The Complex Architecture And Epigenomic Impact Of Plant T-DNA insertions

Salk researchers have mapped the genomes and epigenomes of genetically modified plant lines with the highest resolution ever to reveal exactly what happens at a molecular level when a piece of foreign DNA is inserted. Their findings, published in the journal PLOS Genetics. elucidate the routine methods used to modify plants, and offer new ways to more effectively minimize potential off-target effects.

When a scientist wants to put a new gene into a plant—for basic research purposes or to boost the health or nutrition of a food crop—they usually rely on Agrobacterium tumefaciens to get the job done. Agrobacterium is the bacteria that causes crown gall tumours, large bulges on the trunks of trees. Decades ago, scientists discovered that when the bacteria infected a tree, it transferred some of its DNA to the tree’s genome. Since then, researchers have co-opted this transferability of Agrobacterium for their own purposes, using its transfer-DNA (T-DNA) to move the desired gene into a plant.


“Biotech companies spend a lot of time and effort to characterize transgenic plants and disregard candidates with unwanted changes without understanding—from a basic biological perspective—why these changes may have occurred,” says Ecker. “Our new approach offers a way to better understand these effects and may help to speed up the process.”


Since the T-DNA approach can lead to an integration of many copies of the desired gene into a plant, it can be difficult to study the final result with standard DNA sequencing, as most technologies struggle to sequence highly repetitive stretches of DNA. But Ecker and his colleagues turned to a new combination of approaches—including optical mapping and nanopore sequencing—to look at these long stretches in high resolution. They applied the technologies to four randomly selected T-DNA lines of Arabidopsis thaliana, a commonly used model plant in biology. (These plants are derived from a large population of T-DNA insertional mutants that were created using an Arabidopsis transformation method, called floral dip, to study gene function.)

Optical mapping revealed that the plants had between one and seven distinct insertions or rearrangements in their genomes, ranging in size by almost tenfold. Nanopore sequencing and reconstruction of the genomes of two lines confirmed the insertions to single-letter resolution, including whole segments of DNA that had been exchanged—or translocated—between chromosomes in one of the selected lines. Gene insertions themselves showed a variety of patterns, with the inserted DNA fragment sometimes scrambled, inverted or even silenced.



“Now we have the first high-resolution insights on how T-DNA insertions can shape the local epigenome environment,” says Zander. In an ideal world, the researchers say, T-DNA would insert one single, functional copy of the desired gene with no nearby side effects on a plant’s genome. While their findings show this is rarely the case in Arabidopsis, their methods offer a path to a better understanding and surveillance of the effects.



Source:- PLOS Genetics
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