Transposon based activation tagging in diploid strawberry and monoploid derivatives of potato

Abstract

Diploid strawberry and potato transformed with a transposon tagging construct exhibited either global (strawberry) or local transposition (potato). An activation tagged, compact-sized strawberry mutant overexpressed the gene adjacent to Ds. As major fruit and vegetable crops, respectively, strawberry and potato are among the first horticultural crops with draft genome sequences. To study gene function, we examined transposon-tagged mutant strategies in model populations for both species, Fragaria vesca and Solanum tuberosum Group Phureja, using the same Activation/Dissociation (Ac/Ds) construct. Early somatic transposition during tissue culture occurred at a frequency of 18.5% in strawberry but not in potato transformants. Green fluorescent protein under a monocot promoter was a more reliable selectable marker in strawberry compared to potato. BASTA (gluphosinate herbicide) resistance served as an effective selectable marker for both species (80 and 85% reliable in strawberry and potato, respectively), although the effective concentration differed (0.5% for strawberry and 0.03% for potato). Transposons preferentially reinserted within genes (exons and introns) in both species. Real-time quantitative PCR revealed enhanced gene expression (670 and 298-fold expression compared to wild type in petiole and leaf tissue, respectively) for an activation tagged strawberry mutant with Ds inserted about 0.6 kb upstream from a gene coding for an epidermis-specific secreted glycoprotein EP1. Our data also suggested that endopolyploid (diploid) cells occurring in leaf explants of monoploid potato were the favored targets of T-DNA integration during transformation. Mutants obtained in these studies provide a useful resource for future genetic studies.

Activation-tagging in indica rice identifies a novel transcription factor subunit, NF-YC13 associated with salt tolerance

“…This system uses tetramer copies of cauliflower mosaic virus (CaMV) 35 S enhancers 15 and the enhancers upon integration in the recipient plant genome can function in either orientation at the place of insertion, thereby causing transcriptional activation of nearby genes resulting in dominant gain-of-function mutations 16 . There are several reports citing this technique with the development of AT lines in Arabidopsis 17 , 18 , japonica rice 16 , 19 , indica rice 20 , tomato 21 , 22 , poplar 23 , strawberry, potato 24 , barley 25 and sorghum 26 .…”

DNA Transposition at Work

“…Although successful transposon mutagenesis in different species sometimes entails closely matching the original transposon host with the target species, this is not obligatory. Thus, Ac / Ds has been successfully used to target plant species other than maize, ranging from poplar trees and strawberries to rice and soybeans, yet it has also been reported to work effectively in medaka fish . Similarly, Tol2 , a hAT transposon originally isolated from the medaka fish, , has not only been successfully applied to manipulate the genome of zebrafish − and frogs, , but has been shown to be active in a range of mammalian and human cells.…”

“…In plants, the occurrence of naturally active transposons, not only the hAT elements such as Ac / Ds or Tam3 but also those of the Mutator ( MuDR ) and CMC ( CACTA / En / Spm ) superfamilies, has provided a palette of tools for genetic manipulation. Large-scale screens provide a way to search for interesting phenotypes and desirable growth properties among important crop species, and transposon mutagenesis offers an alternative when other approaches to genetic manipulation (such as those based on bacteria such as Agrobacterium tumefaciens or Agrobacterium rhizogene s that can introduce exogenous DNA“T-DNA” for transfer DNAinto the plant genome) are not practical. , Transposon mutagenesis offers a potential advantage over T-DNA insertional mutagenesis, as once a “launching site” (an initial transposon insertion) has been established, new mutants can be continually generated as long as there is a source of the transposase. Clearly, this is a phenomenon not limited to plants, but is particularly helpful when it is difficult or labor-intensive to introduce foreign DNA into cells.…”

Overexpression of a developing xylem cDNA library in transgenic poplar generates high mutation rate specific to wood formation

“…Nevertheless, dominant insertional mutagenesis approaches, like activation tagging, have been successfully applied to poplar (Busov et al , ) and other nonmodel plants (Aulakh et al , ; Lu et al , ; Mathews et al , ; Zubko et al , ). Thus far, in poplar, generation and screening of activation tagging population of few hundreds to few thousand lines have been undertaken (Busov et al , ; Dash et al , ; Harrison et al , ; Mathews et al , ).…”