Research Group Lahaye
Analysis and application of TAL effectors
Prof. Dr. Thomas Lahaye
ZMBP, Allgemeine Genetik
6th floor, room 6S22
Auf der Morgenstelle 32
Tel. +49-7071 / 29 7 8745
Fax. +49-7071 / 29 50 42
Transcription-Activator Like Effectors and homologous TALE-like proteins – analysis of biological function unlocks potential for applied biotechnology
With an increasing population, environmental degradation and dwindling natural resources, research into crop development has never been more important. Plant pathogens are a major threat to our plant-based food supply. As plant pathologists we work on understanding the molecular basis of plant disease in order to identify promising resistance strategies. During infection microbial plant pathogens secrete diverse effector proteins to block resistance and promote microbial disease. The transcription activator-like effectors (TALEs) of Xanthomonas spp. and their homologs from the bacterial wilt pathogen Ralstonia solanacearum, the RipTALs, are one such class of effectors.
TALEs are unusual in acting as transcription factors inside the host (Bogdanove et al., 2010). After injection into the plant cell they bind specific effector binding elements (EBEs) in plant promoters, triggering expression of downstream susceptibility (S) genes whose products benefit the bacterium. A corresponding defense system has evolved in some plant hosts, whereby EBEs upstream of plant resistance (R) genes allow the bacterial threat to be stopped on detection of the TALE.
EBE binding is carried out by the largest domain of the TALE, the repeat domain. The tandem-arranged, near-identical repeats each bind one DNA base in the target sequence. Base specificity is defined by the amino acid at position 13, the base-specifying residue (BSR) (de Lange et al., 2014). The BSR code has by now been fully elucidated allowing accurate prediction of optimal TALE targets in a host genome.
The Lahaye group was involved in the early characterization of TALEs from Xanthomonas (Boch et al., 2009) and Ralstonia (de Lange et al., 2013) and continues to work with this protein class – studying its distribution, evolution and molecular properties. We also use TALEs both as tools to identify or create resistant crops and as tools for biotechnology.
Promoter-trap R genes – Fighting the pathogen with its own weapons
Through screening of natural pepper accessions the Lahaye lab has identified two resistance genes that are transcriptionally activated by TALEs of Xanthomonas euvesicatoria: Bs3, Capsicum annuum (Römer et al., 2007) and Bs4C C. pubescens, (Strauss et al., 2012). These resistance genes encode two non-homologous proteins and trigger a cell death reaction of the infected tissue within 48 hours.
We are currently exploring the potential of these genes for the production of resistant crop lines as well as working to understand the mechanisms underlying their function.
In addition we have previously demonstrated that several TALE binding boxes can be combined in one “complex” promoter (Römer et al., 2009). Using such promoter constructs upstream of an executor R-gene coding sequence may allow us to create crop accessions with broad-spectrum resistance.
RipTALs of Ralstonia solancearum and their role in bacterial wilt
Ralstonia solanacearum causes bacterial wilting disease in a phylogentically and geographically diverse set of hosts. We recently showed that R. solanacearum effector proteins with similarity to TALEs at the sequence level, and therefore known as RipTALs, mediate molecular functions homologous to those of TALEs (de Lange et al., 2013). With a newly established high-security area for work with the pathogen we are now looking into natural interactions to identify S and R genes. We hope with this knowledge to be able to provide tools to combat the devastating disease caused by this broad-host range pathogen.
Toolkit for TALEs and TALEs as Tools
The Lahaye lab developed a toolkit system for the assembly of TALE DNA binding domains using the straightforward golden-gate cloning strategy (Morbitzer et al., 2011). This toolkit is available on request and has been adapted to be compatible with a suite of in planta golden-gate vectors (Binder et al., 2014) .
We have worked with several collaborators exploring the potential uses of TALEs as molecular tools (as summarized in de Lange, Binder & Lahaye, 2014). Applications have included their use as designer transcription factors (Bultmann et al., 2012), nucleases (Mussolino et al., 2011) and as tags for in vivo imaging of DNA sequences (Thanisch et al., 2014). Further collaborations are always welcome!
Binder, A., Lambert, J., Morbitzer, R., Popp, C., Ott, T., Lahaye, T. and Parniske, M. (2014) A modular plasmid assembly kit for multigene expression, gene silencing and silencing rescue in plants. PLoS One, 9, e88218.
Boch, J., Scholze, H., Schornack, S., Landgraf, A., Hahn, S., Kay, S., Lahaye, T., Nickstadt, A. and Bonas, U. (2009) Breaking the code of DNA binding specificity of TAL-type III effectors. Science, 326, 1509–12.
Bogdanove, A.J., Schornack, S. and Lahaye, T. (2010) TAL effectors: finding plant genes for disease and defense. Curr. Opin. Plant Biol., 13, 394–401.
Bultmann, S., Morbitzer, R., Schmidt, C., Spada, F., Lahaye, T. and Leonhardt, H. (2012) Targeted transcriptional activation of the pluripotency gene oct4 by designer TALEs. Nucleic Acids Res., 40, 5368-77.
Lange, O. de, Binder, A. and Lahaye, T. (2014) From dead leaf, to new life: TAL effectors as tools for synthetic biology. Plant J., (in press).
Lange, O. de, Schreiber, T., Schandry, N., Radeck, J., Braun, K.H., Koszinowski, J., Heuer, H., Strauß, A. and Lahaye, T. (2013) Breaking the DNA-binding code of Ralstonia solanacearum TAL effectors provides new possibilities to generate plant resistance genes against bacterial wilt disease. New Phytol., 199, 773–86.
Morbitzer, R., Elsaesser, J., Hausner, J. and Lahaye, T. (2011) Assembly of custom TALE-type DNA binding domains by modular cloning. Nucleic Acids Res., 39, 5790–9.
Mussolino, C., Morbitzer, R., Lütge, F., Dannemann, N., Lahaye, T. and Cathomen, T. (2011) A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Res., 39, 9283–93.
Römer, P., Hahn, S., Jordan, T., Strauss, T., Bonas, U. and Lahaye, T. (2007) Plant pathogen recognition mediated by promoter activation of the pepper Bs3 resistance gene. Science, 318, 645–8.
Römer, P., Recht, S. and Lahaye, T. (2009) A single plant resistance gene promoter engineered to recognize multiple TAL effectors from disparate pathogens. Proc. Natl. Acad. Sci. U. S. A., 106, 20526–31.
Strauss, T., Poecke, R.M.P. van, Strauss, A., et al. (2012) RNA-seq pinpoints a Xanthomonas TAL-effector activated resistance gene in a large-crop genome. Proc. Natl. Acad. Sci. U. S. A., 109, 19480–5.
Thanisch, K., Schneider, K., Morbitzer, R., Solovei, I., Lahaye, T., Bultmann, S. and Leonhardt, H. (2014) Targeting and tracing of specific DNA sequences with dTALEs in living cells. Nucleic Acids Res., 42, e38.