Transposon-mediated activation tagging

A powerful approach for identifying specific genes that control plant morphology and physiology (i.e., connecting genotype to phenotype) without prior knowledge is mutant screen. We are particularly interested in generating Mimulus mutants affecting phenotypes that are absent in Arabidopsis or other plant model systems, but otherwise widespread in the plant kingdom (e.g., floral carotenoid pigmentation, corolla tube formation and elaboration, petal reflexing, nectar volume, serpentine soil adaptation, copper tolerance). Such mutants can provide key insights into the developmental and physiological mechanisms underlying these important phenotypes.

Figure 1. Transposon-mediated activation tagging. The mPing element from rice is used to carry the figwort mosaic virus (FMV) enhancer. When transposed, mPing has an insertion preference into 5′-regulatory regions of genes, and the “cargo” FMV enhancer could ectopically activate the nearby gene.

While EMS mutagenesis is highly effective in Mimulus and has resulted in many mutants with interesting phenotypic alterations, identification of the causal genes underlying mutant phenotypes can be costly and time-consuming. A complementary approach is T-DNA or transposon insertion mutagenesis. Once a mutant phenotype is discovered, the causal gene can be readily identified by using the insertion sequence as a “tag”. Although our current transformation protocol for Mimulus is sufficiently robust to routinely generate tens of independent transgenic lines for any gene of interest, it is not efficient enough for large-scale T-DNA mutagenesis. Instead, in this project we employ a transposon mutagenesis strategy (Figure 1), which requires only a small number of primary transformants; an endless number of new insertions can be subsequently generated through transposition in a large population. Furthermore, by nesting a strong enhancer inside the transposon (Figure 1), this strategy produces not only recessive, loss-of-function mutations (i.e., when the transposon insertion disrupts coding sequences), but also gain-of-function mutations (i.e., when the enhancer is mobilized to gene regulatory regions) that show effects in even heterozygotes. These gain-of-function mutants are particularly important for characterizing genes with functionally redundant paralogs or genes that are lethal when knocked out.