Effective RNAi-Mediated Silencing of the Mismatch Repair MSH2 Gene Induces Sterility of Tomato Plants but Not an Increase in Meiotic Recombination

Abstract

In plant breeding, the ability to manipulate meiotic recombination aids in the efficient construction of new allelic compositions of chromosomes and facilitates gene transfer from wild relatives of crop plants. The DNA mismatch repair system antagonizes meiotic recombination. In this research, a trial was conducted to evaluate transgenic tomato plants carrying an RNA interference (RNAi) construct designed to inhibit the expression of the mismatch repair MSH2 gene. To drive the RNAi construct, we used either a pro-SmAMP2 promoter from Stellaria media ANTIMICROBIAL PEPTIDE2 or a Cauliflower mosaic virus 35S promoter (CaMV35S). The results of real-time PCR showed that, with a 16 h light/8 h dark photoperiod, MSH2-RNAi tomato transgenic plants exhibited MSH2 gene transcript contents ranging from 0% to 3% in the leaves, relative to untransformed controls. However, with this lighting mode, the MSH2-RNAi transgenic plants grew slowly, flowered poorly, and did not form seed sets. During cultivation with a 12 h light/12 h dark photoperiod, MSH2-RNAi transgenic plants exhibited MSH2 gene transcript contents ranging from 3% to 42%, relative to untransformed controls. Under these conditions, F1 hybrid seed sets formed for most of the MSH2-RNAi transgenic plants with the RNAi construct driven by the CaMV35S promoter, and for one transformant with the RNAi construct driven by the pro-SmAMP2 promoter. Under conditions of a 12 h light/12 h dark photoperiod, most of the F1 transgenic hybrids showed MSH2 gene transcript contents ranging from 3% to 34% and formed F2 offspring sets, which made it possible to assess the meiotic recombination frequency. We showed that the effective inhibition of MSH2 in MSH2-RNAi tomato transgenic plants is not associated with an increase in meiotic recombination compared to the control, but it stimulates the sterility of plants. It was established that the expression of the MSH2 gene in tomato plants is about 50 times higher with a 12 h light/12 h dark than with a 16 h light/8 h dark photoperiod. It is discussed that, in Solanum lycopersicum tomato plants, which are not sensitive to the day length for flowering, changing the lighting time may be a means of controlling the meiotic recombination frequency within certain limits.

Control of meiotic crossing over in plant breeding

“…In particular, none of the three combinations of crossing a cultivated tomato expressing the recA gene and wild tomato species S. cheesmaniae, S. pimpinellifolium, and S. habrochaites showed a significant increase in the frequency of recombination between the marker genes of chromosome 2. It is assumed that the factor limiting recombination between chromosomes from different species is the mismatch repair system, which eliminates mismatched bases in DNA at the DSB repair site (Chambers et al, 1996;Emmanuel et al, 2006;Strelnikova et al, 2021). This assumption is based on the fact that in interspecific tomato hybrids, due to the increased level of DNA polymorphism between chromosomes of different species, one should expect a more active resistance of mismatch repair to meiotic crossing over than in interline hybrids of cultivated tomato.…”

“…These results allowed to apply the strategy of suppressing mismatch repair by inhibiting the expression of the MSH2 and Control of meiotic crossing over in plant breeding MSH7 genes to increase the frequency of crossover exchanges in other plant species. In particular, in cultivated tomato, the inhibition of the expression of the MSH2 and MSH7 genes was performed by three independent scientific groups at different times either using RNA interference (RNAi) (Tam et al, 2011;Sarma et al, 2018;Strelnikova et al, 2021) or using a dominant-negative construct with the mutant MSH2-DN2 protein gene from arabidopsis (Tam et al, 2011).…”

“…The use of a dominant-negative construct or inhibition of the MSH7 transcript by RNAi allowed a non-substantial increase by 17.8 % in the frequency of meiotic recombination between homeologues in a cultivated tomato heterozygous by chromosome 8 from S. lycopersicoides Dunal (Tam et al, 2011). At the same time, silencing of the MSH2 gene transcript with RNAi delivered pronounced negative consequences for the fertility of tomato plants (Sarma et al, 2018;Strelnikova et al, 2021), especially when using the strong pro-SmAMP2 plant promoter to control the expression of the RNAi construct (Strelnikova et al, 2021). In recent experiments, it was convincingly shown that the highly effective RNAi of the MSH2 gene leads to phenotypic anomalies in cultivated tomato plants: growth and flowering retardation and formation of a reduced number of seeds (Sarma et al, 2018;Strelnikova et al, 2021).…”

“…At the same time, silencing of the MSH2 gene transcript with RNAi delivered pronounced negative consequences for the fertility of tomato plants (Sarma et al, 2018;Strelnikova et al, 2021), especially when using the strong pro-SmAMP2 plant promoter to control the expression of the RNAi construct (Strelnikova et al, 2021). In recent experiments, it was convincingly shown that the highly effective RNAi of the MSH2 gene leads to phenotypic anomalies in cultivated tomato plants: growth and flowering retardation and formation of a reduced number of seeds (Sarma et al, 2018;Strelnikova et al, 2021). In cases where the RNAi of the MSH2 gene was moderate, tomato plants were fertile, but no increase in the frequency of meiotic recombination was found (Tam et al, 2011;Strelnikova et al, 2021).…”

“…In recent experiments, it was convincingly shown that the highly effective RNAi of the MSH2 gene leads to phenotypic anomalies in cultivated tomato plants: growth and flowering retardation and formation of a reduced number of seeds (Sarma et al, 2018;Strelnikova et al, 2021). In cases where the RNAi of the MSH2 gene was moderate, tomato plants were fertile, but no increase in the frequency of meiotic recombination was found (Tam et al, 2011;Strelnikova et al, 2021).…”