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I've read this paper where they specifically modify a region in the rice genome to ablate the binding site of a pathogen, Xanthomonas oryzae, and disrupt the hijacking of a gene network in the rice genome to the advantage of the pathogen.

It's an interesting concept but left me wondering how one would determine de novo the binding of these pathogen effectors into the host if one didn't know them. What would be the steps to determine where are the pathogen's TFs binding the host genome?

Edit: there is the possibility of not knowing which proteins bind to the host genome, so what would be the way of developing the antibodies in this case?

Reference:

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You have two answers. I couldn't help but notice your accept rate is 0%. –  David James Jun 25 '12 at 2:34

2 Answers 2

The techniques used to do this are ChIP-seq and ChIP-chip.

Basically, you

  • let the pathogen bind to the (highly replicated) DNA
  • cut up the DNA into little random pieces by sonication
  • enrich (“pull down”) the pathogen-bound DNA fragments by using a known antibody which binds to the pathogen
  • sequence the thus enriched DNA
  • map the sequenced fragments back to their original location on the genome.

Wikipedia has a nice picture explaining this process in a little more detail:

ChIP-seq workflow

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A colleague of mine discovered the cipher that determines TAL effector DNA specificities, which is described in this short paper. These specificities were determined by observing TAL effectors bound to DNA and recording how often a given repeat-variable diresidue (RVD) would correspond to a given nucleotide (using a weight matrix).

Now that the specificities have been determined, identifying TAL effector target sites is simply a matter of measuring the probability of that TAL effector's RVD sequence aligning to a novel sequence. Some applications are described here and here.

The TALENT 2.0 server (invited paper under review) provides tools for designing custom TAL effectors to target specific DNA sequences, or for identifying potential off-target binding sites in a given sequence or genome.

References:

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