phaseolicola a1448sexn / asequencer or array typeillumina hiseq 2000 systemdata formatraw data : fastq files ; analyzed data : txtexperimental factorsphm2-rhpr - ha vs. empty plasmid . in the pseudomonas syringae pv . phaseolicola rhps mutant , a c - terminal ha tagged rhpr was expressed under native promoter in plasmid phm2.experimental featuresusing chip - seq , we obtained a genome - wide dna binding map of rhpr in p. syringae , which revealed 167 loci.consentn/asample source locationchicago , il , usa . the bacterial strains used in this study were the rhps mutant from pseudomonas syringae pv . phaseolicola 1448a strain   containing either an empty phm2 plasmid or phm2-rhpr - ha ( c - terminal hemagglutinin - tagged rhpr ) . the rhpr expression level in the rhps mutant is 10-fold higher than that in the wild - type strain . these strains were grown at 28  c in king 's b medium   containing the appropriate antibiotics to an optical density at 600  nm ( od600 ) of 2.02.5 . the bacteria were centrifuged at 8000  g for 5  min , washed twice with minimal medium ( mm , 50  mm kh2 po4 , 7.6  mm ( nh4)2 so4 , 1.7  mm mgcl2 , 1.7  mm nacl and 10  mm fructose , ph  5.7 ) , resuspended in mm to an od600 of 0.2 and cultured for 6  h at 28  c before subsequent chromatin immunoprecipitation . antibiotics ( in mg / l ) used for the selection of p. syringae pv . phaseolicola strains were : rifampicin , 25 ; kanamycin , 10 ; and spectinomycin , 50 . chip was performed as previously described   with minor changes . after culturing in mm for 6  h , bacteria were treated with 1% formaldehyde ( final concentration ) for 10  min at 37  c with shaking . crosslinking was stopped by adding 125  mm glycine with shaking for additional 5  min . bacterial pellets were centrifuged at 8000  g for 5  min before washed twice with a tris buffer ( 20-mm tris  hcl ph  7.5 , 150  mm nacl ) . the pellets were then re - suspended in 500  l ip buffer ( 50-mm hepes - koh ph  7.5 , 150  mm nacl , 1  mm edta , 1% triton x-100 , 0.1% sodium deoxycholate , 0.1% sds , mini - protease inhibitor cocktail ( roche ) ) and sonicated ( fisher 505 sonic dismembrator ) the dna to sizes of 100300  bp ( 20% total output , 20-second on , 30-second off , for 2  min on ice ) . insoluble cellular debris was removed by centrifugation ( 12,000  g for 10  min at 4  c ) and the supernatant used as the input sample in ip experiments . the protein a beads ( general electric ) protein a beads were resuspended in lysis buffer containing 1  l of thawed 100   bsa ( bovine serum albumin ) for every 100  l total bead suspension volume , before rotated at 4  c for 1  h. after spin for 1  min at 12,000  g at 4  c , the bsa - lysis buffer was removed . the protein fifty microliter prepared beads were added into each ip sample ( 500  l ) , and then rotated for 4  h at 4  c . the mixtures were then incubated by rotating with 50  l agarose - conjugated anti - ha antibodies ( sigma ) in the ip buffer for overnight at 4  c . on the next day one hundred microliter elution buffer 1 ( 10  mm edta , 1% sds , 50  mm tris hcl ph  8.0 ) was added into each sample and incubated for 10  min at 65  c . edta buffer ) was then added into the remaining beads and incubated for overnight at 65  c to reverse crosslinking . on the third day , five microliter 20  mg / ml proteinase k , 10  l 2  mg / ml glycogen , and 235  l te were added into each sample , and incubated for 2  h at 50  c to degrade all proteins . fifty microliter 4  m licl and 500  l phenol  chloroform were added into each sample . after spin at 12,000  g at 4  c for 10  min , the supernatants were collected and precipitated by adding 1  ml cold 100% ethanol . finally the eluted ip - dna were centrifuged at 12,000  g at 4  c for 30  min , and resuspended in 20  l water . each ip reaction was performed in duplicate from two separate cultures , which generates two biological replicates . dna fragments ( 150 to 250  bp ) were selected for library construction and sequencing libraries prepared using the nextflextm chip - seq kit ( bioo scientific ) . the libraries were sequenced using the hiseq 2000 system ( illumina ) with a 50-bp read length . the resulting sequencing reads ( 1.35  gbp including 8.2  m original reads and 3.6  m mapped reads ) were over 200-fold coverage of the p. syringae genome ( 5.93  mbp in length ) . phaseolicola a1448 genome using tophat ( version 2.0.0 , with the following parameters : -o --solexa - quals -p 3 --bowtie1 -n 2 -g 1 ) with two mismatches allowed . only the uniquely mapped reads were kept for the subsequent analyses . the enriched peaks were identified using macs software ( version 2.0.0 , with the following parameters : callpeak -t ip.bam -c ck.bam -f bam -g 6000000 --nomodel --shiftsize  =  75 -n -p 1e-5 ) , which was followed by multiple em for motif elicitation ( meme ) analyses to generate an rhpr - binding motif ( with the parameters : -oc -dna -mod zoops -nmotifs 5 -minw 4 -maxw 20 -revcomp ) . the top - ranked motif was selected ( tgtn[t / a][c / a]n6gatac[a / g ] , p - value  =  1.3e-31 ) . chip was performed as previously described   with minor changes . after culturing in mm for 6  h , bacteria were treated with 1% formaldehyde ( final concentration ) for 10  min at 37  c with shaking . crosslinking was stopped by adding 125  mm glycine with shaking for additional 5  min . bacterial pellets were centrifuged at 8000  g for 5  min before washed twice with a tris buffer ( 20-mm tris  hcl ph  7.5 , 150  mm nacl ) . the pellets were then re - suspended in 500  l ip buffer ( 50-mm hepes - koh ph  7.5 , 150  mm nacl , 1  mm edta , 1% triton x-100 , 0.1% sodium deoxycholate , 0.1% sds , mini - protease inhibitor cocktail ( roche ) ) and sonicated ( fisher 505 sonic dismembrator ) the dna to sizes of 100300  bp ( 20% total output , 20-second on , 30-second off , for 2  min on ice ) . insoluble cellular debris was removed by centrifugation ( 12,000  g for 10  min at 4  c ) and the supernatant used as the input sample in ip experiments . the protein a beads ( general electric ) protein a beads were resuspended in lysis buffer containing 1  l of thawed 100   bsa ( bovine serum albumin ) for every 100  l total bead suspension volume , before rotated at 4  c for 1  h. after spin for 1  min at 12,000  g at 4  c , the bsa - lysis buffer was removed . the protein fifty microliter prepared beads were added into each ip sample ( 500  l ) , and then rotated for 4  h at 4  c . the mixtures were then incubated by rotating with 50  l agarose - conjugated anti - ha antibodies ( sigma ) in the ip buffer for overnight at 4  c . one hundred microliter elution buffer 1 ( 10  mm edta , 1% sds , 50  mm tris  hcl ph  8.0 ) was added into each sample and incubated for 10  min at 65  c . edta buffer ) was then added into the remaining beads and incubated for overnight at 65  c to reverse crosslinking . on the third day five microliter 20  mg / ml proteinase k , 10  l 2  mg / ml glycogen , and 235  l te were added into each sample , and incubated for 2  h at 50  c to degrade all proteins . fifty microliter 4  m licl and 500  l phenol  chloroform were added into each sample . after spin at 12,000  g at 4  c for 10  min , the supernatants were collected and precipitated by adding 1  ml cold 100% ethanol . finally the eluted ip - dna were centrifuged at 12,000  g at 4  c for 30  min , and resuspended in 20  l water . each ip reaction was performed in duplicate from two separate cultures , which generates two biological replicates . dna fragments ( 150 to 250  bp ) were selected for library construction and sequencing libraries prepared using the nextflextm chip - seq kit ( bioo scientific ) . the libraries were sequenced using the hiseq 2000 system ( illumina ) with a 50-bp read length . the resulting sequencing reads ( 1.35  gbp including 8.2  m original reads and 3.6  m mapped reads ) were over 200-fold coverage of the p. syringae genome ( 5.93  mbp in length ) . phaseolicola a1448 genome using tophat ( version 2.0.0 , with the following parameters : -o --solexa - quals -p 3 --bowtie1 -n 2 -g 1 ) with two mismatches allowed . the enriched peaks were identified using macs software ( version 2.0.0 , with the following parameters : callpeak -t ip.bam -c ck.bam -f bam -g 6000000 --nomodel --shiftsize  =  75 -n -p 1e-5 ) , which was followed by multiple em for motif elicitation ( meme ) analyses to generate an rhpr - binding motif ( with the parameters : -oc -dna -mod zoops -nmotifs 5 -minw 4 -maxw 20 -revcomp ) . the top - ranked motif was selected ( tgtn[t / a][c / a]n6gatac[a / g ] , p - value  =  1.3e-31 ) . the tcs rhprs is a necessary regulator of t3ss in the model plant pathogen p. syringae , and its homologs have been implicated in multiple bacterial pathogens . in order to identify all the in vivo binding sites of rhpr , we have recently presented a genome - wide profile of rhpr binding to chromatin in an rhps mutant . our results revealed 167 binding sites including the hrpr promoter , suggesting the direct link between t3ss cascade genes and rhprs . our data as well as microarray analyses strongly suggest that rhpr is not only a master regulator of t3ss , but also a global regulator of multiple pathways in p. syringae . the chip - seq results have provided important information that will help direct the ongoing efforts in determining the regulatory mechanism of rhpr . this work also serves as a framework for further investigations into rhprs functions in p. syringae and other bacteria .
although pseudomonas syringae uses the two - component system rhprs to modulate the expression of type iii secretion system ( t3ss ) genes and pathogenicity , the molecular mechanisms and the regulon of rhprs have yet to be fully demonstrated . we have performed a genome - wide analysis of rhpr binding to dna prepared from p. syringae pv . 
 phaseolicola in order to identify candidate direct targets of rhpr - mediated transcriptional regulation , as described in our recent article [ 1 ] . 
 the data are available from ncbi gene expression omnibus ( geo ) with the accession number gse58533 . here 
 we describe the detailed methods and data analyses of our rhpr chip - seq dataset .