Kunkel+May+4

Topic: Transcription activator—like (TAL) effectors
I highly recommended you read ONE of the first three reviews for background Bogdanove et al, 2010. Curr. Op. Plant Biol. 13:394-401.
 * __Reviews & Commentary__ **

Scholze & Boch, 2011. Curr. Op. Microbiol. 14: 47-53

Boch and Bonas, 2010. Xanthomonas AvrBs3 Family-Type Effectors: Discovery and Function. Annu. Rev. Plant Pathol. 48:419-436

Voytas and Joung, 2009. DNA Binding Made Easy Science 326: pp. 1491-1492

Adam J. Bogdanove and Daniel F. Voytas, 2011. TAL Effectors: Customizable Proteins for DNA Targeting //Science// 333:1843 []

1. Patrick Römer et al, 2007. Plant Pathogen Recognition Mediated by Promoter Activation of the Pepper Bs3 Resistance Gene. Science 318: 645-648.
 * __Primary Research articles to discuss in class:__ **

2. Boch J, et al., 2009. Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326:1509-1512.

Miller J, et al., 2010 A TALE nuclease architecture for efficient genome editing. Nat. Biotech. Online pub.
 * __Supplemental reading on function, structure and biotech applications of TAL Effectors__**

Mak, et al., 2012. The Crystal Structure of TAL Effector PthXo1 Bound to Its DNA Target Science 335: 716-719. http://www.sciencemag.org/content/335/6069/716.full.html

Dong Deng et al, 2012. Structural Basis for Sequence-Specific Recognition of DNA by TAL Effectors Science 335: 720-723.P


 * __ Commercial application of TAL binding motifs __**

General:
 * __Discussion Questions:__**
 * 1) What is the original function, in an evolutionary sense, of TAL effectors? In other words, how does expression of TAL effectors benefit the pathogen, Xanthomonas?
 * 2) How have plants evolved to become resistant to Xanthomonas strains expressing TAL effectors?
 * 3) What does the term “recognitional specificity” mean?
 * 4) What are some remaining questions related to TAL effector structure and function.
 * 5) How can what we have learned about TAL effector proteins be put to use in biotechnology?

Specific questions: 1. Roemer et al, Figure 1A:
 * What causes the dark appearance in the regions of the leaves infiltrated with Agrobacterium expressing AvrBs3 and Bs3 (Fig. 1A)?
 * What are the controls in this panel. Why are so many included?
 * What controls for this panel are not shown? Could the lack of a response in the tissue infiltrated with Bs3 and the various variants of avrBs3 or with avrBs4 be due to a trivial reason?
 * Is this possible “trivial reason” a concern for the AvrBs3Drep16 allele?

2. Roemer et al, FigS9
 * How do you interpret the results of the EMSA assays shown in Fig. S9A (Supplemental material is posted above)? How does this finding fit with the observed difference in “recognitional specificity” of these alleles?

3. How has the cloning of the Bs3 R gene changed the way scientists think about the basis of “recognitional specificity” or “recognition” of a pathogen expressing an AvrBs3-like Avr gene?

4. The Boch 2009 paper presents a very nice example of how careful observation can lead to a simple and testable model regarding AvrBs3 DNA binding specificity.
 * What were these observations?
 * What hypothesis/model did these observations lead the authors to propose? What other information did they need to have to formulate their model? How does having access to variant, yet active alleles of AvrBs3 strengthen their hypothesis?
 * How did they come up with a predicted code for the TAL effector DNA binding specificity?
 * How did they experimentally test this model ? (Focus on Fig. 2, Fig 3C-E).
 * What type of experiments did they carry out to determine the :
 * 1) nucleotide specificity of different repeats?
 * 2) minimum number of repeats necessary to generate an active TAL effector?
 * 3) identify putative target genes ? Are any of these likely to be important in Xanthomonas disease?