TAL Effector PthXo1

TAL Effector PthXo1

Rebecca Hölzel '22 and Joanna van Dyk '22

I. Introduction

Transcription activator-like (TAL) effectors are a class of proteins secreted by Xanthomonas bacteria and function to infect targeted plant cells. Xanthomonas can infect a wide range of species, but the specific TAL effector shown here, PthXo1, is secreted by Xanothomonas oryzae and targets the Os-8N3 gene found in rice.

TAL effectors easily enter the nucleus of the cell and bind DNA, ultimately promoting transcription to the benefit of the bacteria. For example, some TAL effectors target genes involved in glucose transport. The heightened transcription provides more glucose to the bacteria, allowing further growth and infection of the plant (Yang, B., et al).

As TAL effectors have predictable and repetitive structures, engineering artificial TAL effectors is feasible. A TAL effector could be manufactured for a specific gene it would not otherwise target. It can then act as an activator or repressor; it may also act as a nuclease, providing another method for targeting and editing DNA. While promising, research with TAL effectors is still in its early stages (“TAL”).

Mechanism

Figure 1. Xanothomonas oryzae infection of a rice plant cell. A TAL Effector enters the nucleus and binds the promoter region, ultimately acting in tangent with other transcription factors to induce transcription. Image taken from Yuan, Meng, et al, 2016

II. General Structure

PthXo1 consists of a singular domain containing a repeat region as well as the N-Terminal and C-terminal sequences. All TAL effectors, including PthXo1, are composed of , each composed of two alpha helices. Most repeats are 34 amino acids in length:

Each pair of alpha helices is connected by a half-repeat loop that contains the repeat variable diresidue ( ), or amino acids in the 12th and 13th position that differ between tandem repeats. Though more than 20 types of RVDs have been identified in TAL effectors, the are HD, NG, NI, NN, NS, N*, and HG. Each RVD sequence is followed by two glycine residues, though the importance of this conserved sequence is not fully understood.

The are responsible for bacterial type III secretion, which allows Xanothomonas oryzae to inject PthXo1 directly into the target cell's cytoplasm. The C-terminal sequences are required for transcription activation. These sequences also contain positively charged residues required for nuclear localization; they act to "tag" the protein for transport into the nucleus (Mak, A.n.s., et al).

III. Repeat Variable Diresidues and DNA Binding

The RVDs of each tandem repeat are responsible for all DNA interactions; the remaining portion of each repeat is non-sequence specific. Notably, each repeat, in addition to containg the RVD, also contains a in position 27 which creates a kink in the second helix. This kink is essential for association of PthXo1 with the DNA, as it positions the tandem repeat against the major groove.

Only the amino acids of RVDs contact the DNA directly. The way RVDs bind DNA bases is predictable, which is part of the reason artificial TAL effectors can be used to target specific DNA sequences. In PthXo1, RVDs bind cytosine, and RVDs bind thymine (one NG binds cytosine), RVDs bind cytosine or adenine, RVDs bind guanine, and RVDs bind cytosine.

Within the HD RVDs, aspartate contacts the N4 atom of cytosine through both van der Waals and hydrogen bonds. Glycine in both NG and HG RVDs form non polar van der Waals interactions with the methyl group of the thymine base

The isoleucine of NI RVDs makes van der Waals contacts with both the C8 and N7 atoms of adenine's purine ring or C5 in cytosines pyrimidine ring.

Asparagine in NN RVDs hydrogen bonds with the N7 in guanine or adenine; each is equally likely to occur.

Within this structure are two N* RVDs, where the second residue is missing. As a result, this RVD does not contact the major groove directly. The conserved glycines following the deleted residue are significantly further from the DNA bases than usual, meaning N* binding is not as specific as other for RVDs.

For most TAL effectors, the binding site is preceded by a thymine; mutation at this position results in TAL effectors almost entirely unable to bind the DNA. Interactions with this thymine are accomplished by the , where a tryptophan in position 232 makes a nonpolar van der Waals contact with thymine’s methyl carbon. (Mak, A.n.s., et al).

V. References

Mak, A.n.s., et al. “Structure of TAL Effector PthXo1 Bound to Its DNA Target.” Science, vol. 335, Apr. 2012, doi:10.2210/pdb3ugm/pdb.

“TAL Effectors: Function, Structure, Engineering and Applications.”Current Opinion in Structural Biology, Feb. 2013, doi:https://doi.org/10.1016/j.sbi.2012.11.001.

Yang, B., et al. “Os8N3 Is a Host Disease-Susceptibility Gene for Bacterial Blight of Rice.” Proceedings of the National Academy of Sciences, vol. 103, no. 27, 2006, pp. 10503–10508., doi:10.1073/pnas.0604088103.

Yuan, Meng, et al. “A Host Basal Transcription Factor Is a Key Component for Infection of Rice by TALE-Carrying Bacteria.” ELife, vol. 5, 2016, doi:10.7554/elife.19605.

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