1887

Abstract

pv. () causes bacterial leaf streak (BLS) in rice, an emerging and destructive disease worldwide. Identification of key virulence factors is a prerequisite for understanding the pathogenesis of . In this study, a Tn-tagged mutant library of strain RS105 was screened on rice, and 27 Tn mutants were identified that were either non-pathogenic or showed reduced virulence in rice. Fourteen of the non-pathogenic mutants were also unable to elicit the hypersensitive response (HR) in tobacco and were designated Pth/HR mutants; 13 mutants showed attenuated virulence and were able to induce an HR (Vir/HR). Sequence analysis of the Tn-tagged genes indicated that the 14 Pth/HR mutants included mutations in , , , , and . The 13 Vir/HR mutants included (a transcriptional activator-like TAL effector), (regulator of pathogenicity factors), (oxidative stress transcriptional regulator), (disulfide isomerase), (glucan biosynthesis glucosyltransferase H), (glucose-1-phosphate thymidylyltransferase), (aminotransferase), (amidophosphoribosyltransferase), (threonine synthase), (tryptophan synthase alpha subunit) and three genes encoding hypothetical proteins (, and ). Collectively, the 27 Tn insertions are located in 21 different open reading frames. Bacterial growth and virulence assays demonstrated that , , , , , and are candidate virulence genes involved in pathogenesis. Reduced virulence in 13 mutants was restored to wild-type levels when the cognate gene was introduced . Expression profiles demonstrated that the seven candidate virulence genes were significantly induced , although their roles in pathogenesis remain unclear.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.050419-0
2012-02-01
2019-10-15
Loading full text...

Full text loading...

/deliver/fulltext/micro/158/2/505.html?itemId=/content/journal/micro/10.1099/mic.0.050419-0&mimeType=html&fmt=ahah

References

  1. Baker C. J. , Orlandi E. W. . ( 1995; ). Active oxygen in plant pathogenesis. . Annu Rev Phytopathol 33:, 299–321. [CrossRef] [PubMed]
    [Google Scholar]
  2. Chen G. Y. , Zou L. F. , Wu X. M. , Wang J. S. . ( 2005; ). avr/pth13 gene of Xanthomonas oryzae pv. oryzicola, a novel virulence member of avrBs3/PthA family, strengthening virulence of Xanthomonas oryzae pv. oryzae on rice. . Chin J Rice Sci 19:, 291–296.
    [Google Scholar]
  3. Cho H. J. , Park Y. J. , Noh T. H. , Kim Y. T. , Kim J. G. , Song E. S. , Lee D. H. , Lee B. M. . ( 2008; ). Molecular analysis of the hrp gene cluster in Xanthomonas oryzae pathovar oryzae KACC10859. . Microb Pathog 44:, 473–483. [CrossRef] [PubMed]
    [Google Scholar]
  4. Christman M. F. , Storz G. , Ames B. N. . ( 1989; ). OxyR, a positive regulator of hydrogen peroxide-inducible genes in Escherichia coli and Salmonella typhimurium, is homologous to a family of bacterial regulatory proteins. . Proc Natl Acad Sci U S A 86:, 3484–3488. [CrossRef] [PubMed]
    [Google Scholar]
  5. Chu C. C. . ( 1978; ). The N6 medium and its application to anther culture of cereal crops. In Proceedings of the Symposium on Plant Tissue Culture, pp. 43–50. Beijing: Science Press.
  6. Cogez V. , Talaga P. , Lemoine J. , Bohin J. P. . ( 2001; ). Osmoregulated periplasmic glucans of Erwinia chrysanthemi . . J Bacteriol 183:, 3127–3133. [CrossRef] [PubMed]
    [Google Scholar]
  7. Cross S. E. , Kreth J. , Zhu L. , Sullivan R. , Shi W. , Qi F. , Gimzewski J. K. . ( 2007; ). Nanomechanical properties of glucans and associated cell-surface adhesion of Streptococcus mutans probed by atomic force microscopy under in situ conditions. . Microbiology 153:, 3124–3132. [CrossRef] [PubMed]
    [Google Scholar]
  8. Furutani A. , Takaoka M. , Sanada H. , Noguchi Y. , Oku T. , Tsuno K. , Ochiai H. , Tsuge S. . ( 2009; ). Identification of novel type III secretion effectors in Xanthomonas oryzae pv. oryzae . . Mol Plant Microbe Interact 22:, 96–106. [CrossRef] [PubMed]
    [Google Scholar]
  9. Gonçalves L. R. , Soares M. R. , Nogueira F. C. , Garcia C. , Camisasca D. R. , Domont G. , Feitosa A. C. , Pereira D. A. , Zingali R. B. , Alves G. . ( 2010; ). Comparative proteomic analysis of whole saliva from chronic periodontitis patients. . J Proteomics 73:, 1334–1341. [CrossRef] [PubMed]
    [Google Scholar]
  10. Ha U. H. , Wang Y. , Jin S. . ( 2003; ). DsbA of Pseudomonas aeruginosa is essential for multiple virulence factors. . Infect Immun 71:, 1590–1595. [CrossRef] [PubMed]
    [Google Scholar]
  11. He Y. W. , Zhang L. H. . ( 2008; ). Quorum sensing and virulence regulation in Xanthomonas campestris . . FEMS Microbiol Rev 32:, 842–857. [CrossRef] [PubMed]
    [Google Scholar]
  12. Hiniker A. , Bardwell J. C. . ( 2004; ). In vivo substrate specificity of periplasmic disulfide oxidoreductases. . J Biol Chem 279:, 12967–12973. [CrossRef] [PubMed]
    [Google Scholar]
  13. Hiniker A. , Collet J. F. , Bardwell J. C. . ( 2005; ). Copper stress causes an in vivo requirement for the Escherichia coli disulfide isomerase DsbC. . J Biol Chem 280:, 33785–33791. [CrossRef] [PubMed]
    [Google Scholar]
  14. Hsiao Y. M. , Liu Y. F. , Huang Y. L. , Lee P. Y. . ( 2011; ). Transcriptional analysis of pmeA gene encoding a pectin methylesterase in Xanthomonas campestris pv. campestris . . Res Microbiol 162:, 270–278. [CrossRef] [PubMed]
    [Google Scholar]
  15. Jiang B. L. , Liu J. , Chen L. F. , Ge Y. Y. , Hang X. H. , He Y. Q. , Tang D. J. , Lu G. T. , Tang J. L. . ( 2008; ). DsbB is required for the pathogenesis process of Xanthomonas campestris pv. campestris . . Mol Plant Microbe Interact 21:, 1036–1045. [CrossRef] [PubMed]
    [Google Scholar]
  16. Jiang J. , Zou H. S. , Li Y. R. , Chen G. Y. . ( 2009; ). [Expression of the hrcC, hrpE and hpa3 genes is not regulated by the hrpG and hrpX genes in a rice pathogen Xanthomonas oryzae pv. oryzicola.]. Wei Sheng Wu Xue Bao 49:, 1018–1025 (in Chinese).[PubMed]
    [Google Scholar]
  17. Kim S. Y. , Kim J. G. , Lee B. M. , Cho J. Y. . ( 2009; ). Mutational analysis of the gum gene cluster required for xanthan biosynthesis in Xanthomonas oryzae pv. oryzae . . Biotechnol Lett 31:, 265–270. [CrossRef] [PubMed]
    [Google Scholar]
  18. Kingsbury J. M. , McCusker J. H. . ( 2010; ). Fungal homoserine kinase (thr1Δ) mutants are attenuated in virulence and die rapidly upon threonine starvation and serum incubation. . Eukaryot Cell 9:, 729–737. [CrossRef] [PubMed]
    [Google Scholar]
  19. Kriechbaumer V. , Weigang L. , Fießelmann A. , Letzel T. , Frey M. , Gierl A. , Glawischnig E. . ( 2008; ). Characterisation of the tryptophan synthase alpha subunit in maize. . BMC Plant Biol 8:, 44. [CrossRef] [PubMed]
    [Google Scholar]
  20. Kullik I. , Toledano M. B. , Tartaglia L. A. , Storz G. . ( 1995; ). Mutational analysis of the redox-sensitive transcriptional regulator OxyR: regions important for oxidation and transcriptional activation. . J Bacteriol 177:, 1275–1284.[PubMed]
    [Google Scholar]
  21. Łasica A. M. , Jagusztyn-Krynicka E. K. . ( 2007; ). The role of Dsb proteins of Gram-negative bacteria in the process of pathogenesis. . FEMS Microbiol Rev 31:, 626–636.[CrossRef]
    [Google Scholar]
  22. Leach J. E. , White F. F. , Rhoads M. J. , Leung H. . ( 1990; ). A repetitive DNA sequence differentiates Xanthomonas campestris pv. oryzae from other pathovar of X. campestris . . Mol Plant Microbe Interact 3:, 238–246. [CrossRef]
    [Google Scholar]
  23. Lee C. , Lee S. M. , Mukhopadhyay P. , Kim S. J. , Lee S. C. , Ahn W. S. , Yu M. H. , Storz G. , Ryu S. E. . ( 2004; ). Redox regulation of OxyR requires specific disulfide bond formation involving a rapid kinetic reaction path. . Nat Struct Mol Biol 11:, 1179–1185. [CrossRef] [PubMed]
    [Google Scholar]
  24. Lee B. M. , Park Y. J. , Park D. S. , Kang H. W. , Kim J. G. , Song E. S. , Park I. C. , Yoon U. H. , Hahn J. H. . & other authors ( 2005; ). The genome sequence of Xanthomonas oryzae pathovar oryzae KACC10331, the bacterial blight pathogen of rice. . Nucleic Acids Res 33:, 577–586. [CrossRef] [PubMed]
    [Google Scholar]
  25. Lee Y. , Kim Y. , Yeom S. , Kim S. , Park S. , Jeon C. O. , Park W. . ( 2008; ). The role of disulfide bond isomerase A (DsbA) of Escherichia coli O157:H7 in biofilm formation and virulence. . FEMS Microbiol Lett 278:, 213–222. [CrossRef] [PubMed]
    [Google Scholar]
  26. Lerouge I. , Vanderleyden J. . ( 2002; ). O-antigen structural variation: mechanisms and possible roles in animal/plant-microbe interactions. . FEMS Microbiol Rev 26:, 17–47. [CrossRef] [PubMed]
    [Google Scholar]
  27. Li Y. R. , Che Y. Z. , Zou H. S. , Cui Y. P. , Guo W. , Zou L. F. , Biddle E. M. , Yang C. H. , Chen G. Y. . ( 2011a; ). Hpa2 required by HrpF to translocate Xanthomonas oryzae transcriptional activator-like effectors into rice for pathogenicity. . Appl Environ Microbiol 77:, 3809–3818. [CrossRef] [PubMed]
    [Google Scholar]
  28. Li Y. R. , Zou H. S. , Che Y. Z. , Cui Y. P. , Guo W. , Zou L. F. , Chatterjee S. , Biddle E. M. , Yang C. H. , Chen G. Y. . ( 2011b; ). A novel regulatory role of HrpD6 in regulating hrp-hrc-hpa genes in Xanthomonas oryzae pv. oryzicola . . Mol Plant Microbe Interact 24:, 1086–1101. [CrossRef] [PubMed]
    [Google Scholar]
  29. Maddocks S. E. , Oyston P. C. . ( 2008; ). Structure and function of the LysR-type transcriptional regulator (LTTR) family proteins. . Microbiology 154:, 3609–3623. [CrossRef] [PubMed]
    [Google Scholar]
  30. Magne Ø. , Driscoll B. T. , Finan T. M. . ( 1997; ). Increased pyruvate orthophosphate dikinase activity results in an alternative gluconeogenic pathway in Rhizobium (Sinorhizobium) meliloti . . Microbiology 143:, 1639–1648. [CrossRef] [PubMed]
    [Google Scholar]
  31. Mailloux R. J. , Bériault R. , Lemire J. , Singh R. , Chénier D. R. , Hamel R. D. , Appanna V. D. . ( 2007; ). The tricarboxylic acid cycle, an ancient metabolic network with a novel twist. . PLoS ONE 2:, e690. [CrossRef] [PubMed]
    [Google Scholar]
  32. Makino S. , Sugio A. , White F. , Bogdanove A. J. . ( 2006; ). Inhibition of resistance gene-mediated defense in rice by Xanthomonas oryzae pv. oryzicola . . Mol Plant Microbe Interact 19:, 240–249. [CrossRef] [PubMed]
    [Google Scholar]
  33. Mann S. , Ploux O. . ( 2011; ). Pyridoxal-5′-phosphate-dependent enzymes involved in biotin biosynthesis: Structure, reaction mechanism and inhibition. . Biochim Biophys Acta 1814:, 1459–1466.[PubMed] [CrossRef]
    [Google Scholar]
  34. Messens J. , Collet J. F. . ( 2006; ). Pathways of disulfide bond formation in Escherichia coli . . Int J Biochem Cell Biol 38:, 1050–1062. [CrossRef] [PubMed]
    [Google Scholar]
  35. Miller J. H. . ( 1972; ). Experiments in Molecular Genetics. Cold Spring Harbor:: Cold Spring Harbor Laboratory;.
    [Google Scholar]
  36. Minsavage G. V. , Mudgett M. B. , Stall R. E. , Jones J. B. . ( 2004; ). Importance of opgHXcv of Xanthomonas campestris pv. vesicatoria in host-parasite interactions. . Mol Plant Microbe Interact 17:, 152–161. [CrossRef] [PubMed]
    [Google Scholar]
  37. Mukhopadhyay P. , Williams J. , Mills D. . ( 1988; ). Molecular analysis of a pathogenicity locus in Pseudomonas syringae pv. syringae . . J Bacteriol 170:, 5479–5488.[PubMed]
    [Google Scholar]
  38. Newman M. A. , von Roepenack-Lahaye E. , Parr A. , Daniels M. J. , Dow J. M. . ( 2002; ). Prior exposure to lipopolysaccharide potentiates expression of plant defenses in response to bacteria. . Plant J 29:, 487–495. [CrossRef] [PubMed]
    [Google Scholar]
  39. Niño-Liu D. O. , Ronald P. C. , Bogdanove A. J. . ( 2006; ). Xanthomonas oryzae pathovars: model pathogens of a model crop. . Mol Plant Pathol 7:, 303–324. [CrossRef] [PubMed]
    [Google Scholar]
  40. Ochiai H. , Inoue Y. , Takeya M. , Sasaki A. , Kaku H. . ( 2005; ). Genome sequence of Xanthomonas oryzae pv. oryzae suggests contribution of large numbers of effector genes and insertion sequences to its race diversity. . Jpn Agric Res Q 39:, 275–287.[CrossRef]
    [Google Scholar]
  41. Oh M. K. , Rohlin L. , Kao K. C. , Liao J. C. . ( 2002; ). Global expression profiling of acetate-grown Escherichia coli . . J Biol Chem 277:, 13175–13183. [CrossRef] [PubMed]
    [Google Scholar]
  42. Ou S. H. . ( 1985; ). Rice Diseases. Slough, UK:: Commonwealth Agricultural Bureau;.
    [Google Scholar]
  43. Park D. H. , Mirabella R. , Bronstein P. A. , Preston G. M. , Haring M. A. , Lim C. K. , Collmer A. , Schuurink R. C. . ( 2010; ). Mutations in γ-aminobutyric acid (GABA) transaminase genes in plants or Pseudomonas syringae reduce bacterial virulence. . Plant J 64:, 318–330. [CrossRef] [PubMed]
    [Google Scholar]
  44. Penaloza-Vazquez A. , Sreedharan A. , Bender C. L. . ( 2010; ). Transcriptional studies of the hrpM/opgH gene in Pseudomonas syringae during biofilm formation and in response to different environmental challenges. . Environ Microbiol 12:, 1452–1467.[PubMed]
    [Google Scholar]
  45. Qian W. , Jia Y. T. , Ren S. X. , He Y. Q. , Feng J. X. , Lu L. F. , Sun Q. H. , Ying G. , Tang D. J. . & other authors ( 2005; ). Comparative and functional genomic analyses of the pathogenicity of phytopathogen Xanthomonas campestris pv. campestris . . Genome Res 15:, 757–767. [CrossRef] [PubMed]
    [Google Scholar]
  46. Qu H. , Xin Y. , Dong X. , Ma Y. F. . ( 2007; ). An rmlA gene encoding d-glucose-1-phosphate thymidylyltransferase is essential for mycobacterial growth. . FEMS Microbiol Lett 275:, 237–243. [CrossRef] [PubMed]
    [Google Scholar]
  47. Salzberg S. L. , Sommer D. D. , Schatz M. C. , Phillippy A. M. , Rabinowicz P. D. , Tsuge S. , Furutani A. , Ochiai H. , Delcher A. L. . & other authors ( 2008; ). Genome sequence and rapid evolution of the rice pathogen Xanthomonas oryzae pv. oryzae PXO99A. . BMC Genomics 9:, 204–221. [CrossRef] [PubMed]
    [Google Scholar]
  48. Sambrook J. , Fritsch E. F. , Maniatis T. . ( 1989; ). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY:: Cold Spring Harbor Laboratory;.
    [Google Scholar]
  49. Shimaoka M. , Takenaka Y. , Kurahashi O. , Kawasaki H. , Matsui H. . ( 2007; ). Effect of amplification of desensitized purF and prs on inosine accumulation in Escherichia coli . . J Biosci Bioeng 103:, 255–261. [CrossRef] [PubMed]
    [Google Scholar]
  50. Soto-Suárez M. , González C. , Piégu B. , Tohme J. , Verdier V. . ( 2010; ). Genomic comparison between Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola, using suppression-subtractive hybridization. . FEMS Microbiol Lett 308:, 16–23. [CrossRef] [PubMed]
    [Google Scholar]
  51. Stenson T. H. , Weiss A. A. . ( 2002; ). DsbA and DsbC are required for secretion of pertussis toxin by Bordetella pertussis . . Infect Immun 70:, 2297–2303. [CrossRef] [PubMed]
    [Google Scholar]
  52. Tang J. L. , Feng J. X. , Li Q. Q. , Wen H. X. , Zhou D. L. , Wilson T. J. , Dow J. M. , Ma Q. S. , Daniels M. J. . ( 1996; ). Cloning and characterization of the rpfC gene of Xanthomonas oryzae pv. oryzae: involvement in exopolysaccharide production and virulence to rice. . Mol Plant Microbe Interact 9:, 664–666. [CrossRef] [PubMed]
    [Google Scholar]
  53. Tang D. J. , He Y. Q. , Feng J. X. , He B. R. , Jiang B. L. , Lu G. T. , Chen B. , Tang J. L. . ( 2005; ). Xanthomonas campestris pv. campestris possesses a single gluconeogenic pathway that is required for virulence. . J Bacteriol 187:, 6231–6237. [CrossRef] [PubMed]
    [Google Scholar]
  54. Tinsley C. R. , Voulhoux R. , Beretti J. L. , Tommassen J. , Nassif X. . ( 2004; ). Three homologues, including two membrane-bound proteins, of the disulfide oxidoreductase DsbA in Neisseria meningitidis: effects on bacterial growth and biogenesis of functional type IV pili. . J Biol Chem 279:, 27078–27087. [CrossRef] [PubMed]
    [Google Scholar]
  55. Toledo M. A. , Schneider D. R. , Azzoni A. R. , Favaro M. T. , Pelloso A. C. , Santos C. A. , Saraiva A. M. , Souza A. P. . ( 2011; ). Characterization of an oxidative stress response regulator, homologous to Escherichia coli OxyR, from the phytopathogen Xylella fastidiosa . . Protein Expr Purif 75:, 204–210. [CrossRef] [PubMed]
    [Google Scholar]
  56. Turner P. E. . ( 2004; ). Phenotypic plasticity in bacterial plasmids. . Genetics 167:, 9–20. [CrossRef] [PubMed]
    [Google Scholar]
  57. Urban A. , Leipelt M. , Eggert T. , Jaeger K. E. . ( 2001; ). DsbA and DsbC affect extracellular enzyme formation in Pseudomonas aeruginosa . . J Bacteriol 183:, 587–596. [CrossRef] [PubMed]
    [Google Scholar]
  58. Vincent-Sealy L. V. , Thomas J. D. , Commander P. , Salmond G. P. . ( 1999; ). Erwinia carotovora DsbA mutants: evidence for a periplasmic-stress signal transduction system affecting transcription of genes encoding secreted proteins. . Microbiology 145:, 1945–1958. [CrossRef] [PubMed]
    [Google Scholar]
  59. Wang L. , Makino S. , Subedee A. , Bogdanove A. J. . ( 2007; ). Novel candidate virulence factors in rice pathogen Xanthomonas oryzae pv. oryzicola as revealed by mutational analysis. . Appl Environ Microbiol 73:, 8023–8027. [CrossRef] [PubMed]
    [Google Scholar]
  60. Wang Y. P. , Zou L. F. , Zhou D. , Chen G. Y. . ( 2009; ). Key roles of hrpE gene of Xanthomonas oryzae pv. oryzicola in formation of Hrp pilus and pathogenicity in rice. . Acta Phytopathologica Sin 39:, 392–398.
    [Google Scholar]
  61. Wren B. W. . ( 2000; ). Microbial genome analysis: insights into virulence, host adaptation and evolution. . Nat Rev Genet 1:, 30–39. [CrossRef] [PubMed]
    [Google Scholar]
  62. Yang B. , White F. F. . ( 2004; ). Diverse members of the AvrBs3/PthA family of type III effectors are major virulence determinants in bacterial blight disease of rice. . Mol Plant Microbe Interact 17:, 1192–1200. [CrossRef] [PubMed]
    [Google Scholar]
  63. Ye J. , McGinnis S. , Madden T. L. . ( 2006; ). blast: improvements for better sequence analysis. . Nucleic Acids Res 34: (Suppl. 2), W6–W9. [CrossRef] [PubMed]
    [Google Scholar]
  64. Yuan L. , Li Y. R. , Zhang X. F. , Guo W. , Che Y. Z. , Chen G. Y. . ( 2009; ). Quick Identification of pathogenicity-related genes in Xanthomonas oryzae pv. oryzicola by thermal asymmetric interlaced PCR (TAIL-PCR) and Tn5 transposon rescue. . J Agric Biotechnol 17:, 1089–1095.
    [Google Scholar]
  65. Zhao B. , Ardales E. Y. , Raymundo A. , Bai J. , Trick H. N. , Leach J. E. , Hulbert S. H. . ( 2004; ). The avrRxo1 gene from the rice pathogen Xanthomonas oryzae pv. oryzicola confers a nonhost defense reaction on maize with resistance gene Rxo1 . . Mol Plant Microbe Interact 17:, 771–779. [CrossRef] [PubMed]
    [Google Scholar]
  66. Zhao Y. C. , Qian G. L. , Yin F. Q. , Fan J. Q. , Zhai Z. W. , Liu C. H. , Hu B. S. , Liu F. Q. . ( 2011; ). Proteomic analysis of the regulatory function of DSF-dependent quorum sensing in Xanthomonas oryzae pv. oryzicola . . Microb Pathog 50:, 48–55. [CrossRef] [PubMed]
    [Google Scholar]
  67. Zheng M. , Aslund F. , Storz G. . ( 1998; ). Activation of the OxyR transcription factor by reversible disulfide bond formation. . Science 279:, 1718–1722. [CrossRef] [PubMed]
    [Google Scholar]
  68. Zou L. F. , Chen G. Y. , Wu X. M. , Wang J. S. . ( 2005; ). Cloning and sequence analysis of diverse members of avrBs3/PthA family of Xanthomonas oryzae pv. oryzicola . . Scientia Agricultura Sinica 38:, 929–935.
    [Google Scholar]
  69. Zou L. F. , Wang X. P. , Xiang Y. , Zhang B. , Li Y. R. , Xiao Y. L. , Wang J. S. , Walmsley A. R. , Chen G. Y. . ( 2006; ). Elucidation of the hrp clusters of Xanthomonas oryzae pv. oryzicola that control the hypersensitive response in nonhost tobacco and pathogenicity in susceptible host rice. . Appl Environ Microbiol 72:, 6212–6224. [CrossRef] [PubMed]
    [Google Scholar]
  70. Zou H. S. , Yuan L. , Guo W. , Li Y. R. , Che Y. Z. , Zou L. F. , Chen G. Y. . ( 2011; ). Construction of a Tn5-tagged mutant library of Xanthomonas oryzae pv. oryzicola as an invaluable resource for functional genomics. . Curr Microbiol 62:, 908–916. [CrossRef] [PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.050419-0
Loading
/content/journal/micro/10.1099/mic.0.050419-0
Loading

Data & Media loading...

Supplements

Supplementary material 

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error