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Supplementary material
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Construction of R. solanacearum mutants
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Construction of a eps mutant. The disruption of an eps gene in R. solanacearum
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RS1002 was performed using the marker-exchange plasmid pK18mobsacB (Schäfer et
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al., 1994). A 2.4 kb fragment including the epsP gene was amplified by polymerase
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chain
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(5'-CGTAAGCATTCCGAGTATCC-3')
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(5'-TTGCGCTTGATGTTCTGGGC-3') and RS1002 genomic DNA as the template.
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PCR was performed using the GC-RICH PCR system (Roche). The PCR fragment was
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cloned into pK18mobsacB to yield pRS183. Next, deletion was carried out in the epsP
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gene by excluding a 0.4 kb EcoRV-NruI fragment from pRS189. Using pRS189, the
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EPS- strain RS1085 was constructed from RS1002 by the marker-exchange method
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(Schäfer et al., 1994).
reaction
(PCR)
using
the
primer
and
set
of
P26
P29
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Construction of (hrpB-hrpD) mutants. First, a hrp gene cluster was cloned from R.
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solanacearum RS1002. A part of the popA gene was amplified by PCR using primers P1
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(5'-AGGTGCCCGCTCGTTAC-3') and P2 (5'-CCATCGCCATTACATCGGCT-3') and
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RS1002 genomic DNA. The 1.1 kb fragment was cloned into a suicide vector pARO191
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(Parke, 1990) to yield pINT101. pINT101 was integrated into the genome of RS1002
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through conjugation with S17-1. Genomic DNA was purified from a transconjugant and
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the integrated plasmid was rescued to include the surrounding hrp region. The partial
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sequencing of the rescued plasmid pRS103 revealed that the hrp gene cluster of RS1002
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has the same gene organization as that of GMI1000 (Van Gijsegem et al., 1995). A 4.0
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kb EcoRI fragment containing the hrpB-hrcT-hrpD-hrcN-hrpF region from pRS103 was
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subcloned into pK18mobsacB, and a 1.3 kb XhoI fragment including the
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hrpB-hrcT-hrpD region was replaced with a gentamycin resistance (Gmr) gene cassette,
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yielding pRS121. Using pRS121, (hrpB-hrpD) strains were constructed by the
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marker-exchange method.
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Construction of hpaB mutants. A 0.9 kb fragment upstream of hpaB was PCR
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amplified
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(5'-GGGAAGCTTGTGTGCAACGGACTTATCCC-3')
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(5'-GGGGGATCCGTGTGCAACGGACTTATCCC-3') with HindIII (underlined) and
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BamHI (double underlined) restriction sites, respectively. Similarly, a 1.0 kb fragment
from
RS1002
genomic
DNA
using
primers
and
P691
P694
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downstream
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(5'-GGGGGATCCAGACCACCGACGACATGTT-3')
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(5'-GGGGGTACCAACAGGTTCATGAACCGCGAGC-3') with BamHI (underlined)
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and KpnI (double underlined) sites, respectively. The two fragments were tandemly
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inserted into pK18mobsacB. The resulting pRS769 (Fig. 3) contains a 340 bp deletion in
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hpaB. hpaB strains were constructed by marker exchange using pRS769. Primers P691
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and P698 were used for the selection of hpaB mutants by PCR.
of
hpaB
was
amplified
using
primers
and
P695
P698
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Construction of hrpY mutants. A 0.8 kb EcoRI-PstI fragment containing the hrpY gene
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was cloned from pRS103 into pHSG398, yielding pRS771. pRS771 was digested with
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SalI and EcoO109I, treated by Klenow and self-ligated to result in a 160 bp deletion in
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hrpY. The EcoRI-PstI fragment containing hrpY was subcloned into pK18mobsacB to
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yield pRS828. hrpY strains were constructed by marker exchange using pRS828.
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Primers P691 and P694 were used for the selection of hrpY mutants.
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Disruption of the fliC gene by plasmid insertion. A 0.4 kb SalI fragment including a
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central part of the fliC gene was cloned from a PCR fragment amplified from RS1002
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genomic DNA using primers P881 (5'-AGTCGCTGCAGCGCCTGTCGA-3') and P882
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(5'-AAGGTCGACATGTTGACGTTG-3') into pARO191 to generate pRS845. pRS845
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was integrated into the genome of R. solanacearum strains through conjugation with
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S17-1. The transconjugants contain the pRS845 insertion into the chromosomal fliC
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locus, resulting in mutants with disruptions of the fliC gene.
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Construction of pilA mutants. A 0.5 kb fragment containing a 5'-half of the pilA gene
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was
PCR-amplified
from
RS1002
genomic
DNA
using
primers
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(5’-GGAATTCAAGTCGATGCCGGTCAAT-3’)
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(5’-CGGGATCCGTCAGACTGTGCATTGGC-3’) with EcoRI (underlined) and BamHI
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(double underlined) sites, respectively. Similarly, a 0.5 kb fragment containing a 3'-half
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of
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(5’-CGGGATCCGTCAGATCACCGTCACCT-3’)
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(5’-AACTGCAGACGACCAGTTGCACGATC-3’) with BamHI (underlined) and PstI
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(double underlined) sites, respectively. The two fragments were tandemly inserted into
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pK18mobsacB, and a 2.9 kb BamHI Gmr gene cassette was inserted between them to
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yield pRS844. Using pRS844, the pilA::Gmr mutation was introduced into R.
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solanacearum strains by the marker-exchange method. The inactivation of pilA was
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confirmed by the loss of twitching motility on plates (Kang et al., 2002).
pilA
was
amplified
and
P814
using
primers
and
P815
P818
P819
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Construction of a popA-lacZYA fusion. Plasmid pINT102, which has the same
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construction as that of pINT101 except for the opposite insertional direction, was used
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to clone the prhA region as described above. The 2.3 kb SmaI fragment containing a part
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of prhA was subcloned into pARO191. The resulting pINT103 was used to clone the
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popA region from RS1002. The 3.3 kb SphI-BglII fragment including popA was
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subcloned into pK18mobsacB to yield pRS188. Then, the promoterless lacZYA fragment
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from pUC-lacZYA was inserted into the BamHI site in popA, yielding pRS192.
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pUC-lacZYA was constructed as follows. The BamHI Kanr cassette was inserted into
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pRS415 (Simons, 1987) to yield pRS415-BK. The promoterless lacZYA fragment was
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excised using SalI from pRS415-BK and cloned into the multicloning site of pKRP10 to
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yield pUC-lacZYA. RS1087 was constructed by introducing the popA-lacZYA fusion
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into RS1085 by the marker-exchange method using pRS192.
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Construction of hpx'-'lacZ fusions. To construct in-frame hpx'-'lacZ fusions, a
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plasmid-based method was applied as follows. Approximately 1 kb restriction fragment
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or PCR-ampliphied fragment containing a part of the coding sequence of hpx gene was
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inserted into the SmaI site of plasmid pMC1871 to yield an in-frame hpx'-'lacZ fusion.
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pMC1871 was contructed by inserting a 2.2 kb EcoRV-SmaI  (Smr/Spr) cassette
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from pHRP315 into the SmaI site of pMC1871 (Shapira et al., 1983). The resulting
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plasmid carrying the hpx'-'lacZ fusion was integrated into the genome of R.
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solanacearum strains through electroporation of the plasmid DNA (2 g). The
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transformants contain the plasmid insertion into the chromosomal hpx locus, resulting in
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hpx'-'lacZ fusions.
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References
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Parke, D. (1990) Construction of mobilizable vectors derived from plasmids RP4,
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pUC18 and pUC19. Gene 93: 135-137.
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Schäfer, A., Tauch, A., Jäger, W., Kalinovski, J., Thierbach, G., and Pühler, A. (1994)
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Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli
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1
plasmids pK18 and pK19: selection of defined deletions in the chromosome of
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Corynebacterium glutamican. Gene 145: 69-73.
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Shapira, S.K., Chou, J., Richard, F.V., and Casadavan, M.J. (1983) New versatile
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plasmid vectors for expression of hybrid proteins coded by a cloned gene fused to lacZ
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gene sequences encoding an enzymatically active carvoxyl-terminal protein of
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-galactosidase. Gene 25: 71-82.
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