Xanthomonas spp. are plant pathogens that modulate their host’s gene expression in order to facilitate infection. They do this using transcription activator-like effectors (TALEs). Two domains are conserved in TALEs: an N-terminus, required for type III secretion into the plant cells; and a C-terminus with transcription factor activity. In the middle is a set of tandem repeats of amino acids, which mediates binding to host DNA.
As the binding and effector domains of TALEs can be customised, the possibility of using them for molecular and synthetic biology has been explored for some time. They have been used to change gene expression in plants, yeast, and even human cells.
TALEs have been adapted by researchers to make TALE nucleases (TALENs) – TALEs attached to a FOK1 nuclease domain. TALENs work in pairs that flank either side of the target site so that the nuclease domains meet at the point of cleavage. The nucleases cause a double-stranded DNA break, which is fixed imperfectly, causing an insertion or deletions.
In May this year, a paper was published demonstrating the huge impact TALEs could have on agriculture. Li et al. prove that transcription activator-like effector nucleases (TALENs) can be used to render rice resistant to the major agricultural pathogen, Xanthomonas oryzae pv. Oryzae (Xoo).
Highlighted Article:Ting Li, Bo Liu, Martin Spalding, Donald Weeks and Bing Yang. High-efficiency TALEN-based gene editing produces disease-resistant rice. Nature Biotechnology 30: 390-392.
Xanthomonas oryzae is the cause of bacterial leaf blight in rice, an important rice disease. It controls the expression of a number of host plant genes using TALEs. One of these genes is a SWEET sucrose efflux transporter, OS11N3, which is hijacked by TALEs AvrXa7 or PthXo3. It has sites on its promotor which these TALEs bind to, allowing them to redirect the plant’s sugar to the bacterial cells.
Li et al. made two pairs of designer TALENs containing 24 repeat units for recognition of a specific set of 24 contiguous nucleotides at the two TALE binding sites on the OS11N3 promotor. Each pair of TALENs was put into a plasmid containing promotors for each TALEN and a hygromycin resistance gene.
The team used Agrobacterium transformation to insert the plasmids into rice embryonic cells. They genotyped the resulting plants and infected them with Xoo. 47% of the first generation were resistant to Xoo, including homozygous and heterozygous plants. Plants from the second generation were also resistant to Xoo, and all plants were morphologically the same as wildtype plants.
Significance: As pointed out in Biofortified, this technology in no way introduces transgenes. The rice product at the end of this experiment contained a genome with less than 100 missing base pairs, and no foreign DNA. A gene is inactivated, not induced. While I doubt that anti-GM campaigners will embrace crops with improvements caused by missing DNA, many of the reasons cited for dis-trusting GM plant products are avoided by this technology, which could perform the same function in generating crops resistant to pathogens or harsh environmental conditions.
There are online tools and resources for working with TALEs – see TAL effectors for protocols, links to other resources, and a good technical introduction to TALEs!