MSH1-Derived Epigenetic Breeding Potential In Tomato

MutS HOMOLOG1(MSH1) encodes a dual targeted protein that is localized to the mitochondrion, where it suppresses illegitimate DNA recombination, and to the plastid. Its role in the plastid is less well defined, but mutation of the MSH1gene in Arabidopsis is known to condition plastid-associated changes in plant growth behavior. The mshl mutant is markedly altered in growth rate, branching behavior, flowering time, juvenility-maturity growth transition, perennial growth behavior, and abiotic stress response. This developmental reprogramming (DR) is accompanied by altered genome methylation, with intensity of altered phenotype showing apparent association with enhanced non-CG hypermethylation of pericentromeric genomic intervals. This complex DR phenotype has been recapitulated in several crop species, both monocot and dicot, by RNAi suppression of MSH1, prompting changes in growth rate, tillering, flowering time, leaf morphology, and abiotic stress tolerance. The DR state conditioned by mutation or silencing of MSH1is unusual. Crossing of the modified plant, either Arabidopsis mshl mutant or sorghum MSHl-RNAi suppression line, with its isogenic wild type counterpart produces heritable enhanced growth vigor in subsequent progeny generations.To investigate the potential for exploiting non-genetic variation in a directed crop breeding effort, we introduced an MSHl-RNAi transgene construction to the tomato cultivar ’Rutgers’. Here we demonstrated that:1) MSH1-RNAi suppression in tomato leads to developmental reprogramming. Suppression of MSH1expression resulted in a wide range of altered phenotypes, including changes in leaf morphology, variegation, dwarfing, male sterility, flower development and flower timing. We observed variation in phenotype in both transgene positive and transgene null selections, so the altered phenotypes were subsequently inherited independent of the transgene. In each cycle of self-pollination, progeny produce a range in phenotype intensity.2) Isogenic crosses using MSH1-RNAi transgene nulls result in enhanced growth. Reciprocal crosses of the transgene-null lines to wild type resulted in F1progeny showing normal growth, and F2progeny (designated epiF2) showing a range of enhanced growth vigor. Early growth of the epiF2plants was more rapid than Rutgers wild type.Single plant selections from the broad range of epiF2phenotypes resulted in both epiF3families that yielded higher and those yielding lower than wild type. These results suggest that MSH1-associated non-genetic variation responds to selection.3) Enhanced-growth tomato phenotypes show enhanced field performance. Results in the field included enhanced seedling growth vigor relative to wildtype, evident within the first2-3weeks, and early transition to reproduction. Rutgers wild type plants were slightly enhanced in photosynthetic rate and produced greater overall vegetative growth with lower fruit set than the epi-lines. Flower and fruit set showed steady increases in the epiF2, epiF3and epiF4over wild type, with epiF4productivity consistently the highest. Average fruit size was lower in the epi-lines relative to wild type, although seed number was greater and fruit sugar content was unchanged. Overall fruit ripening was more rapid in the epi-lines, resulting in a greater proportion of red fruit at a single harvest time.4) We generated developmental reprogramming population in Nicotiana benthamiana and tomato by using virus induced gene silencing.This point to the possibility that we could use multiple technologies to get the developmental reprogramming population and then get the enhanced growth population in various species.5) The enhanced growth effects appear to be graft-transmissible in tomato. We carried out grafting experiments between Rutgers wild type and Rutgers transgenic MSH1-RNAi lines. While we detected no significant growth change in progeny coming from wild type grafted to wild type, progeny from wild type scion grafted to the MSH1-RNAi transgenic line as rootstock showed markedly enhanced early growth rate, resembling the epiF3effect.6) The enhanced growth effect is partially reversed by a methylation inhibitor. The enhanced growth vigor phenotype was partially obviated with the exogenous application of the DNA methylation inhibitor5-azacytidine. EpiF4seedlings growing in the presence of the inhibitor were reduced in plant height relative to the untreated epiF4control as well as the treated wild type seedlings.7) RNA-Seq profiling reveals a common process underlying developmental reprogramming caused by MSH1suppression in Arabidopsis and tomato, and provides an intrinsic perspective to understand the MSH1-derived development reprogramming and growth vigor.8) Based on current available data, a putative model for the MSH1-derived development reprogramming and growth vigor is presented.