Supplementary Materials

Supplementry Mterils Title: A functionl 4-hydroxyenzote degrdtion pthwy in the phytopthogen Xnthomons cmpestris is required for full pthogenicity Authors: Ji-Yun Wng, Lin Zhou, Bo Chen, Shung Sun, Wei Zhng, Ming Li, Hongzhi Tng, Bo-Le Jing, Ji-Ling Tng, Y-Wen He Contents Supplementry Fig. S1 Supplementry Fig. S2 Supplementry Fig. S3 Supplementry Fig. S4 Supplementry Fig. S5 Supplementry Fig. S6 Supplementry Fig. S7 Supplementry Fig. S8 Supplementry Fig. S9 Supplementry Tle S1 Supplementry Tle S2 Supplementry Tle S3

E.coli ex eb ea Xcc 41% nodt 31% Xcc4168 Xcc4169 Xcc4170 Xcc4171 PP PP1271 PP1271 PP1273 Xcc 23% 31% 34% Xcc1398 Xcc1399 Xcc1400 Xcc4152 Xcc4153 Xcc4154 (ene) Xcc1684 Xcc1685 Xcc1686 (MFS) Xcc0348 Xcc0349 Xcc0350 (vnk) Supplementry Fig. S1 Puttive genes or gene clusters for 4-HBA trnsporttion in Xnthomons cmpestris pv. cmpestris. () Puttive gene clusters for 4-HBA efflux pumping; () Puttive genes for 4-HBA uptke. The percentge indictes mino cid identity. All sequences were downloded from the Microil Genome Dtse of NCBI.

0.6 XOLN+0.5 mm 4-HBA OD 600 0.4 ΔpcHG 0.2 ΔpoA ΔpoA(poA) ΔpcHG(HG) 0.0 0 12 24 36 Time fter inocultion (h) 3 NYG 2 OD 600 1 ΔpoA ΔpoA (poa) ΔpcHG ΔpcHG(HG) 0 0 12 24 36 48 Time (h) Supplementry Fig. S2 Growth of Xcc strins in () XOLN medium or () NYG medium supplemented with 0.5 mm 4-HBA.

0.8 XOLN + 0.5mM 4HBA 0.6 OD 600 0.4 0.2 Δ0357 Δ 0358-61 Δ0372 0.0 0 6 12 18 24 Time(h) 0.6 XOLN + 0.5mM 4HBA c 4-HBA conc.(mm) 0.4 0.2 Δ0357 Δ0358-61 Δ0372 XOLN 0.0 0 6 12 18 24 Time(hour) XOLN + 1.5 mm 4-HBA Δ0357 Δ0358-61 Δ0372 Supplementry Fig. S3 The genes Xcc0357, Xcc0358-61, nd Xcc0372 re not required for 4-HBA degrdtion. () The growth of Xcc strins s determined t n opticl density of 600 nm. () The 4-HBA levels in the cell cultures of Xcc strins during growth in XOLN medium. (c) Bcteril growth of Xcc strins on n XOLN plte supplemented with 1.5 mm 4-HBA.

PcI c pci pci pci pci pci MA-DIVSLSEAVAQLIADGDCVAMEGFTHL-IPHAAGHEVIRQRKRNLR----------- MINKTYESIASAVEGITDGSTIMVGGFGTAGMPSELIDGLIATGARDLTIISNNAGNGEI *.. :..: *:**. : : ** :*. :* *:* Glycine cluster -----------------LVRMTPDLIYDQLIGAGCASALRFSWGGNPGVGSLHRLRDAVE GLAALLMAGSVRKVVCSFPRQSDSYVFDELYRAGKIE----------------------- : * :. ::*:* **. HGWPAPLQLREHSHADMANAYVAGASGLP-FAVLRGYV-----GSDLPKVNDSIRFLQCP --------LEVVPQGNLAERIRAAGSGIGAFFSPTGYGTLLAEGKETREID--------- *. :.::*: *..**: * ** *.: ::: YTGQTLATSPAVNPDVTVIHAQQADRRGNVLLWGIL-GVQKEAALAARKVIVTVEEIVDT --GRMYVLEMPLHADFALIKAHKGDRWGNLTYRKAARNFGPIMAMAAKTAIAQVDQVVEL *:.. :. *.::*:*::.** **:. *:**:..*. *:::*: LDAPPNACILPRWVVSAVCVVPGGAAPSYAHGYSARDNRFYLAWDAMARDRARFQAWIQA GELDPEHIITPGIFVQRVVAVTGAAASSIAKAV--------------------------- : *: * *.*. *.* *.** * *:. HILDTDDFAGFQRVYAGSQHAEAA pci ------------------------ Supplementry Fig. S4 Domin orgniztion nd multiple sequence lignment of nd PcI. () Domin orgniztion of PcI in Pseudomons putid. () Domin orgniztion of in Xcc. (c) Sequence lignment of PcI nd. Domin orgniztion ws conducted using the SMART progrm (http://smrt.eml-heidelerg.de). Sequence lignment ws conducted using the progrm Clustl Omeg (http://www.ei.c.uk/tools/ms/ clustlo/).

PcJ c pcj pcj pcj pcj pcj MTITKKLSRTEMAQRVAADIQEGAYVNLGIGAPTLVANYL---GDKEVFLHSENGLLGMG --MSDYSTNEMMTVAAARRLPDGAVCFVGIGLPSTAANLARLTHAPEVTLIYESGPIGAR ::. :. *:.* : :** :*** *:.** ** * *.* :* SENG motif PSPAPGEEDDDLINAGKQHVTLLTGGAFFHHADS-FSMMRGGHLDIAVLGAFQVSVKGDL PQVLPLSIGDGELAETAD--------TVVSTPEIFRYWLQGGRVDVGFLGAAQIDRHANL *. *. * : : :.. : ::**::*:..*** *:. :.:* ANWH----TGAEGSIPAVGGAMDLATGARQVFVMMDHLTKTGESKLVP------------ NTTVIGPYAAPKTRLPGAGGAPEIATSAKQVFIIMRQSTRSFVQTLDFITTVGHLDGGDA. :. : :*..*** ::**.*:***::* : *::..* --ECTYPLTGIACVSRIYTDLAVLEVTPEGLKVV--EICADIDFDELQKLSGVPL----- RTRAGLPGAGP---TVVVTDLCVMEPDPVSRELTVTALHPGVTQAQVTAATGWAVRFAAT.. * :* : : ***.*:* *. ::. : : :: :* : --------IK------------------------- LTETAAPTATELHALRALQARTAAAHGTQAAGAEA. Supplementry Fig. S5 Domin orgniztion nd multiple sequence lignment of nd PcJ. () Domin orgniztion of PcJ in Pseudomons putid. () Domin orgniztion of in Xcc. (c) Sequence lignment of PcJ nd. Domin orgniztion ws conducted using the SMART progrm (http://smrt.eml-heidelerg.de). Sequence lignment ws conducted using the progrm Clustl Omeg (http://www.ei.c.uk/tools/ms/ clustlo/).

XCC0364 homologues in Bcteri MADIVSLSEAVAQLIADGDCVAMEGFTHLIPHAAGHEVIRQRKRNLRLVRMTPDLIYDQL SMB20587 MARIMSLAEAVEENVRDGDTVAMEGFTHLIPYAAGHEVIRQGRKDLFLVRMTPDILYDQL ** *:**:*** : : *** ***********:********* :::* *******::**** IGAGCASALRFSWGGNPGVGSLHRLRDAVEHGWPAPLQLREHSHADMANAYVAGASGLPF SMB20587 IGVGAARGMKFSWGGNPGVGSLHRFRDAVENQWPRPLEIEEHSHAAMANAYEAGAGNLPF **.*.*.::**************:*****. ** **::.***** ***** ***. *** AVLRGYVGSDLPKVNDSIRFLQCPYTGQTLATSPAVNPDVTVIHAQQADRRGNVLLWGIL SMB20587 AAFRGYIGADLPKVNLNIRSVTCPFTGEVLAAVPAIRPDVTIIHAQRADRNGNVLIEGIV *.:***:*:******.** : **:**:.**: **:.****:****:***.****: **: GVQKEAALAARKVIVTVEEIVDTLDAP-PNACILPRWVVSAVCVVPGGAAPSYAHGYSAR SMB20587 GVQKEAVLAARRSIVTVEEIVDELSPPSPNSVVLPGWAVTAVAHVPGGAFPSYAHGYYPR ******.****: ********* *. * **: :** *.*:**. ***** ******* * DNRFYLAWDAMARDRARFQAWIQAHILDTD--DFAGFQRVYAGSQHAEAA SMB20587 SNAFYIRWDEIARDRETFTAWIRENVLAAKPEDFARHAAKTAAKV----A.* **: ** :**** * ***:.:* :. ***. *.. * Species Homologues of Amino cid identity (%) NCBI ccess No. Xc XAC0364 92 AAM35256.1 Xcr XCR_4151 99 AEL09008.1 Xff XFF4834R_chr03 480 92 CDF60017.1 Xoc XOC_4548 91 AEQ98605.1 Lysocter cpsici AZ78_14030 79 EYR67734.1 Pseudomons eruginos PA0226 73 AAG03615.1 Pseudomons knckmussii PKB_2952 72 CDF84299.1 Sinorhizoium meliloti SMB20587 67 CAC49899.1 Mesorhizoium loti MAFF_RS17350 58 BAB50895.1 Supplementry Fig. S6 Homologs of in other cteril species. () Multiple sequence lignment nlysis of nd SMB20588. () Puttive homologs of in other cteril species.

-------MSDYSTNEMMTVAAARRLPDGAVCFVGIGLPSTAANLARLTHAPEVTLIYESG SMB20588 MLEGKTKMTHFTPTEMMTVAAARALSNDDVCFVGIGAPSAACNVARLTNAPDITLIYESG *:.::.********* * : ******* **:*.*:****.**::******* PIGARPQVLPLSIGDGELAETADTVVSTPEIFRYWLQGGRVDVGFLGAAQIDRHANLNTT SMB20588 TVGTKPDVLPLSIGDGELCDTALFTVSVPEMFRYWLQGGRITTGFLGGAQIDRFANLNTT :*::*:***********.:**. **.**:*********:.****.*****.****** VIGPYAAPKTRLPGAGGAPEIATSAKQVFIIMRQSTRSFVQTLDFITTVGHLDGGDARTR SMB20588 VVGPYDHPKVRLPGGGGAPEIASNCGRIFITMALTKRGFVERLPFVTSMGHGEGGNHRER *:*** **.****.*******:.. ::** * :.*.**: * *:*::** :**: * * AGLPGAGPTVVVTDLCVMEPDPVSRELTVTALHPGVTQAQVTAATGWAVRFAATLTETAA SMB20588 LGLTTSGPTRVITDLCILEPDPETKELTVVSMHPGVARDQIAGNCGWPIKFAETVIETPA ** : *** *:****::**** ::****.::****:: *::. ** ::** *: ** * PTATELHALRALQARTAAAHGTQAAGAEA SMB20588 PTETELVVLRDINARTKKAHKAAGKEAA- ** ***.** ::*** ** :. * Species Homologues of Amino cid identity (%) NCBI ccess No. X. xonopodis pv. citri XAC0365 89 AAM35257.1 Xcr XCR_4150 100 AEL09007.1 Xff XFF4834R_chr0 3490 89 CDF60018.1 Xoc XOC_4547 90 AEQ98604.1 Lysocter cpsici AZ78_14025 76 EYR67733.1 Pseudomons eruginos PA0227 72 AAG03616.1 Pseudomons knckmussii PKB_2951 74 CDF84298.1 Sinorhizoium meliloti SMB20588 60 CAC49900.1 Mesorhizoium loti MAFF_RS17345 63 BAB50894.1 Supplementry Fig. S7 Homologs of in other cteril species. () Multiple sequence lignment nlysis of nd SMB20588. () Puttive homologs of in other cteril species.

ΔpcGH ΔpoA ΔpcI ΔpoA ΔpcGH ΔpcI c ΔpoA ΔpcGH ΔpcI d EPS production (mg/ml) 5 4 3 2 1 0 poa pcgh pci Supplementry Fig. S8 Production of extrcellulr enzymes nd polyscchride (EPS) in Xcc strins. () Cellulse ctivity. Aout 1 μl of the cell culture (OD 600 1.0) ws spotted onto NYG contining 0.5% CMC. () Amylse ctivity. Xcc strins were grown on n NYG plte supplemented with 0.1% solule strch. (c) Protese ctivity. The strins were grown on n NYG plte supplemented with 1.0% skim milk. (d) EPS production in NYG medium.

Are -ESI Scn (50.171 min) 2-HBA -ESI Scn (11.680 min) 4-HBA -ESI Scn (15.879 min) 3-HBA 1.2E+7 1.0E+7 2-HBA 3-HBA 4-HBA 8.0E+6 6.0E+6 4.0E+6 2.0E+6 0.0E+0 0 10 20 30 40 50 60 Concentrtion of HBAs (µm) Supplementry Fig. S9 LC-MS nlysis of rdish hydrolystes. () Moleculr weights of the puttive 2-HBA, 3-HBA nd 4-HBA detected in rdish hydrolystes. () The estlished stndrd curves for determining the concentrtion of 2-HBA, 3-HBA nd 4-HBA.

Supplementry Tle S1. Bcteril strins used in this study Strins Relevnt chrcteristics Refernce/source Xcc Wild type, Rif R Lortory collection Δ0355 xcc0355 in-frme deletion mutnt of Δ0356 xcc0356 in-frme deletion mutnt of Δ0357 xcc0357 in-frme deletion mutnt of Δ0358-61 xcc0358, xcc0359, xcc0360, nd xcc0361 in-frme deletion mutnt of Δ0364 xcc0364 in-frme deletion mutnt of Δ0365 xcc0365 in-frme deletion mutnt of Δ0364-65 xcc0364 nd xcc0365 in-frme deletion mutnt of Δ0367-68 xcc0367 nd xcc0368 in-frme deletion mutnt of Δ0370 xcc0370 in-frme deletion mutnt of Δ0372 xcc0372 in-frme deletion mutnt of Δ0373 xcc0373 in-frme deletion mutnt of Δ0356C Δ0356 mutnt contining pbbr_0356 which contins full length xcc0356 Δ0364C Δ0364 mutnt contining pbbr_0364 which contins full length xcc0364 Δ0365C Δ0365 mutnt contining pbbr_0365 which contins full length xcc0365 Δ0364(pcI) Δ0364 mutnt contining pbbr_pci which contins full length pci Δ0365(pcJ) Δ0365 mutnt contining pbbr_pcj which contins full length pcj Δ0367-68C Δ0367-68 mutnt contining pbbr_0367-68 which contins full length xcc0367 nd xcc0368 P.putid KT2440 Wild type Prof. Ping Xu l collection E.coli DH5α F - φ80lcz M15 enda1hsdr17 (r k - m k- ) supe44 thi -1 gyra96 (lczya-rgf) S17-1 reca pro (RP4-2Tet::Mu Kn::Tn7) Lortory collection Simon et l., 1983 Simon, R., Priefer, U., nd Puhler, A. 1983. A rod host rnge moiliztion system for in vivo genetic engineering: trnsposon mutgenesis in grm negtive cteri. Nt Biotech 1:784-791

Supplementry Tle S2. Plsmids used in this study Plsmids Relevnt chrcteristics Refernce/source pk18moscb prk2013 pbbr1mcs2 Suicide plsmid in XCC, Km R ColE1 replicon TrRK+ Mo+; Helper plsmid for moilistion of non-self-trnsmissile plsmids, Km R Plsmid for gene complementtion, Km R Schfer et l., 1994 Figurski nd Helinski, 1979 Kovch et l.,1995 pk18-0355 Sucide construct for in-frme deletion of xcc0355 pk18-0356 Sucide construct for in-frme deletion of xcc0356 pk18-0357 Sucide construct for in-frme deletion of xcc0357 pk18-0358-61 Sucide construct for in-frme deletion of xcc0358, xcc0359, xcc0360, nd xcc0361 pk18-0364 Sucide construct for in-frme deletion of xcc0364 pk18-0365 Sucide construct for in-frme deletion of xcc0365 pk18-0364-65 Sucide construct for in-frme deletion of xcc0364 nd xcc0365 pk18-0367-68 Sucide construct for in-frme deletion of xcc0367 nd xcc0368 pk18-0370 Sucide construct for in-frme deletion of xcc0370 pk18-0372 Sucide construct for in-frme deletion of xcc0372 pk18-0373 Sucide construct for in-frme deletion of xcc0373 pbbr0356 Complementry vector contining whole length of xcc0356 pbbr0364 Complementry vector contining whole length of xcc0364 pbbr0365 Complementry vector contining whole length of xcc0365 pbbr0367-68 Complementry vector contining whole length of xcc0367 nd xcc0368 pbbr-pci Complementry vector contining whole length of pci pbbr-pcj Complementry vector contining whole length of pcj Figurski, D.H., nd Helinski, D.R. 1979. Repliction of n origin-contining derivtive of plsmid RK2 dependent on plsmid function provided in trns. Proceedings of the Ntionl Acdemy of Sciences of the United Sttes of Americ 76:1648-1652. Kovch, M.E., Elzer, P.H., Hill, D.S., Roertson, G.T., Frris, M.A., Roop, R.M., 2nd, nd Peterson, K.M. 1995. Four new derivtives of the rod-host-rnge cloning vector pbbr1mcs, crrying different ntiiotic-resistnce cssettes. Gene 166:175-176. Schfer, A., Tuch, A., Jger, W., Klinowski, J., Thierch, G., nd Puhler, A. 1994. Smll moilizle multi-purpose cloning vectors derived from the Escherichi coli plsmids pk18 nd pk19: selection of defined deletions in the chromosome of Corynecterium glutmicum. Gene 145:69-73.

Supplementry Tle S3. Primers used in this study Appliction Primers Sequence(5'-3') In-frme deletion Δ0355 0355-F1 cccgcccccgcggtccc 0355-R1 cgctcgccgcgtcttcgc 0355-F2 tctgcggctgtgcggtggctgccgccttggtttttc 0355-R2 cccccgcggcctgttcttgc Δ0356 0356-F1 ggttctcggcggctggcgcg 0356-R1 cctgggtgcgctcgggt 0356-F2 tgcgccccggttgcggcggtgttgcgcgtttc 0356-R2 cgggtcccgccgggcgcggggc Δ0357 0357-F1 gcccgccgcgccgttctcct 0357-R1 cgctcgcccgctgcgctttg 0357-F2 tctgcgggcggcggcccgcgtgtccgtccg 0357-R2 ccgggctggcggccctt Δ0358-61 0361-F1 cgcgcggcgtgtgggtt 0361-R1 cgccgctcgccggttctcc 0358-F2 ctggcgttgcggcgcgcggcccgcggcct 0358-R2 ccgccccccccccg Δ0364 0364-F1 ggttccggccctgcgcgttgtgcc 0364-R1 ggcgtgcgggtcggtgggt 0364-F2 ctgtcccgccgccgcggggcggctggcgct 0364-R2 cgggtcctggctgccgctgtgtggc Δ0365 0365-F1 ggttcccccggcctgtttcgccc 0365-R1 gccgtgccgcgcccc 0365-F2 ttcgtcggctcggcgggccgggctggcggg 0365-R2 cgggtcctccggcgcggttgtggc Δ0364-65 0364-F1 ggttccggccctgcgcgttgtgcc 0364-R1 ggcgtgcgggtcggtgggt 0365-F3 ctgtcccgccgccgggccgggctggcggg 0365-R2 cgggtcctccggcgcggttgtggc Δ0367-68 0367-F1 cgcgcgggtgctggggtttg 0367-R1 gcgccgtgccggcggct 0368-F2 ctcggcttcggcgcctcgtgttctgcgcgggctgt 0368-R2 gtggcctgggcggctgcc Δ0370 0370-F1 gcggcgtgtggtgctgctg 0370-R1 gcgtgtgtcggcctggg 0370-F2 ttgccgcctcgccgcgtgctgctgggttttctc 0370-R2 tcgggtgcgtgtctttgtgttg

Δ0372 0372-F1 gcctgcccgccccctcg 0372-R1 cggcgccgcgcccccc 0372-F2 tggtcgcggctgccgctgctgctgcgcggtgtggtg 0372-R2 cctggcggccctgggtttgtg Δ0373 0373-F1 ggttccgccggcgtccccgttg Complementtion 0373-R1 gggcgtttgcggcggggg 0373-F2 cgccgccgtcccctgcgtggcgggtgttgc 0373-R2 cgggtcctgttgtcggcgctggtgctgttg 0356 0356-F cccgctttcggcggctggcgcg 0356-R cgggtcccgctcgcccgctgcgctttgc 0364 0364-F cccgcttcggccctgcgcgttgtgcc 0364-R gctctggccgtgccgcgcccc 0365 0365-F cccgctttccgtttcctgcgtgcccctc 0365-R gctctgctggcgtgcgccgccgt 0367-68 0367-68-F cccgcttcgcgcgggtgctggggtttg 0367-68-R gctctgccctggcgcctgtgccctc pci 3951-F ggttcctgctcgccctgggcg 3951-R cgggtcccggtgcgggggctttttggtg pcj 3952-F ggttcccctggccggcgttccg 3952-R cgggtcctcggcggctgtggtgtctcg qrt-pcr 0364 RT0364-F cttttcctggggcggctcct RT0364-4 cctgtccgcgtggctgtgttc 0365 RT0365-1 cccccgggtctttcgctttgg RT0365-2 cgtgccgcggtggtgtg tpd tpd-f gcgtggtgtcggccgtg tpd-r cggccgcgtgtgcggtgtgt