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causes soft rot disease of potatoes, was investigated for 169
production of a catechol siderophore. Catechol production was
detected colorimetrically, by bioassay, and by homology to THE HYPERSENSITIVE RESPONSE
genes
IS
determining
ELICITED BY ESCHERICHIA
biosynthesis
COLI
of enterochelin, the catechol CONTAINING A CLUSTER OF PATHOGENICITY
siderophore
GENES
of
FROM
Escherichia
ERWINIA
coli. Results suggested that strain AMYLOVORA. S. V.
W3C105
BEER;
produces
C. H. ZUMOFF;
a catzechol
D. W.
siderophore
BAUER; B. J.
with
SNEATH;
functional and R. J. LABY. Department of Plant Pathology,
similarity
Cornell
to enterochelin. Genes involved in catechol University, Ithaca, NY 14853 U.S.A.
production were identified from a genomic library of strain
W3C105 in E. coli strain AN192, which is deficient in Hrp mutants of E. amylovora
enterochelin
are deficient
production.
in
Three
both pathogenicity
clones producing a catechol to pear fruit and the ability to
were
elicit
detected
the hypersensitive
on a universal siderophore-detection medium, response in tobacco.
These
A cosmid,
clones
pCPP430,
provided
containing
iron to enterochelin-deficient
wild-type
indicator DNA of E. amylovora, was
strains.
identified
The
that
role
restores
of catechol
pathogenicity
siderophore production in the and HR-eliciting ability to 18 transposon-induced
pathogenicity and
Hrpecology
and two
of E. carotovora will be evaluated, naturally occurring Hrp- mutants. pCPP430 contains a cluster of
hrp genes, dispersed throughout a 45 kb region of chromosomal
DNA. When Escherichia coli, strain DH5, containing pCPP430, was
infiltrated into tobacco-leaf tissue, strong collapse occurred
extremely rapidly. In addition, the cosmid conferred
166 HR-eliciting ability (in tobacco), to several other species of
CHARACTERIZATION OF AN rcsA-LIKE GENE OF
Erwinia.
ERWINIA
Theseresults
AMYLOVORA THAI
clearly indicate
all the
that
genes
pCPP430
needed
contains
for elicitation
STIMULATES
of
EXTRACELLULAR
the HR, and that
POLYSACCHARIDE
they
(EPS) PRODUCTION IN are expressed in E. coli and in other Erwinia species.
ERWINIA SPP. AND OTHER ENTEROBACTERIA. W. Chun, A. Chatterjee.
R. N. Goodman and A. K. Chatterjee, Department of Plant
Pathology, University of Missouri-Columbia, Columbia, MO 65211. 170
EPS production by E. amylovora is required in the elicitation of CHARACTERIZATION OF PROMOTER-ACTIVE FRAGMENTS FROM
the fire-blight disease in apples and pears. To examine the XANTHOMONAS USING A NEW BROAD HOST RANGE PROMOTER
regulation of EPS biosynthesis, we obtained from an E. amylovora SELECTION VECTOR. S. Swarup, R. DeFeyter and D.W. Gabriel.
cosmid library, several E. coli (HB1OI) clones that were mucoid Plant Pathology Dept., University of Florida, Gainesville, FL 32611.
on agar plates. The complementation of an rcsA mutation in E.
coli and the stimulation of EPS production in various A broad host-range (Inc Q) promoter selection vector, pUFC600, was
enterobacteria (E. amylovora, E. stewartii, E. coli, and
Salmonella typhimurium) associate the mucoid phenotype conferred constructed that enables a direct selection of promoter-active fragments.
by the cloned DNA to an E. amylovora regulator gene. This pUFC600 is 9.4 kb in size, Knr, has a promoterless Smr gene, multiple
rcsA-like gene along with its own promoter has been localized on cloning sites and tandem transcriptional terminators upstream of the cloning
a-2.2 kb DNA segment. Nucleotide sequence homology determined by sites. In the absence of any promoter-active fragment, E. coil strain DH5x
Southern hybridizations and functional complementation of the and Xanthomonas strains containing the plasmid were sensitive to
mucoid phenotype by the cloned DNA indicate that the genes for streptomycin at 15 pg/ml in minimal and 25 pg/ml in complete media. A
the regulation of EPS biosynthesis have been conserved in these genomic library (-4 kb insert size) of X. citri strain 3213 was constructed
enterobacteria. in pUFC600. Clones were introduced into X.c. pv. citrumelo strain 3048 Spr
at an average frequency of 10-4 per recipient. Promoter activity was
selected or screened on both minimal and complete media with
streptomycin and various classes of promoter active fragments were
identified. The relationship of pathogenicity genes to promoter-active
167 fragments which were induced on minimal media will be discussed.
CLONING OF AN AVIRULENCE GENE FROM PSEUDOMONAS SOLANACEARUI
STRAIN AWl AND ITS INVOLVEMENT IN HOST RANGE. B. F. Carney and
T. P. Denny, Dept. of Plant Pathology, UGA, Athens, GA 30602. 171
A locus responsible for the hypersensitive response (HR) in
P. solanacearum strain AWl (avirulent on tobacco, but pathogenic
on tomato) was cloned by complementation in P. solanacearum
strain K601 (pathogenic on tobacco and tomato). Pseudomonas
solanacearum K601 transconjugants [ K601(pBC73) and K601(pBC62)
] were nonpathogenic on tobacco, suggesting that cloned locus had
the potential to restrict the host range of K601. DNA analysis
of the clones pBC73 and pBC62 indicated that a common 4.2 Kb
EcoRI/BamHI fragment was responsible for the induction of HR in
K601 transconjugants. Transposon mutagenesis of the wild type
locus in strain AWl resulted in the loss of HR on tobacco but
retention of pathogenicity on tomato, indicating that this locus
contains an avirulence gene. Pathogenicity on tobacco was not
acquired upon inactivation of this avirulence gene in AWl,
suggesting that AWl lacks positive acting host range genes that
are required for pathogenicity on tobacco,
168
1682
CLONING OF TWO GENES FOR PRODUCTION OF
POLYSACCHARIDE
EXTRAC ELLULAR
FROM PSEUDOMONAS SOLANACEARUM AND THEIR
CONTRIBUTION TO VIRULENCE. S. -R. Baek and T. P. Denny. Dept.
of Plant Pathology, University of Georgia, Athens, GA 30602.
Tn5-induced mutations of Xanthomonas campestris pv. citrumelo
affecting pathogenicity and host-species specificity. .
Kingsley and D.W. Gabriel. University of Florida, Gainesville,
Florida 32611
Tn5-induced mutations affecting pathogenicity (PATH-) and host
species specificity (HSS-) were recovered in X. campestris pv.
citrumelo. All 3048::Tn5 exconjugants (including auxotrophs)
were screened on both bean and citrus. Auxotrophic mutations
had significant effects in planta. For example, uracil
auxotrophy resulted in a path- phenotype, while isoleucine-
valine auxotrophs were hss- in bean but were relatively
unaffected in citrus. None of the Tn5-inserts affecting
pathogenicity or host range appeared to be clustered by
hybridization analyses, nor did the affected DNA regions
hybridize with the P. solanacearum hrp cluster (ie. pVir2). A
high level of polymorphism was observed between, but not
within 12 species or pathovars of Xanthomonas probed with
HSS-related DNA fragments, which may indicate a lack of
conservation of the gene(s) present at the affected loci.
17
MOLECULAR CHARACTERIZATION OF A LOCUS REGULATING PRODUCTION OF
EXTRACELLULAR POLYSACCHARIDE SLIME AND VIRULENCE IN PSEUDOMONAS
SOLANACEARUM. S. M. Brumbley and T. P. Denny, The University of
Previous research had marked two loci of P. solanacearum
AWl that
strain
are involved in the production of extracellular polysaccharide
(EPS) with Tn5 insertions. These Tn5 insertions and
flanking DNA of P. solanacearum were cloned and used to locate
eight cosmid clones that contained homologous sequences in a
genomic library of P. solanacearum AWl. The cosmids were
restriction mapped and found to contain unique and overlapping
regions that spanned a total of 55 kilobases; the Tn5 insertions
in the two loci were centrally located and 12.5 kb apart. The
Tn5 inactivated genes in strains AWl-l and AWI-41 were
designated epsA and epsB, respectively. Seven of the eight
cosmids completely restored EPS production to strain AWI-41.
Three cosmids partially restored EPS production to strain AWl-l
and these transconjugants were more virulent on tomato than was
strain AWl-l. These results support the idea that EPS has a
major role in P. solanacearum causing wilt symptoms on tomato.
Georgia, Athens, GA 30602.
A well known phenomenon of P. solanacearum is the
spontaneous mutation from a mucoid to a nonmucoid form. A
variety of other traits, including virulence, are affected. A
previously isolated Tn5 mutant (AWI-80) is identical to the
naturally occurring spontaneous mutant AWl-A in every way tested.
An EcoRI fragment containing the Tn5 plus flanking DNA from AWl-
80 was cloned and used as a hybridization probe to identify two
cosmids from a genomic library of the wild type P. solanacearum
strain (AWl). These cosmids restored wild type traits when
conjugated into AWI-80, AWl-A and several other spontaneous
avirulent mutants strains of P. solanacearum. These results
suggest that this locus (designated rpc) contains a regulatory
element(s) with global functions. This system does not appear
to be the same as that regulating the production of alginate in
Pseudomonas aeruginosa.
1156 PHYTOPATHOLOGY