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

The Mismatch Repair System (MMR) is a highly conserved pathway for the maintenance of genomic stability in many organisms. In plants this is particularly important due to their fixed lifestyle and the lack of a reserved germ line. Suppression of MMR leads to a random occurrence of mutations in the genome and an accumulation over successive generations, and thus maximizes genetic diversity. MMR deficiency has already 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 that is applicable in Solanaceae in general. This amiRNA was inserted in a transformation vector and placed under control of the CaMV 35S promoter and the meiosis active AtDMC1 promoter. Introduction of this amiRNA construct in Nicotiana tabacum and Nicotiana plumbaginifolia reduced the MSH2 transcript levels to 20-30 %.
Aberrant phenotypes appeared in the transformed Nicotiana lines at higher frequencies than in the negative control lines. However the mutations were not transmitted to subsequent generations, since a higher mutation frequency was not observed.
The designed amiRNA was capable of suppressing the MSH2 activity, which caused the occurrence of somatical mutations. Apparently the silencing of MSH2 was not strong enough in the germ line to cause inheritable mutations. MMR shows a remarkable robustness in these cells, where mutations can be transmitted to succeeding generations.