Plant Biotechnology Reports

, Volume 7, Issue 4, pp 467–480

Genomic stability in Nicotiana plants upon silencing of the mismatch repair gene MSH2

Original Article

Abstract

The Mismatch Repair (MMR) system is a highly conserved pathway for the maintenance of genomic stability in many organisms. In plants, this is particularly important because of the lack of a reserved germline. Suppression of MMR leads to an accumulation of random mutations in the genome over successive generations, and thus maximizes genetic diversity. MMR deficiency has been shown to be a useful technique in plant breeding, complementary to chemical or physical mutagenesis. We have developed an artificial microRNA (amiRNA) targeting the MSH2 gene, which is generally applicable in Solanaceae. Two amiRNA precursors were inserted in a transformation vector, under the control of the CaMV 35S promoter and the meiosis active AtDMC1 promoter, respectively. Introduction of this amiRNA construct in Nicotiana tabacum and N. plumbaginifolia reduced the MSH2 transcript levels to 20–30 %. Morphological and developmental abnormalities and plants with white sectors on the first pair of leaves or on the cotyledons (referred to as ‘chimeric albinos’) appeared in the transformed Nicotiana lines at higher frequencies than in the control lines. Also, some plants which show an increased tolerance for the herbicide chlorsulfuron were found. However, the mutant phenotypes were not transmitted to subsequent generations. We conclude that the designed amiRNA was capable of suppressing the MSH2 activity, which caused the occurrence of somatic mutations. Apparently, the silencing of MSH2 was not strong enough in the germline to cause inheritable mutations.

Keywords

Mismatch repair MSH2 Mutagenesis Nicotiana Solanum tuberosum 

Supplementary material

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References

  1. Adé J, Haffani Y, Belzile FJ (2001) Functional analysis of the Arabidopsis thaliana mismatch repair gene MSH2. Genome 44:651–657PubMedGoogle Scholar
  2. Alvarez JP, Pekker I, Goldshmidt A, Blum E, Amsellem Z, Eshed Y (2006) Endogenous and synthetic microRNAs stimulate simultaneous, efficient, and localized regulation of multiple targets in diverse species. Plant Cell 18:1134–1151PubMedCrossRefGoogle Scholar
  3. Bollmann SR, Tominey CM, Hoffman PD, Hoffman TMC, Hays JB (2011) Reversion-reporter transgenes to analyze all six base-substitution pathways in Arabidopsis. Plant Physiol 155:1286–1300PubMedCrossRefGoogle Scholar
  4. Cecchetti V, Pomponi M, Altamura MM, Pezzotti M, Marsilio S, D’Angeli S, Tornielli GB, Costantino P, Cardarelli M (2004) Expression of rolB in tobacco flowers affects the coordinated processes of anther dehiscence and style elongation. Plant J 38:512–525PubMedCrossRefGoogle Scholar
  5. Chao Q, Sullivan CD, Getz JM, Gleason KB, Sass PM, Nicolaides NC, Grasso L (2005) Rapid generation of plant traits via regulation of DNA mismatch repair. Plant Biotechnol J 3:399–407PubMedCrossRefGoogle Scholar
  6. Chong-Pérez B, Kosky RG, Reyes M, Rojas L, Ocaña B, Tejeda M, Pérez B, Angenon G (2012) Heat shock induced excision of selectable marker genes in transgenic banana by the Cre-lox site-specific recombination system. J Biotechnol 159:265–273PubMedCrossRefGoogle Scholar
  7. Colbert T, Till BJ, Tompa R, Reynolds S, Steine MN, Yeung AT, McCallum CM, Comai L, Henikoff S (2001) High-throughput screening for induced point mutations. Plant Physiol 126:480–484PubMedCrossRefGoogle Scholar
  8. Crouse GF (2010) An end for mismatch repair. Proc Natl Acad Sci USA 107:20851–20852PubMedCrossRefGoogle Scholar
  9. Cuellar W, Gaudin A, Solorzano D, Casas A, Nopo L, Chudalayandi P, Medrano G, Kreuze J, Ghislain M (2006) Self-excision of the antibiotic resistance gene nptII using a heat inducible Cre-loxP system from transgenic potato. Plant Mol Biol 62:71–82PubMedCrossRefGoogle Scholar
  10. Culligan KM, Hays JB (2000) Arabidopsis MutS homologs—AtMSH2, AtMSH3, AtMSH6, and a novel AtMSH7—form three distinct protein heterodimers with different specificities for mismatched DNA. Plant Cell 12:991–1002PubMedGoogle Scholar
  11. Culligan KM, Meyer-Gauen G, Lyons-Weiler J, Hays JB (2000) Evolutionary origin, diversification and specialization of eukaryotic MutS homolog mismatch repair proteins. Nucleic Acids Res 28:463–471PubMedCrossRefGoogle Scholar
  12. Dong C, Whitford R, Langridge P (2002) A DNA mismatch repair gene links to the Ph2 locus in wheat. Genome 45:116–124PubMedCrossRefGoogle Scholar
  13. Doutriaux MP, Couteau F, Bergounioux C, White C (1998) Isolation and characterisation of the RAD51 and DMC1 homologs from Arabidopsis thaliana. Mol Gen Genet 257:283–291PubMedCrossRefGoogle Scholar
  14. Duggleby RG, McCourt JA, Guddat LW (2008) Structure and mechanism of inhibition of plant acetohydroxyacid synthase. Plant Physiol Biochem 46:309PubMedCrossRefGoogle Scholar
  15. Emmanuel E, Yehuda E, Melamed-Bessudo C, Avivi-Ragolsky N, Levy AA (2006) The role of AtMSH2 in homologous recombination in Arabidopsis thaliana. EMBO Rep 7:100–105PubMedCrossRefGoogle Scholar
  16. Endo M, Osakabe K, Ono K, Handa H, Shimizu T, Toki S (2007) Molecular breeding of a novel herbicide-tolerant rice by gene targeting. Plant J 52:157–166PubMedCrossRefGoogle Scholar
  17. Fukui K (2010) DNA mismatch repair in eukaryotes and bacteria. J Nucleic Acids. doi:10.4061/2010/260512
  18. Gómez R, Spampinato CP (2013) Mismatch recognition function of Arabidopsis thaliana MutSγ. DNA Repair 12:257–264PubMedCrossRefGoogle Scholar
  19. Greene EA, Codomo CA, Taylor NE, Henikoff JG, Till BJ, Reynolds SH, Enns LC, Burtner C, Johnson JE, Odden AR, Comai L, Henikoff S (2003) Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis. Genetics 164:731–740PubMedGoogle Scholar
  20. Harfe BD, Jinks-Robertson S (2000) DNA mismatch repair and genetic instability. Annu Rev Genet 34:359–399PubMedCrossRefGoogle Scholar
  21. Haughn GW, Smith J, Mazur B, Somerville C (1988) Transformation with a mutant Arabidopsis acetolactate synthase gene renders tobacco resistant to sulfonylurea herbicides. Mol Gen Genet 211:266–271CrossRefGoogle Scholar
  22. Higgins JD, Armstrong SJ, Franklin FCH, Jones GH (2004) The Arabidopsis MutS homolog AtMSH4 functions at an early step in recombination: evidence for two classes of recombination in Arabidopsis. Genes Dev 18:2557–2570PubMedCrossRefGoogle Scholar
  23. Higgins JD, Vignard J, Mercier R, Pugh AG, Franklin FCH, Jones GH (2008) AtMSH5 partners AtMSH4 in the class I meiotic crossover pathway in Arabidopsis thaliana, but is not required for synapsis. Plant J 55:28–39PubMedCrossRefGoogle Scholar
  24. Hoffman PD, Leonard JM, Lindberg GE, Bollmann SR, Hays JB (2004) Rapid accumulation of mutations during seed-to-seed propagation of mismatch-repair-defective Arabidopsis. Genes Dev 18:2676–2685PubMedCrossRefGoogle Scholar
  25. Hollingsworth NM, Ponte L, Halsey C (1995) MSH5, a novel MutS homolog, facilitates meiotic reciprocal recombination between homologs in Saccharomyces cerevisiae but not mismatch repair. Genes Dev 9:1728–1739PubMedCrossRefGoogle Scholar
  26. Hombauer H, Srivatsan A, Putnam CD, Kolodner RD (2011) Mismatch repair, but not heteroduplex rejection, is temporally coupled to DNA replication. Science 334:1713–1716PubMedCrossRefGoogle Scholar
  27. Hraška M, Rakouský S, Čurn V (2008) Tracking of the CaMV-35S promoter performance in GFP transgenic tobacco, with a special emphasis on flowers and reproductive organs, confirmed its predominant activity in vascular tissues. Plant Cell Tiss Organ Cult 94:239–251CrossRefGoogle Scholar
  28. Iyer RR, Pluciennik A, Burdett V, Modrich PL (2006) DNA mismatch repair: functions and mechanisms. Chem Rev 106:302–323PubMedCrossRefGoogle Scholar
  29. Kim J, Somers DE (2010) Rapid assessment of gene function in the circadian clock using artificial microRNA in Arabidopsis mesophyll protoplasts. Plant Physiol 154:611–621PubMedCrossRefGoogle Scholar
  30. Klimyuk VI, Jones JDG (1997) AtDMC1, the Arabidopsis homologue of the yeast DMC1 gene: characterization, transposon-induced allelic variation and meiosis-associated expression. Plant J 11:1–14PubMedCrossRefGoogle Scholar
  31. Kovalchuk I, Kovalchuk O, Hohn B (2000) Genome-wide variation of the somatic mutation frequency in transgenic plants. EMBO J 19:4431–4438PubMedCrossRefGoogle Scholar
  32. Lafleuriel J, Degroote F, Depeiges A, Picard G (2007) Impact of the loss of AtMSH2 on double-strand break-induced recombination between highly diverged homeologous sequences in Arabidopsis thaliana germinal tissues. Plant Mol Biol 63:833–846PubMedCrossRefGoogle Scholar
  33. Lazo GR, Stein PA, Ludwig RA (1991) A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Nat Biotechnol 9:963–967CrossRefGoogle Scholar
  34. Leonard JM, Bollmann SR, Hays JB (2003) Reduction of stability of Arabidopsis genomic and transgenic DNA-repeat sequences (microsatellites) by inactivation of AtMSH2 mismatch-repair function. Plant Physiol 133:328–338PubMedCrossRefGoogle Scholar
  35. Li L, Jean M, Belzile F (2006) The impact of sequence divergence and DNA mismatch repair on homeologous recombination in Arabidopsis. Plant J 45:908–916PubMedCrossRefGoogle Scholar
  36. Li J, Farmer AD, Lindquist IE, Dukowic-Schulze S, Mudge J, Li T, Retzel EF, Chen C (2012) Characterization of a set of novel meiotically-active promoters in Arabidopsis. BMC Plant Biol 12:104PubMedCrossRefGoogle Scholar
  37. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408PubMedCrossRefGoogle Scholar
  38. Lloyd AH, Milligan AS, Langridge P, Able JA (2007) TaMSH7: a cereal mismatch repair gene that affects fertility in transgenic barley (Hordeum vulgare L.). BMC Plant Biol 7:67PubMedCrossRefGoogle Scholar
  39. McElhinny SAN, Kissling GE, Kunkel TA (2010) Differential correction of lagging-strand replication errors made by DNA polymerases a and d. Proc Natl Acad Sci USA 107:21070–21075CrossRefGoogle Scholar
  40. Nicolaides NC, Littman SJ, Modrich P, Kinzler KW, Vogelstein B (1998) A naturally occurring hPMS2 mutation can confer a dominant negative mutator phenotype. Mol Cell Biol 18:1635–1641PubMedGoogle Scholar
  41. Ossowski S, Schwab R, Weigel D (2008) Gene silencing in plants using artificial microRNAs and other small RNAs. Plant J 53:674–690PubMedCrossRefGoogle Scholar
  42. Ossowski S, Schneeberger K, Lucas-Lledó JI, Warthmann N, Clark RM, Shaw RG, Weigel D, Lynch M (2010) The rate and molecular spectrum of spontaneous mutations in Arabidopsis thaliana. Science 327:92–94PubMedCrossRefGoogle Scholar
  43. Parsons R, Li GM, Longley M, Modrich P, Liu B, Berk T, Hamilton SR, Kinzler KW, Vogelstein B (1995) Mismatch repair deficiency in phenotypically normal human cells. Science 268:738–740PubMedCrossRefGoogle Scholar
  44. Paterson AH, Bowers JE, Burow MD, Draye X, Elsik CG, Jiang CX, Katsar CS, Lan TH, Lin YR, Ming R, Wright RJ (2000) Comparative genomics of plant chromosomes. Plant Cell 12:1523–1539PubMedGoogle Scholar
  45. Pluciennik A, Dzantiev L, Iyer RR, Constantin N, Kadyrov FA, Modrich P (2010) PCNA function in the activation and strand direction of MutLα endonuclease in mismatch repair. Proc Natl Acad Sci USA 107:16066–16071PubMedCrossRefGoogle Scholar
  46. Ross-Macdonald P, Roeder GS (1994) Mutation of a meiosis-specific MutS homolog decreases crossing over but not mismatch correction. Cell 79:1069–1080PubMedCrossRefGoogle Scholar
  47. Sachadyn P (2010) Conservation and diversity of MutS proteins. Mutat Res 694:20–30PubMedCrossRefGoogle Scholar
  48. Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D (2006) Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell 18:1121–1133PubMedCrossRefGoogle Scholar
  49. Shi R, Yang C, Lu S, Sederoff R, Chiang VL (2010) Specific down-regulation of PAL genes by artificial microRNAs in Populus trichocarpa. Planta 232:1281–1288PubMedCrossRefGoogle Scholar
  50. Stevens R, Grelon M, Vezon D, Oh J, Meyer P, Perennes C, Domenichini S, Bergounioux C (2004) A CDC45 homolog in Arabidopsis is essential for meiosis, as shown by RNA interference-induced gene silencing. Plant Cell 16:99–113PubMedCrossRefGoogle Scholar
  51. Sunilkumar G, Mohr LA, Lopata-Finch E, Emani C, Rathore KS (2002) Developmental and tissue-specific expression of CaMV 35S promoter in cotton as revealed by GFP. Plant Mol Biol 50:463–474PubMedCrossRefGoogle Scholar
  52. Tam SM, Samipak S, Britt A, Chetelat RT (2009) Characterization and comparative sequence analysis of the DNA mismatch repair MSH2 and MSH7 genes from tomato. Genetica 137:341–354PubMedCrossRefGoogle Scholar
  53. Tam SM, Hays JB, Chetelat RT (2011) Effects of suppressing the DNA mismatch repair system on homeologous recombination in tomato. Theor Appl Genet 123:1445–1458PubMedCrossRefGoogle Scholar
  54. Tian L, Gu L, Li GM (2009) Distinct nucleotide binding/hydrolysis properties and molar ratio of MutSα and MutSβ determine their differential mismatch binding activities. J Biol Chem 284:11557–11562PubMedCrossRefGoogle Scholar
  55. Till BJ, Cooper J, Tai TH, Colowit P, Greene EA, Henikoff S, Comai L (2007) Discovery of chemically induced mutations in rice by TILLING. BMC Plant Biol 7:19PubMedCrossRefGoogle Scholar
  56. Toppino L, Kooiker M, Lindner M, Dreni L, Rotino GL, Kater MM (2011) Reversible male sterility in eggplant (Solanum melongena L.) by artificial microRNA-mediated silencing of general transcription factor genes. Plant Biotechnol J 9:684–692PubMedCrossRefGoogle Scholar
  57. Tran HT, Keen JD, Kricker M, Resnick MA, Gordenin DA (1997) Hypermutability of homonucleotide runs in mismatch repair and DNA polymerase proofreading yeast mutants. Mol Cell Biol 17:2859–2865PubMedGoogle Scholar
  58. Umar A, Kunkel TA (1996) DNA-replication fidelity, mismatch repair and genome instability in cancer cells. Eur J Biochem 238:297–307PubMedCrossRefGoogle Scholar
  59. Van der Auwera G, Baute J, Bauwens M, Peck I, Piette D, Pycke M, Asselman P, Depicker A (2008) Development and application of novel constructs to score C:G- to -T:A transitions and homologous recombination in Arabidopsis. Plant Physiol 146:22–31PubMedCrossRefGoogle Scholar
  60. Verweire D (2008) Characterization of putative germline-specific promoters from Arabidopsis thaliana and their application in DNA modification strategies. PhD thesis. Department of Applied Biological Sciences. Vrije Universiteit Brussel, BrusselsGoogle Scholar
  61. Verweire D, Verleyen K, De Buck S, Claeys M, Angenon G (2007) Marker-free transgenic plants through genetically programmed auto-excision. Plant Physiol 145:1220–1231PubMedCrossRefGoogle Scholar
  62. Wu SY, Culligan K, Lamers M, Hays J (2003) Dissimilar mispair-recognition spectra of Arabidopsis DNA-mismatch-repair proteins MSH2·MSH6 (MutSα) and MSH2·MSH7 (MutSγ). Nucleic Acids Res 31:6027–6034PubMedCrossRefGoogle Scholar
  63. Xu J, Li M, Chen L, Wu G, Li H (2012) Rapid generation of rice mutants via the dominant negative suppression of the mismatch repair protein OsPMS1. Theor Appl Genet 125:975–986PubMedCrossRefGoogle Scholar

Copyright information

© Korean Society for Plant Biotechnology and Springer Japan 2013

Authors and Affiliations

  1. 1.Laboratory of Plant Genetics, Institute for Molecular Biology and BiotechnologyVrije Universiteit Brussel (VUB)BrusselsBelgium

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