Since the beginning of genetic engineering and its use in plant biology, the microorganism Agrobacterium tumefaciens has been a prominent figure in scientific endeavors. The natural processes of the species involves them modifying their cellular surroundings inside tree hosts to provide them with the nutrients they need. As a side effect of this process, they have the capability to transfer genetic material into the genome of their host. Indeed, there is plenty of evidence that they have been doing so with plants around the world for millions of years, resulting in not only transgenes from the bacteria themselves, but also far-flung sources like insects and fungi.
This process involves the movement of a small fragment of DNA referred to as transfer DNA (T-DNA) and it has become common to use this system to transfer desired sequences into available plant cultivars. In the model organism Arabidopsis alone, there are over 700,000 known variant lines in the scientific literature with a different T-DNA insertion used for a number of research prospects. Of these, around 325,000 have had their genomes fully sequenced to study the differences in T-DNA insertion and the kinds of ways the DNA addition can take place.
The Methods of Transgenesis
Normally, the insertion process of Agrobacterium involves a tumor-inducing plasmid gene that causes large lumps known as crown galls to form in the infected part of the tree. The bacteria used by scientists, however, has had these sequences removed so that only the desired gene to be inserted is used and nothing else. The replacement for them is what is known as a cassette, which is a set of genes to help facilitate the gene of interest. These extra parts include a promoter sequence and often a selectable marker, whether that is resistance to a specific antibiotic or color change via a fluorescent protein.
The reason why there is at least some uncertainty during insertion events is that the T-DNA sequence is placed at locations in the genome where a double strand break naturally occurs from some other regular form of damage. Since this can happen in most places of the genome, the insertion place is variable and can also happen more than once if the T-DNA is available when more than just a single double strand break occurs.
After the insertion of the target gene, expression testing and overall sequencing is done to properly visualize where in the genome the events happened and it is that data which makes up the database information on Arabidopsis. Since it is also possible for the T-DNA to be inserted in an inverted direction and cause some chromosomal rearrangement from that, along with epigenetic responses, all avenues of change must be notated and considered. Including cases where cellular systems, such as RNA silencing, prevent the inserted transgene from being expressed, while still keeping the insertion. But the capability to visualize and resolve such changes in the data has often been lacking in the past, especially when it came to epigenetics.
Secrets of T-DNA and Epigenetics
A team of researchers at the Salk Institute for Biological Studies decided to use several data processing methods to analyze this available material and determine specifics on T-DNA insertion attempts using Agrobacterium and how this changes the genetic environment around the insertion point. While a single inserted copy would be preferable, the fact that multiple copies are added often isn’t a hindrance based on what they found, as it is very common for these insertions to be completely silenced or not expressed. This is true whether through the gene not being properly transcribed or through more active cellular measures. Those measures include epigenetic modifications that silence the inserted gene through methylation of the markers found on every gene sequence.
It is actually rare for an inserted gene to be active and functional in the first place, which is why several transgenic lines see no expression at all. This also explains why transgenesis using Agrobacterium involves making a large amount of cultivar lines in order to find one that is functionally using the gene. The floral dip method that involves dipping the flowers of plants into a mixture containing Agrobacterium may also play a role in the selective silencing of the target gene.
The selectable marker nptII that confers kanamycin resistance appears to be heavily silenced by Arabidopsis and any successful transcription is swiftly degraded. This does mean that such a marker isn’t exactly useful in showcasing that the cassette containing the gene was inserted and expressed. In comparison, another line using the marker bar that gives a particular herbicide resistance showed no evidence of silencing. So this difference appears to be marker specific and should be noted for future research.
Advanced Sequencing and New Technologies
They hope that this more in-depth information can not only improve usage of Agrobacterium in the future, but also that their publishing of what this sort of single nucleotide-level resolution mapping can figure out will enable greater use of it by others to find the specific insertion lines that are both actively expressed and, preferably, have just a single copy of the transgene. It is a far more efficient method than the currently used screening of large numbers of plants and the researchers noted in their press release that the technology to do this didn’t exist just a year ago. The new nanopore sequencing technology has opened up a lot of doors to finding previously hidden changes in genetic material.
This also shows that next generation sequence machinery continues to expand at a rapid speed, gaining higher levels of functionality and faster ways of resolving entire genomes of data. Who knows where such technology will be just a few years further down the road. We’ll all have to wait and see.
Photo CCs: Anther of thale cress (Arabidopsis thaliana), an artefact from Wikimedia Commons
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