. With respect to the results published here it would be interesting to know whether a second exbD2 gene is present in other HR-inducing bacteria. Mutational analysis revealed that although the tonB, exbB, exbD1 and exbD2 genes are likely to form one operon, the exbD1 and exbD2 genes can be independently expressed from the tonB and exbB genes (Wiggerich et al., 1997
). Since tonB, exbB and exbD1 mutant strains, like the exbD2 mutant strain, were unable to induce an HR on pepper, one can speculate that the ExbD2 protein interacts with the central structure of the TonB system, consisting of the TonB, ExbB and ExbD1 proteins, to increase the scope of the TonB system. Further investigations of the TonB system might give new insights into the signal exchange between phytopathogenic bacteria and plants.
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ACKNOWLEDGEMENTS |
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Received 6 January 2000;
accepted 31 January 2000.
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