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Journal of Bacteriology, June 2000, p. 3498-3507, Vol. 182, No. 12
Department of Genetics, Harvard Medical
School, and Department of Molecular Biology, Massachusetts General
Hospital, Boston, Massachusetts 02114,1 and
Department of Entomology and Plant Pathology, Oklahoma
State University, Stillwater, Oklahoma 740782
Received 4 November 1999/Accepted 10 March 2000
We cloned the rpoN (ntrA and
glnF) gene encoding The rpoN gene encodes the
alternate sigma factor For some phytopathogenic bacteria, rpoN has been implicated
indirectly as a regulator of pathogenicity-related genes known as the
hrp gene cluster (17, 18). Pseudomonas
syringae pv. syringae strain 61, for example, contains a 25-kb
hrp cluster consisting of several complementation groups
comprising at least 27 genes (8, 26). Several hrp
genes encode proteins that have a high degree of homology to components
of the type III secretory pathway of Yersinia species which
are responsible for translocating Yersinia outer membrane
proteins into mammalian cells (13, 15, 55, 83). By analogy,
it is proposed that hrp-encoded proteins in phytopathogenic
bacteria are involved in the transport of pathogenicity-related factors
into plant cells.
The acronym hrp stands for hypersensitive response and
pathogenicity. hrp genes are required not only for
pathogenicity of a virulent pathogen but also for the elicitation of
the hypersensitive response (HR) which occurs on some hosts (44,
45). The HR involves rapid, but localized, programmed plant cell
death and is believed to restrict pathogen spread (1, 37).
There is mounting evidence that the elicitation of an HR is mediated by the specific interaction between the products of a plant resistance gene (R gene) and a pathogen avirulence (avr)
gene (43, 80, 85). It appears likely that at least some
avr genes encode pathogenicity-related factors (34, 47,
71, 84) that are transported into plant cells via the
hrp-encoded transport machinery (58, 59, 90). In
the absence of a corresponding R gene product, the
avr product enhances virulence; however, in hosts which have
the corresponding R gene, recognition of the avr
gene product enhances host resistance. Interestingly, most
avr genes are also coordinately regulated with genes in the
hrp cluster (27, 29, 46, 67, 71, 77, 97).
The HR is accompanied by the induction of defense-related genes
(7, 91) that are differentially expressed depending on the
particular pair of avr and R gene products
eliciting the HR (70, 72). Defense gene induction also
occurs in the absence of the HR during compatible pathogen-host
interactions, although usually later and at lower levels than those
occurring during an HR (11, 32, 66). Furthermore,
hrp mutations that presumably block the export of
avr gene products have been found to reduce, but not
eliminate, defense gene induction (60). Collectively, these
results suggest that there are a variety of signaling pathways that
activate host responses.
In P. syringae the circuitry of hrp regulation
appears to involve a transcriptional activation cascade. At the top of
the cascade are two regulatory genes, hrpR and
hrpS, which are required for expression of the remaining
hrp genes in the cluster (12, 18). Both
hrpR and hrpS encode proteins consist almost
exclusively of the domain conserved among transcriptional activators
such as NtrC, DctD, and NifA that work in concert with
Despite the highly conserved and clustered nature of hrp
genes among phytopathogenic bacteria, transcriptional regulation of the
hrp genes is achieved by different mechanisms in different species. In Ralstonia solanacearum, HrpB, an AraC-like
transcriptional activator, controls hrp gene expression
(14). Similarly, in Xanthomonas campestris pv.
vesicatoria, hrp gene expression is regulated by an OmpR
homolog, HrpG, which in turn activates HrpX, another AraC-like
activator that activates the remaining hrp genes (79,
92). This latter regulatory cascade is consistent with the fact
that rpoN is not required for hrp expression or
pathogenicity in X. campestris pv. vesicatoria
(25).
The experiments described here utilize a pathogenicity system that
involves the infection of Arabidopsis thaliana with P. syringae pv. maculicola strain ES4326. Strain ES4326 belongs to the leaf spotting class of phytopathogenic pseudomonads
(78), proliferates extensively in Arabidopsis
ecotype Columbia leaves, and causes the development of water-soaked
disease lesions (9, 11). In contrast, ES4326 carrying the
avirulence gene avrRpt2 elicits a visible HR about 16 h
after infiltration and proliferates 50- to 100-fold less than the
wild-type strain ES4326 (11). Using this system, we describe
experiments that examine the role of Bacterial strains and media.
Bacterial strains and plasmids
used in this work are listed in Table 1.
P. syringae pv. maculicola strain ES4326 and its derivatives
were grown at 28°C in L broth (50), minimal M9 salts media
(50), or King's B medium (36). Escherichia
coli, Klebsiella aerogenes, and P. aeruginosa strains were grown at 37°C in L broth or M9 minimal
salts medium. For clarity, ES4326 carrying plasmid pLH12 (which carries
the avirulence gene avrRpt2) is referred to as ES4326
(avrRpt2). Carbon and nitrogen source utilization tests for
ES4326 rpoN mutants were performed in M9 salts minimal medium by providing a carbon source at 10 mM and by replacing ammonium
chloride with an alternative nitrogen source at 5 mM when required.
Bacterial motility was tested on "swarm plates" consisting of 0.3%
agar, 0.5% NaCl and 0.5% tryptone (38). Antibiotic concentrations for E. coli and P. syringae
strains were as follows: streptomycin, 150 µg/ml; kanamycin, 25 µg/ml; tetracycline, 12 µg/ml; gentamicin, 20 µg/ml; and
spectinomycin, 20 µg/ml. Interspecies complementation tests of the
E. coli rpoN mutant by ES4326 rpoN were carried
out on M9 minimal salts agar supplemented with 0.2% glutamine and 20 µg of 5-bromo-4-chloro-3-indolyl-
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Virulence of the Phytopathogen Pseudomonas
syringae pv. Maculicola Is rpoN Dependent
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
54 from the
phytopathogen Pseudomonas syringae pv. maculicola strain
ES4326. The P. syringae ES4326 rpoN gene
complemented Pseudomonas aeruginosa, Escherichia
coli, and Klebsiella aerogenes rpoN mutants for a variety of rpoN mutant phenotypes, including the inability
to utilize nitrate as sole nitrogen source. DNA sequence analysis of
the P. syringae ES4326 rpoN gene revealed that
the deduced amino acid sequence was most similar (86% identity; 95%
similarity) to the 54 protein encoded by the
Pseudomonas putida rpoN gene. A marker exchange protocol
was used to construct an ES4326 rpoN insertional mutation,
rpoN::Kmr. In contrast to wild-type
ES4326, ES4326 rpoN::Kmr was
nonmotile and could not utilize nitrate, urea,
C4-dicarboxylic acids, several amino acids, or
concentrations of ammonia below 2 mM as nitrogen sources.
rpoN was essential for production of the phytotoxin
coronatine and for expression of the structural genes encoding
coronamic acid. In addition, ES4326
rpoN::Kmr did not multiply or elicit
disease symptoms when infiltrated into Arabidopsis thaliana
leaves, did not elicit the accumulation of several
Arabidopsis defense-related mRNAs, and did not elicit a
hypersensitive response (HR) when infiltrated into tobacco
(Nicotiana tabacum) leaves. Furthermore, whereas P. syringae ES4326 carrying the avirulence gene avrRpt2
elicited an HR when infiltrated into Arabidopsis ecotype
Columbia leaves, ES4326 rpoN::Kmr
carrying avrRpt2 elicited no response. Constitutive
expression of ES4326 hrpL in ES4326
rpoN::Kmr partially restored
defense-related mRNA accumulation, showing a direct role for the
hrp cluster in host defense gene induction in a compatible
host-pathogen interaction. However, constitutive expression of
hrpL in ES4326 rpoN::Kmr
did not restore coronatine production, showing that coronatine biosynthesis requires factors other than hrpL.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
54, which works in conjunction
with the NtrC class of transcriptional activators to control the
expression of many genes in response to nutritional and environmental
conditions (2, 54). For example, genes involved in nitrogen,
hydrogen, and catabolite utilization are frequently regulated by
54 (6, 35, 48, 95). In the case of pathogenic
bacteria, rpoN mediates expression of virulence-related
factors such as pilin in Pseudomonas aeruginosa and
flagellin in Vibrio anguillarum (24, 61, 86).
54 (21, 22; reviewed in references
2 and 54). The hrpR product activates hrpS expression, and the hrpS
product activates hrpL transcription (17, 18).
HrpL is an alternate sigma factor homologous to AlgU of P. aeruginosa and is thought to activate transcription of the
remaining genes in the hrp cluster (96, 97). The
factor(s) involved in the regulation of hrpRS remains obscure. Nevertheless, the central role of hrpS in this
cascade and the HrpS-NtrC homology predicts that rpoN would
be required for activation of the hrp gene cluster in
P. syringae.
54 in the
pathogenicity of P. syringae ES4326. Our results indicate that 54 is an important virulence factor for P. syringae and is required for the elicitation of an HR by P. syringae in both host and nonhost plants.
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
-D-galactopyranoside (X-Gal) per ml (Sigma).
TABLE 1.
Strains and plasmids used in this study
Bacterial genetics. pLAFR3 derivatives were introduced into Pseudomonas strains via triparental matings with MM294(pRK2013) (10). Aspartate-utilizing pseudorevertants of strain ES4326 rpoN::Kmr were obtained by plating approximately 109 CFU on M9 agar plates containing succinate and aspartate as carbon sources at 10 mM and the appropriate antibiotics (streptomycin and kanamycin).
Plant pathogenicity assays.
Arabidopsis ecotype
Columbia was germinated, grown, and inoculated with ES4326 strains.
Bacterial strains were grown overnight in King's B, subcultured and
grown to mid-log phase, resuspended in 10 mM MgSO4, and
inoculated into the underside of the leaf at a titer of 104
CFU/cm2 using a disposable syringe. Growth of P. syringae strains in leaves was measured by individually grinding
four to six 0.2-cm2 leaf punches (excised with a no. 2 cork
borer) in 10 mM MgSO4, plating appropriate dilutions on
King's B medium containing the appropriate antibiotics, and counting
the CFU. For RNA blot analysis, entire Arabidopsis leaves
infiltrated with ES4326 bacterial suspensions were harvested, frozen in
liquid nitrogen at the indicated times, and stored at 
80°C until
needed (see below). Nicotiana tabacum (tobacco) cultivar
Xanthi was grown under greenhouse conditions and inoculated with ES4326
strains and assayed for the HR as previously described (82).
Cosmid library constructions. Total bacterial genomic DNA was prepared from strain ES4326 as described previously (3), partially digested with Sau3A, and size fractionated on a 14-ml sucrose gradient (50). DNA fragments of approximately 20 kb were purified and ligated with linearized pLAFR3 that had been prepared to promote the formation of concatemers (50). Packaging, infection, and plating of the cosmid clones were performed using the Giga Gold packaging kit according to the manufacturer's specifications (Stratagene, La Jolla, Calif.).
Nucleic acid manipulations. Routine manipulations such as DNA blots and plasmid DNA isolation were performed as described earlier (3). Restriction enzymes, T4 DNA ligase, and calf intestine phosphatase were purchased from Boehringer Mannheim and New England BioLabs and used according to the manufacturer's specifications. Deletions in plasmids were created using the Erase-a-Base kit (Promega, Madison, Wis.). Isolation of Arabidopsis mRNA and RNA blot analysis was carried out as described previously (11). Hybridizations were performed at stringent conditions (2× SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 65°C) as described earlier (50). 32P-labeled DNA probes for use in hybridizations were prepared as described previously for the Pal1, PR1, BGL2 (PR2), PR5, and GST1 pathogen-induced genes (16, 73).
Cloning and sequencing the ES4326 rpoN gene. DNA blot analysis data indicated that ES4326 contained a single 4.5-kb HindIII fragment that hybridized to a 4.2-kb XhoI fragment in plasmid pKI11 containing the P. aeruginosa strain PAK rpoN gene (31). Approximately 1,400 clones from a cosmid library of strain ES4326 DNA were screened by colony hybridization using the rpoN probe derived from P. aeruginosa. A hybridizing clone, pPG101, carrying a 17.2-kb insert was identified and shown to contain the 4.5-kb HindIII fragment previously detected by Southern blot analysis (data not shown).
E. coli strain TH1
gln101, which contains deletions in
rpoN and lacZ and a glnA-lacZ reporter
construct, was used as an assay for functional ES4326 rpoN
clones by plating subclones of pPG101 onto M9 medium containing 0.2%
glutamine and 20 µg of X-Gal per ml. The 4.5-kb
HindIII fragment from pPG101 contained a functional rpoN gene that complemented the E. coli rpoN
mutation in TH1 gln101. The rpoN gene in this construct
presumably contained its own promoter since this fragment activated the
glnA-lacZ fusion in TH1 gln101 when cloned in the
HindIII site of pBluescript SK(+) in either orientation
(plasmids pRN5 and pRN9). For subsequent use in Pseudomonas spp., the 4.5-kb fragment containing ES4326 rpoN was
subcloned into cosmid pLAFR3. A 4.5-kb HindIII fragment
from pRN5 was subcloned into pGem7Zf. Using the pGem7Zf polylinker
sites, the rpoN gene was recloned as a 4.5-kb
EcoRI-BamHI fragment in pLAFR3 to produce pPG102.
Plasmids pRN5 and pRN9 were used to derive a series of nested deletions
starting from either end of the 4.5 HindIII fragment. This analysis showed that the ES4326 rpoN gene was located
near the left end of the 4.5-kb fragment (data not shown).
DNA sequence analysis was initiated at the middle XhoI site
in Fig. 1 and continued in both
directions using synthetic oligonucleotides for a total of
approximately 1,900 bp. A single large open reading frame that encodes
a protein that is highly homologous to 54 in other
bacterial species was identified in the 1,900-bp region.
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Insertional mutagenesis of the ES4326 rpoN gene. pGem7Zf containing the 4.5-kb rpoN fragment was digested with PstI, and a 1.24-kb fragment encoding the aminoglycoside 3'-phosphotransferase activity of Tn903 (Pharmacia) from pUC4K was ligated to the PstI-digested ends. A 5.8-kb EcoRI-BamHI fragment from this plasmid was then subcloned into pLAFR3. The resulting plasmid (pLAFR-RK) was used to recombine the mutated rpoN gene (referred to as rpoN::Kmr) into strain ES4326 by first mobilizing pLAFR-RK into ES4326 and then introducing plasmid pPH1, which confers gentamicin (Gm) resistance. Cultures were grown under selection for Kmr and Gmr, and individual colonies were screened for tetracycline sensitivity (76). Southern blot analysis of chromosomal DNA prepared from a putative rpoN mutant confirmed the insertion of Kmr into the rpoN gene (data not shown).
COR preparation and assay. Coronatine (COR) synthesis by strain ES4326 was assayed using two approaches. In the first procedure (74), a 5-ml culture of ES4326 grown overnight in King's B medium was used to inoculate 50 ml of Woolley's liquid medium (94), where potassium nitrate was replaced with 5 mM arginine to facilitate rapid growth of the ES4326 rpoN-Kmr mutant. Cultures (50 ml) were shaken at 20°C for 6 days, at which point the optical density at 600 nm (OD600) was measured, and the cells were pelleted and weighed. The supernatants were acidified to pH 2.0 with HCl and extracted with 50 ml of ethyl acetate. The organic phase was lyophilized to dryness, and the residue was resuspended in 2.0 ml of H2O/g of wet bacterial pellet. Then, 10-µl droplets containing dilutions of either purified COR or the COR preparation described above were inoculated into Arabidopsis and tomato leaves. Elicitation of red anthocyanin pigments on Arabidopsis leaves and chlorosis on tomato leaves was assayed 3 to 7 days later.
In the second method, P. syringae strains were grown at 18°C in Hoitink-Sinden medium optimized for COR production (HSC) (63), and the supernatants were analyzed for COR production by high-pressure liquid chromatography (HPLC) 5 days after inoculation (63). Each strain was inoculated into three replicate aliquots (10 ml) of HSC medium for evaluation of COR production, and each experiment was repeated.Nucleotide sequence accession number. The rpoN sequence from P. syringae pv. maculicola has been assigned accession number AF199600 in the GenBank database.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cloning the strain ES4326 rpoN gene.
An
interspecies hybridization strategy was used to isolate pPG101, a
cosmid clone that carried a presumptive P. syringae ES4326 rpoN gene. Plasmid pPG101 complemented the inability of
E. coli, K. aerogenes, and P. aeruginosa
rpoN mutants (strains TH1 gln101, HG63 rpoN, and
PAKN1, respectively) to utilize 10 mM nitrate and 10 mM ammonia as sole
nitrogen sources; the inability of the E. coli rpoN mutant
to utilize histidine, arginine, or proline as sole nitrogen sources;
and the lack of motility of the P. aeruginosa rpoN mutant.
As described in Materials and Methods and as illustrated in Fig. 1, the
ES4326 rpoN gene on pPG101 was mapped to a
4.5-kb-HindIII fragment, and a 1,900-bp region
containing the presumptive rpoN gene was sequenced. DNA
sequence analysis (Fig. 2) revealed that the presumptive ES4326 rpoN gene encodes a protein with 86%
identity and 95% similarity to the rpoN gene of P. putida.
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Construction and metabolic phenotypes of a strain ES4326
rpoN insertional mutant.
An ES4326 rpoN
mutant was constructed by subcloning rpoN from pPG101,
inserting a DNA cassette conferring kanamycin resistance into the
PstI site (located at codon 162) of rpoN,
transferring the mutated rpoN gene to pLAFR3, and marker
exchanging the mutant gene into the ES4326 genome (see Materials and
Methods). ES4326 rpoN::Kmr exhibited
an array of phenotypes typical of rpoN mutants, including the inability to grow on nitrate and urea as sole nitrogen sources, lack of motility, and inability to grow on a variety of
C4-dicarboxylic acids as sole carbon sources, including
aspartate, succinate, and fumarate, as well as the tricarboxylic acid
intermediate 
-ketoglutarate (data not shown). Unlike rpoN
mutants of enteric bacteria (49, 86), however, ES4326
rpoN::Kmr was able to grow on glucose
and ammonia as the sole carbon and nitrogen sources if ammonia was
present at concentrations higher than 2 mM.
Strain ES4326 rpoN is nonpathogenic on
Arabidopsis and cannot elicit an HR.
As described
previously (11), infiltration of Arabidopsis
leaves with ES4326 at a titer of 103 to 104
CFU/cm2 of leaf area resulted in the development of
characteristic disease symptoms, including spreading chlorosis,
water-soaked lesions, and growth of the infiltrated bacteria to a titer
of approximately 107 CFU/cm2 (Fig.
3A). In contrast, Arabidopsis
leaves infiltrated with ES4326 rpoN::Kmr exhibited no symptoms, even
when inoculated with 108 CFU/cm2. Furthermore,
the titer of ES4326 rpoN::Kmr in
Arabidopsis leaves remained consistently low for the
duration of the experiment (Fig. 3A). ES4326
rpoN::Kmr carrying pPG102, which
carries wild-type rpoN, exhibited the same pathogenic
phenotype as ES4326, and its growth in Arabidopsis leaves
was indistinguishable from that of the wild type (Fig. 3A). This result
indicated that the nonpathogenic phenotype of ES4326
rpoN::Kmr was due to the disruption of
the rpoN gene. However, because the complementing plasmid
contains about 2,000 bp downstream of rpoN, these data do
not rule out the possibility that the insertion in rpoN
exerts polarity on a downstream gene and that this downstream gene is
required for pathogenicity.
|
,
ES4326(pLAFR3); 
, ES4326 rpoN::Kmr
(pLAFR3); 
, ES4326 rpoN::Kmr
(pPG102) (rpoN+). The experiment was repeated
with similar results. (B) Symbols: 
, ES4326(pLAFR3); The pathogenicity defect of ES4326
rpoN::Kmr is not solely due to the
inability to assimilate aspartate.
Because genes encoding
dicarboxylic acid permease are regulated by DctD, an NtrC homolog,
rpoN mutants in a variety of species cannot utilize
dicarboxylic acids as carbon or nitrogen sources. R. meliloti mutants defective in aspartate aminotransferase cannot utilize aspartate and are defective in symbiosis, suggesting that aspartate may be a major carbon source for symbiotic bacterial cells
(69). Therefore, we tested whether ES4326
rpoN::Kmr is nonpathogenic solely
because it cannot assimilate aspartate. We selected a ES4326
rpoN::Kmr pseudorevertant, as
described in Materials and Methods, that was able to utilize aspartate
as a carbon source. This revertant still had an RpoN
phenotype with respect to its inability to use nitrate and succinate and its lack of motility. The Asp+ revertant failed to grow
in A. thaliana or to elicit an HR in tobacco or
Arabidopsis (data not shown).
Strain ES4326 rpoN::Kmr fails to
synthesize the phytotoxin coronatine.
One possible explanation for
the reduced virulence of ES4326
rpoN::Kmr is the inability to produce
virulence factors such as toxins. Many P. syringae
pathovars, including ES4326, produce a chlorosis-inducing phytotoxin,
coronatine, which is composed of an ethyl cyclopropyl amino acid linked
to a polyketide moiety (19). COR production is regulated by
a modified two-component regulatory system that controls the expression
of essential COR biosynthetic genes. The regulators CorR and CorP are
related to response regulators of the ROIII group, while CorS is
similar to the corresponding histidine protein kinase sensors. To
determine whether COR biosynthesis requires rpoN, crude COR
was extracted from strains ES4326, ES4326 rpoN::Kmr, the COR-producing strain
P. syringae pv. tomato DC3000, and DC3661, a
COR mutant of DC3000. The COR extracted from DC3000 and
ES4326 elicited typical COR-induced symptoms, chlorosis and anthocyanin
accumulation, respectively, on tomato and Arabidopsis
leaves. These symptoms were not detected with organic acids extracted
from ES4326 rpoN::Kmr or DC3661.
Further characterization was carried out by quantitatively analyzing
COR production using HPLC. As shown in Table
2, ES4326 produced 51 mg of COR/g of
protein, a level comparable to that produced by P. syringae
pv. glycinea strain PG4180.N9, a high-yielding COR producer which has
been used in many genetic investigations (65, 88). However,
ES4326 rpoN::Kmr produced only 0.3 mg
of COR, a level comparable to PG4180.P2, a corR mutant of
PG4180.N9 which is considered completely defective in COR production
(65).
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-glucuronidase activity from pRGMU7, a
construct containing the cmaABT promoter fused to
uidA (87). As shown in Table 2, when pRGMU7 was
introduced into the two COR-producing strains, ES4326 and PG4180.N9,
transcriptional activity from the cmaABT promoter was
comparable (347 and 390 U GUS, respectively). However,
-glucuronidase activity in the rpoN mutant containing
pRGMU7 was extremely low and was comparable to the low level of
expression in PG4180.P2(pRGMU7). It is important to note that PG4180.P2
is defective in corR, a gene which encodes a positive
transcriptional activator of the cmaABT transcript (64). The present data suggest that a functional
rpoN is required for expression of the cmaABT
transcript in P. syringae pv. maculicola.
In the accompanying study (23), we demonstrate that
rpoN in ES4326 is required for the expression of
hrpL, which encodes an alternative sigma factor and is
required for expression of the ES4326 hrp genes and
avrRpt2 (12, 17, 29, 97). We also show that
constitutive expression of hrpL on plasmid pHRPLC restores
the ability of ES4326 rpoN::Kmr to
elicit disease symptoms in A. thaliana and an HR in tobacco. However, pHRPLC did not restore COR production to ES4326
rpoN::Kmr (Table 2), indicating that
COR biosynthesis is not dependent on hrpL in ES4326 but on a
separate regulatory pathway that also requires rpoN.
Strain ES4326 rpoN::Kmr fails to
activate high-level expression of Arabidopsis
defense-related genes.
The infiltration of Arabidopsis
leaves with ES4326 normally leads to the accumulation of mRNAs
corresponding to a variety of Arabidopsis defense-related
genes, including PR2 (BGL2), GST1, PR5 and PR1, which encode -1,3-glucanase,
glutathione S-transferase, a thaumatin-like protein, and a
protein with unknown activity, respectively (42, 51, 52,
91). Each of these genes shows a different induction pattern. In
general, GST1 and PR5 are induced within several
hours after infection, whereas PR1 and BGL2 are induced later than 24 h postinfection. In contrast to ES4326, very
little accumulation of the BGL2, GST1,
PR1, and PR5 transcripts was seen following
infiltration with ES4326 rpoN::Kmr
(Fig. 4), which gave results similar to
those for the MgSO4 control. This demonstrated that a
factor under rpoN control is necessary for defense gene
induction.
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, ES4326; 
, ES4326
rpoN::Kmr (pHRPLC).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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ES4326 rpoN gene.
The amino acid sequence of the
ES4326 54 protein is most closely related to the
P. putida 54 protein, and the phenotype of
the ES4326 rpoN mutant resembles the phenotype of P. putida rpoN mutants (38). Both P. syringae and P. putida rpoN mutants grow more slowly than their
wild-type counterparts in all media tested and were unable to utilize
several uncharged amino acids as substrates. Neither is a glutamine
auxotroph, and both can utilize NH4 as a sole nitrogen
source. They can also utilize lysine as a nitrogen source though not as
a carbon source. In P. putida, the inability of the
rpoN mutant to grow on lysine is probably due to the fact
that lysine decarboxylase is under 54 control
(38). The major phenotypic difference that we observed between the P. putida and the P. syringae rpoN
mutants was that the P. syringae rpoN mutant could not
utilize proline and histidine as nitrogen sources, whereas the P. putida rpoN mutant could.
Lack of growth of strain ES4326 rpoN on low
concentrations of ammonia and other nitrogen sources.
The
inability of ES4326 rpoN::Kmr to grow
on low concentrations of ammonia (<1 mM) and various amino acids is
presumably caused by the lack of glnA expression (which
encodes glutamine synthetase), which is under 54 control
in several other bacterial species (54, 86). On the other
hand, the ES4326 rpoN mutant is not a glutamine auxotroph, since it grew well when high concentrations of ammonia (>20 mM) or
several amino acids other than glutamine were supplied as the sole
nitrogen source. One explanation for these results is that P. syringae, like Rhizobium and Bradyrhizobium
species, has more than one gene encoding glutamine synthetase, one that
is expressed at high levels under 54 control and a
second copy that is expressed at low levels and is 54
independent (41). Alternatively, P. syringae
could have a single gene encoding glutamine synthetase, which requires
54 for the high-level expression needed for ammonia
assimilation but which has sufficient basal expression to prevent
auxotrophy. rpoN mutants of most soil bacteria, including
Azotobacter vinelandii, P. putida, and
Agrobacterium tumefaciens, are not glutamine auxotrophs (39, 40, 53, 74, 75).
Growth rate of strain ES4326 rpoN. The ES4326 rpoN mutant displayed slower growth rates than the wild type in each medium examined, including M9 supplemented with 0.2 mM glutamine. Thus, it appears unlikely that the slower growth of the rpoN mutant can be explained solely on the basis of decreased levels of glnA expression. While it is possible that the growth deficit is due to a secondary mutation, plasmid pPG102, which carries the wild-type rpoN gene, fully complemented every rpoN-related phenotype tested, including growth and pathogenesis in Arabidopsis leaves.
Nonpathogenic phenotype of strain ES4326 rpoN.
Given the
pleiotropic phenotype of rpoN mutants, it is not possible to
state precisely why the ES4326 rpoN mutant failed to elicit
disease symptoms and to grow in Arabidopsis leaves or to elicit an HR. In the accompanying study we show that the absence of a
functional 54 in ES4326 blocks the transcription of
hrp genes downstream of hrpRS (23),
which would account for the nonpathogenic and HR-deficient phenotypes.
However, we also report that the constitutive expression of
hrpL in ES4326 rpoN::Kmr
restored the elicitation of disease symptoms but failed to restore growth of ES4326 rpoN::Kmr in planta,
implying that the absence of hrp functions is not the sole
reason for the nonpathogenic phenotype of ES4326
rpoN::Kmr.
rpoN-mediated regulation of COR synthesis.
The
data in Table 2 demonstrate that ES4326
rpoN::Kmr does not produce COR, which
contributes to lesion expansion, chlorosis, and bacterial
multiplication in Arabidopsis (56). Although a COR mutant is not available for P. syringae pv. maculicola, pHRPLC, which expresses
hrpL constitutively, restored some disease symptoms but not
COR production to ES4326 rpoN::Kmr.
However, because pHRPLC failed to restore in planta growth to the
mutant (23), it remains possible that some of the growth defect in ES4326 rpoN::Kmr could be
caused by loss of COR production.
24(GG)/12(GC)
motif is lacking upstream of the cmaA transcriptional start
site (87). Thus, 54 control of
cmaABT expression is probably mediated indirectly through
another regulatory gene whose expression is directly controlled by
54. Possible candidates for 54 control
inside the COR gene cluster include corP and
corR, which encode response regulators with uncharacterized
upstream sequences. Alternatively, 54 might control the
expression of regulatory genes unlinked to the COR biosynthetic gene cluster.
Reduced induction of the host defense response by ES4326
rpoN.
ES4326
rpoN::Kmr failed to induce defense
gene induction in Arabidopsis during both compatible and
incompatible interactions. These results contrast with those reported
previously for hrp mutants. A nonpathogenic hrp
deletion mutant of a compatible X. campestris pv. campestris
strain elicited the expression of a variety of defense genes in the
turnip to approximately 50% of their normal expression levels
(60). Similarly, an incompatible X. campestris
pv. armoraciae strain with an hrp deletion did not induce an
HR in the turnip but still induced defense gene expression (60). In the P. syringae pv. phaseolicola-bean
interaction, approximately the same level of defense gene induction
occurred with incompatible wild-type and hrp deletion
strains (32). One way to explain the discrepancy observed in
defense gene induction by rpoN and hrp mutants is
that important factors for defense gene induction may lie outside of
the hrp pathway but under rpoN control. While
there are reasons to believe that phytotoxins and avr genes
may contribute to defense gene induction, they seem unlikely
explanations for this phenomenon (5, 34, 47, 71). Although
production of the phytotoxin COR is rpoN dependent, in ES4326, COR mutants of DC3000 elicited more defense gene
induction than the wild-type strain (56). Similarly,
avr gene products are thought to require a functioning
hrp cluster for activity and are therefore unlikely
candidates for hrp-independent defense induction factors (34, 47, 71, 84). Finally, the fact that X. campestris pv. vesicatoria rpoN mutants are fully
pathogenic (25) indicates that, at least in the case of this
species, rpoN does not regulate any essential pathogenicity factors.
| |
ACKNOWLEDGMENTS |
|---|
Erik L. Hendrickson and Pablo Guevara contributed equally to this work.
This work was supported by NIH grant GM48707 awarded to F.M.A. and NSF grant MCB-9603618 awarded to C.L.B.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Massachusetts General Hospital, Department of Molecular Biology, Wellman 10, Boston, MA 02114. Phone: (617) 726-5969. Fax: (617) 725-5949.
Present address: Department of Microbiology, University of
Washington, Seattle, WA 98195.
| |
REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Bacteriology, June 2000, p. 3498-3507, Vol. 182, No. 12
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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