Transcriptional expression of aminoacyl tRNA synthetase genes of Xanthomonas oryzae pv. oryzae (Xoo) on rice-leaf extract treatment and crystal structure of Xoo glutamyl-tRNA synthetase
Thien-Hoang Ho A F , Myoung-Ki Hong A F , Seunghwan Kim B , Jeong-Gu Kim B , Jongha Lee A , Kyoungho Jung A , Inho Lee A , Munyoung Choi A , Hyunjae Park A , Sanghee Lee C , Yeh-Jin Ahn D and Lin-Woo Kang A E
+ Author Affiliations
- Author Affiliations
A Department of Biological Sciences, Konkuk University, 1 Hwayang dong, Gwangjin-gu, Seoul, 05029, Republic of Korea.
B Genomics Division, National Institute of Agricultural Sciences, Rural Development Administration (RDA), Jeonju 03016, Republic of Korea.
C Department of Biological Sciences, Myongji University, 116 Myongjiro, Yongin, Gyeonggido 449-728, Republic of Korea.
D Department of Life Science, Sangmyung University, 7 Hongji-dong, Jongno-gu, Seoul 03016, Korea.
E Corresponding author. Email: lkang@konkuk.ac.kr
F These authors contributed equally to this work.
Crop and Pasture Science 68(5) 434-441 https://doi.org/10.1071/CP16435
Submitted: 26 November 2016 Accepted: 2 May 2017 Published: 31 May 2017
Abstract
Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of bacterial blight of rice, one of the most devastating rice diseases. We analysed the time-resolved transcriptional expression of aminoacyl-tRNA synthetase (aaRS) genes in Xoo cells treated with rice-leaf extract. Most aaRS genes showed decreased expression in the initial 30 min and recovered or increased expression in the later 30 min. The protein-synthetic machinery of bacterial cells is an important target for developing antibiotic agents; aaRSs play an essential role in peptide synthesis by attaching amino acids onto the corresponding tRNA. In bacteria, glutaminyl-tRNA (Gln-tRNAGln) is synthesised in two steps by glutamyl-tRNA synthetase (GluRS) and tRNA-dependent aminotransferase, the indirect biosynthetic mechanism of which is not present in eukaryotes. We determined the crystal structure of GluRS from Xoo (XoGluRS) at resolution of 3.0 Å, this being the first GluRS structure from a plant pathogen such as Xoo. The XoGluRS structure consists of five domains, which are conserved in other bacterial GluRS structures. In the bacterial GluRS structures, the Rossmann-fold catalytic domain and the stem-contact domain are most conserved in both sequence and structure. The anticodon-binding domain 1 is less conserved in sequence but overall structure is conserved. The connective-polypeptide domain and the anticodon-binding domain 2 show various conformations in structure. The XoGluRS structure could provide useful information to develop a new pesticide against Xoo and bacterial blight.
Additional keywords: crop disease, genomics, plant pathology, plant–microbe interactions.
References
Ataide SF, Ibba M (2006) Small molecules: big players in the evolution of protein synthesis. ACS Chemical Biology 1, 285–297.| Small molecules: big players in the evolution of protein synthesis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XmtFSiurw%3D&md5=0f7d59c84c91eab9c6991cc5f7afb623CAS |
Brown MJ, Mensah LM, Doyle ML, Broom NJ, Osbourne N, Forrest AK, Richardson CM, O’Hanlon PJ, Pope AJ (2000) Rational design of femtomolar inhibitors of isoleucyl tRNA synthetase from a binding model for pseudomonic acid-A. Biochemistry 39, 6003–6011.
| Rational design of femtomolar inhibitors of isoleucyl tRNA synthetase from a binding model for pseudomonic acid-A.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXislyhtbo%3D&md5=476d4a23cae323fba976e8d6967e5a0aCAS |
Cavarelli J, Delagoutte B, Eriani G, Gangloff J, Moras D (1998) L-arginine recognition by yeast arginyl-tRNA synthetase. The EMBO Journal 17, 5438–5448.
| L-arginine recognition by yeast arginyl-tRNA synthetase.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXmsFOgsL4%3D&md5=24988f353e4a2dc8fd2685402f10088cCAS |
Cusack S (1995) Eleven down and nine to go. Nature Structural Biology 2, 824–831.
| Eleven down and nine to go.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2MXosFWjtLw%3D&md5=f7c87b709fdabccea3c3617c9b0a8585CAS |
Cusack S (1997) Aminoacyl-tRNA synthetases. Current Opinion in Structural Biology 7, 881–889.
| Aminoacyl-tRNA synthetases.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXjtl2ltw%3D%3D&md5=6957e2f9869f95ef069cb940a98f97f3CAS |
Doan TT, Natarajan S, Kim H, Ahn YJ, Kim JG, Lee BM, Kang LW (2009) Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of glutamyl-tRNA synthetase (Xoo1504) from Xanthomonas oryzae pv. oryzae. Acta Crystallographica. Section F, Structural Biology and Crystallization Communications 65, 51–54.
| Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of glutamyl-tRNA synthetase (Xoo1504) from Xanthomonas oryzae pv. oryzae.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXmvVyjug%3D%3D&md5=03cdf560150125d747ee5a21fbe35fecCAS |
Doan TT, Kim JK, Ngo HP, Tran HT, Cha SS, Min Chung K, Huynh KH, Ahn YJ, Kang LW (2014) Crystal structures of d-alanine-d-alanine ligase from Xanthomonas oryzae pv. oryzae alone and in complex with nucleotides. Archives of Biochemistry and Biophysics 545, 92–99.
| Crystal structures of d-alanine-d-alanine ligase from Xanthomonas oryzae pv. oryzae alone and in complex with nucleotides.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2cXjsVWmt74%3D&md5=bdd95638b378a4861465e96c27931dc0CAS |
Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta Crystallographica. Section D, Biological Crystallography 66, 486–501.
| Features and development of Coot.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXksFKisb8%3D&md5=9d4e0e0c370c38f74bbfa240d7e8c233CAS |
Eriani G, Delarue M, Poch O, Gangloff J, Moras D (1990) Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs. Nature 347, 203–206.
| Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK3MXpslensg%3D%3D&md5=15dc3338ee1da4144dde45d473a50945CAS |
Gouet P, Courcelle E, Stuart DI, Métoz F (1999) Espript: analysis of multiple sequence alignment in Postcript. Bioinformatics 15, 305–308.
| Espript: analysis of multiple sequence alignment in Postcript.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXjslCmtrY%3D&md5=8143f25886ec0b8dee9c4e3766c4c941CAS |
Green A (2017) PPDB: Pesticide Properties DataBase. Available at: http://sitem.herts.ac.uk/aeru/ppdb/en/ (accessed 2 March 2017).
Ito T, Yokoyama S (2010) Two enzymes bound to one transfer RNA assume alternative conformations for consecutive reactions. Nature 467, 612–616.
| Two enzymes bound to one transfer RNA assume alternative conformations for consecutive reactions.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXht1ajtbnP&md5=34032571420df1abc5dc922aec7bdb07CAS |
Kim SH, Lee SE, Hong MK, Song NH, Yoon B, Viet P, Ahn YJ, Lee BM, Jung JW, Kim KP, Han YS, Kim JG, Kang LW (2011) Homologous expression and quantitative analysis of T3SS-dependent secretion of TAP-tagged XoAvrBs2 in Xanthomonas oryzae pv. oryzae induced by rice leaf extract. Journal of Microbiology and Biotechnology 21, 679–685.
| Homologous expression and quantitative analysis of T3SS-dependent secretion of TAP-tagged XoAvrBs2 in Xanthomonas oryzae pv. oryzae induced by rice leaf extract.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXhtFaiurbF&md5=f975be7e0b3c898c8592de74b860ce66CAS |
Kim JK, Natarajan S, Park H, Huynh KH, Lee SH, Kim JG, Ahn YJ, Kang LW (2013a) Crystal structure of XoLAP, a leucine aminopeptidase, from Xanthomonas oryzae pv. oryzae. Journal of Microbiology 51, 627–632.
| Crystal structure of XoLAP, a leucine aminopeptidase, from Xanthomonas oryzae pv. oryzae.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXhslajtbfF&md5=61b3d968aba4a0dea49677dc8b31a078CAS |
Kim S, Nguyen TD, Lee J, Hong MK, Pham TV, Ahn YJ, Lee BM, Han YS, Kim DE, Kim JG, Kang LW (2013b) Homologous expression and T3SS-dependent secretion of TAP-tagged Xo2276 in Xanthomonas oryzae pv. oryzae induced by rice leaf extract and its direct in vitro recognition of putative target DNA sequence. Journal of Microbiology and Biotechnology 23, 22–28.
| Homologous expression and T3SS-dependent secretion of TAP-tagged Xo2276 in Xanthomonas oryzae pv. oryzae induced by rice leaf extract and its direct in vitro recognition of putative target DNA sequence.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXjtlGntrs%3D&md5=dc40bd9704c68fab34bc4540167a16b6CAS |
Kim S, Cho YJ, Song ES, Lee SH, Kim JG, Kang LW (2016a) Time-resolved pathogenic gene expression analysis of the plant pathogen Xanthomonas oryzae pv. oryzae. BMC Genomics 17, 345
| Time-resolved pathogenic gene expression analysis of the plant pathogen Xanthomonas oryzae pv. oryzae.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2MXhsFSltb3F&md5=3ffd2f1f5113e57a84c7d27c81631d7bCAS |
Kim S, Cho YJ, Song ES, Lee SH, Kim JG, Kang LW (2016b) Time-resolved pathogenic gene expression analysis of the plant pathogen Xanthomonas oryzae pv. oryzae. BMC Genomics 17, 345
| Time-resolved pathogenic gene expression analysis of the plant pathogen Xanthomonas oryzae pv. oryzae.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2MXhsFSltb3F&md5=3ffd2f1f5113e57a84c7d27c81631d7bCAS |
Krissinel E (2012) Enhanced fold recognition using efficient short fragment clustering. Journal of Molecular Biochemistry 1, 76–85.
Lamour V, Quevillon S, Diriong S, N’Guyen VC, Lipinski M, Mirande M (1994) Evolution of the Glx-tRNA synthetase family: the glutaminyl enzyme as a case of horizontal gene transfer. Proceedings of the National Academy of Sciences of the United States of America 91, 8670–8674.
| Evolution of the Glx-tRNA synthetase family: the glutaminyl enzyme as a case of horizontal gene transfer.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2cXmt1ClsL0%3D&md5=fca9b8bd7877a92f8c94eae2a075d585CAS |
Laskowski R, MacArthur M, Moss D, Thornton J (1993) PROCHECK: a program to check the sterochemical quality of protein structures. Journal of Applied Crystallography 26, 283–291.
| PROCHECK: a program to check the sterochemical quality of protein structures.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK3sXit12lurY%3D&md5=aa2afb3039145aa663cbd36566a9a3ccCAS |
Lee LW, Ravel JM, Shive W (1967) A general involvement of acceptor ribonucleic acid in the initial activation step of glutamic acid and glutamine. Archives of Biochemistry and Biophysics 121, 614–618.
| A general involvement of acceptor ribonucleic acid in the initial activation step of glutamic acid and glutamine.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaF2sXkvVOrt7s%3D&md5=949224dc6f36f0c55b1f17a470e51c46CAS |
Lee BM, Park YJ, Park DS, Kang HW, Kim JG, Song ES, Park IC, Yoon UH, Hahn JH, Koo BS, Lee GB, Kim H, Park HS, Yoon KO, Kim JH, Jung CH, Koh NH, Seo JS, Go SJ (2005) The genome sequence of Xanthomonas oryzae pathovar oryzae KACC10331, the bacterial blight pathogen of rice. Nucleic Acids Research 33, 577–586.
| The genome sequence of Xanthomonas oryzae pathovar oryzae KACC10331, the bacterial blight pathogen of rice.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXhtV2qtrs%3D&md5=7fdc3eaa5edf5a59e077a0801879cd59CAS |
Li JY, Wang J, Zeigler RS (2014) The 3,000 rice genomes project: new opportunities and challenges for future rice research. GigaScience 3, 8
| The 3,000 rice genomes project: new opportunities and challenges for future rice research.Crossref | GoogleScholarGoogle Scholar |
Liu J, Lin SX, Blochet JE, Pezolet M, Lapointe J (1993) The glutamyl-tRNA synthetase of Escherichia coli contains one atom of zinc essential for its native conformation and its catalytic activity. Biochemistry 32, 11390–11396.
| The glutamyl-tRNA synthetase of Escherichia coli contains one atom of zinc essential for its native conformation and its catalytic activity.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK3sXmt1Snt7Y%3D&md5=a8ceb0e915a580f33a7fec830b31e1afCAS |
Mew TW, Alvarez AM, Leach JE, Swings J (1993) Focus on bacterial-blight of rice. Plant Disease 77, 5–12.
| Focus on bacterial-blight of rice.Crossref | GoogleScholarGoogle Scholar |
Murshudov GN, Skubak P, Lebedev AA, Pannu NS, Steiner RA, Nicholls RA, Winn MD, Long F, Vagin AA (2011) REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallographica. Section D, Biological Crystallography 67, 355–367.
| REFMAC5 for the refinement of macromolecular crystal structures.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXktFWqtbk%3D&md5=ee4cd528e24fd8a8e39d261214cfd2fdCAS |
Ngo HP, Ho TH, Lee I, Tran HT, Sur B, Kim S, Kim JG, Ahn YJ, Cha SS, Kang LW (2016) Crystal structures of peptide deformylase from rice pathogen Xanthomonas oryzae pv. oryzae in complex with substrate peptides, actinonin, and fragment chemical compounds. Journal of Agricultural and Food Chemistry 64, 7307–7314.
| Crystal structures of peptide deformylase from rice pathogen Xanthomonas oryzae pv. oryzae in complex with substrate peptides, actinonin, and fragment chemical compounds.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC28XhsVyqtbrE&md5=73a33600255d3c19c8929cca719c8a2dCAS |
Nureki O, Vassylyev DG, Katayanagi K, Shimizu T, Sekine S, Kigawa T, Miyazawa T, Yokoyama S, Morikawa K (1995) Architectures of class-defining and specific domains of glutamyl-tRNA synthetase. Science 267, 1958–1965.
| Architectures of class-defining and specific domains of glutamyl-tRNA synthetase.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2MXkslOktb4%3D&md5=1064999713de07ad110139a83ee2305bCAS |
Nureki O, Vassylyev DG, Tateno M, Shimada A, Nakama T, Fukai S, Konno M, Hendrickson TL, Schimmel P, Yokoyama S (1998) Enzyme structure with two catalytic sites for double-sieve selection of substrate. Science 280, 578–582.
| Enzyme structure with two catalytic sites for double-sieve selection of substrate.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXjtVWqurs%3D&md5=92fd2195699321cba63824718033379bCAS |
Nureki O, O’Donoghue P, Watanabe N, Ohmori A, Oshikane H, Araiso Y, Sheppard K, Soll D, Ishitani R (2010) Structure of an archaeal non-discriminating glutamyl-tRNA synthetase: a missing link in the evolution of Gln-tRNAGln formation. Nucleic Acids Research 38, 7286–7297.
| Structure of an archaeal non-discriminating glutamyl-tRNA synthetase: a missing link in the evolution of Gln-tRNAGln formation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXhsVeksbbK&md5=3f3776698c408900fa0cc9162bff3a0eCAS |
Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods in Enzymology 276, 307–326.
| Processing of X-ray diffraction data collected in oscillation mode.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2sXivFehsbw%3D&md5=04865915b3dd73b7d4fee86472880896CAS |
Rock FL, Mao W, Yaremchuk A, Tukalo M, Crepin T, Zhou H, Zhang YK, Hernandez V, Akama T, Baker SJ, Plattner JJ, Shapiro L, Martinis SA, Benkovic SJ, Cusack S, Alley MR (2007) An antifungal agent inhibits an aminoacyl-tRNA synthetase by trapping tRNA in the editing site. Science 316, 1759–1761.
| An antifungal agent inhibits an aminoacyl-tRNA synthetase by trapping tRNA in the editing site.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXms1Wgsbo%3D&md5=78d60e92477315120c08cf65b1a70137CAS |
Rould MA, Perona JJ, Soll D, Steitz TA (1989) Structure of E. coli glutaminyl-tRNA synthetase complexed with tRNA(Gln) and ATP at 2.8 A resolution. Science 246, 1135–1142.
| Structure of E. coli glutaminyl-tRNA synthetase complexed with tRNA(Gln) and ATP at 2.8 A resolution.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK3cXltFahsA%3D%3D&md5=77764f272d9ded6c35c9f5c1697bb055CAS |
Schrödinger LLC (2010) ‘The PyMOL Molecular Graphics System. Version 1.3r1.’ (Schrödinger LLC: New York)
Schulze JO, Masoumi A, Nickel D, Jahn M, Jahn D, Schubert WD, Heinz DW (2006) Crystal structure of a non-discriminating glutamyl-tRNA synthetase. Journal of Molecular Biology 361, 888–897.
| Crystal structure of a non-discriminating glutamyl-tRNA synthetase.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XotV2kt70%3D&md5=1ada53f8d1a6d7e32e49902fe478c9e5CAS |
Sekine S, Shichiri M, Bernier S, Chenevert R, Lapointe J, Yokoyama S (2006) Structural bases of transfer RNA-dependent amino acid recognition and activation by glutamyl-tRNA synthetase. Structure 14, 1791–1799.
| Structural bases of transfer RNA-dependent amino acid recognition and activation by glutamyl-tRNA synthetase.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XhtlShsrvJ&md5=c3dd6a893652c1caa93dd66d3062f054CAS |
Siatecka M, Rozek M, Barciszewski J, Mirande M (1998) Modular evolution of the Glx-tRNA synthetase family–rooting of the evolutionary tree between the bacteria and archaea/eukarya branches. European Journal of Biochemistry 256, 80–87.
| Modular evolution of the Glx-tRNA synthetase family–rooting of the evolutionary tree between the bacteria and archaea/eukarya branches.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXlslCmtbw%3D&md5=17bc6d23d175b0b223f77152e0e6f05cCAS |
Silvian LF, Wang J, Steitz TA (1999) Insights into editing from an ile-tRNA synthetase structure with tRNAile and mupirocin. Science 285, 1074–1077.
| Insights into editing from an ile-tRNA synthetase structure with tRNAile and mupirocin.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXlt1Gms7k%3D&md5=3e81bf6723054fe42b07b4443d733781CAS |
Sugiura I, Nureki O, Ugaji-Yoshikawa Y, Kuwabara S, Shimada A, Tateno M, Lorber B, Giege R, Moras D, Yokoyama S, Konno M (2000) The 2.0 Å crystal structure of Thermus thermophilus methionyl-tRNA synthetase reveals two RNA-binding modules. Structure 8, 197–208.
| The 2.0 Å crystal structure of Thermus thermophilus methionyl-tRNA synthetase reveals two RNA-binding modules.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXhsVygtbo%3D&md5=37915875b0fa1085059668d2601854d9CAS |
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The Clustal_X woindows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25, 4876–4882.
| The Clustal_X woindows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXntFyntQ%3D%3D&md5=f613ea216c72016ef607484cbf56788eCAS |
Vondenhoff GH, Van Aerschot A (2011) Aminoacyl-tRNA synthetase inhibitors as potential antibiotics. European Journal of Medicinal Chemistry 46, 5227–5236.
| Aminoacyl-tRNA synthetase inhibitors as potential antibiotics.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXhtlKktbrE&md5=4908817a2c561dd546b8c6f7911ea46fCAS |
Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nature Reviews. Genetics 10, 57–63.
| RNA-Seq: a revolutionary tool for transcriptomics.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXhsFWis7bL&md5=5aa29b145188aa30edafa934175c83c0CAS |
Wilcox M, Nirenberg M (1968) Transfer RNA as a cofactor coupling amino acid synthesis with that of protein. Proceedings of the National Academy of Sciences of the United States of America 61, 229–236.
| Transfer RNA as a cofactor coupling amino acid synthesis with that of protein.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaF1MXhtlc%3D&md5=1ad988cde6925d074af0f3dba8592a5cCAS |
Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan RM, Krissinel EB, Leslie AG, McCoy A, McNicholas SJ, Murshudov GN, Pannu NS, Potterton EA, Powell HR, Read RJ, Vagin A, Wilson KS (2011) Overview of the CCP4 suite and current developments. Acta Crystallographica. Section D, Biological Crystallography 67, 235–242.
| Overview of the CCP4 suite and current developments.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXktFWqt70%3D&md5=308b1a33dc7c92fafe17d6d13eacc4b9CAS |
