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

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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.

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Acknowledgments

We thank Martine Claeys for assistance with plant growth and maintenance. This research is funded by the Agency for Innovation by Science and Technology in Flanders (IWT-Vlaanderen) through a PhD scholarship to I.V.M. and by Research Foundation-Flanders (FWO; research project G.0067.09).

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  1. Laboratory of Plant Genetics, Institute for Molecular Biology and Biotechnology, Vrije Universiteit Brussel (VUB), Pleinlaan 2, 1050, Brussels, Belgium

    Inke Van Marcke & Geert Angenon

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  1. Inke Van Marcke
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  2. Geert Angenon
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Correspondence to Geert Angenon.

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Fig. S1: a. Alignment of the cDNA sequences of MSH2 from Oryza sativa, Arabidopsis thaliana, Petunia hybrida, Nicotiana tabacum, Nicotiana plumbaginifolia, and Solanum lycopersicum and the genomic DNA sequence from Solanum tuberosum. Position in the sequences is indicated between parentheses. The numbering for the N. plumbaginifolia and S. tuberosum sequences is according to a sequenced fragment and not to the full sequence. The position of the target site of the amiRNA is indicated with a grey bar. Yellow and blue backgrounds indicate identical nucleotides; b. Alignment of the protein sequences of MSH2 from O. sativa, A. thaliana, P. hybrida, N. tabacum, S. lycopersicum, and S. tuberosum. Position in the sequences is indicated between parentheses. The numbering for the S. tuberosum sequence is according to a sequenced fragment and not to the full sequence.

Fig. S2: a. Quantitative real-time RT-PCR analysis on homozygous T3-generation Ntami lines; b. Quantitative real-time analysis RT-PCR on some T4-generation Ntami lines, which showed an increased frequency of mutant phenotypes (see Sect. 3.3); c. Quantitative real-time RT-PCR analysis on some T5-generation Ntami lines, which showed an increased frequency of mutant phenotypes (see Sect. 3.3) or a slightly increased tolerance for the herbicide chlorsulfuron (Ntami-CS line, see Sect. 3.4). Total RNA was extracted from 2-weeks-old plants and the relative expression levels of the MSH2 gene were determined in the Ntami lines compared to the corresponding negative controls NtL2K-1.19 (T3-generation), NtL2K-1.19.M5 (T4-generation) and NtL2K-1.19.x.A3 (T5-generation). * significantly different from the control (p = 0.05).

Fig. S3: Examples of aberrant phenotypes observed during successive generations of Ntami lines: a. Morphological and developmental mutant phenotypes; b. Chimeric albinos (white sectors on the first pair of leaves or on the cotyledons); c. Callus-like tissues.

Fig. S4: Observed mutation frequencies (%) during in vitro screenings for aberrant phenotypes in the T5-progeny of chimeric albinos in the T4-generation (observed during in vivo screening). About 3000 plants per line were screened. * significantly different from NtL2K-1.19.A1 (p = 0.05 when only the chimeric albinos are taken into account; p = 0.09 when the total number of mutant phenotypes was taken into account).

Fig. S5: a. Example of Ntami-CS plants that survived the treatment with chlorsulfuron; b. Example of plants in the progeny of Ntami-CS plants that survived the treatment with chlorsulfuron; c. Nursery tray with progeny plants from Ntami-CS plants after chlorsulfuron treatment. Only a few plants survived the treatment; d. Nursery tray with wild type plants after chlorsulfuron treatment. None of the plants survived the treatment.

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Van Marcke, I., Angenon, G. Genomic stability in Nicotiana plants upon silencing of the mismatch repair gene MSH2 . Plant Biotechnol Rep 7, 467–480 (2013). https://doi.org/10.1007/s11816-013-0285-0

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