Refined Requirements for Protein Regions Important for Activity of the TALE AvrBs3

Abstract

AvrBs3, the archetype of the family of transcription activator-like (TAL) effectors from phytopathogenic Xanthomonas bacteria, is translocated by the type III secretion system into the plant cell. AvrBs3 localizes to the plant cell nucleus and activates the transcription of target genes. Crucial for this is the central AvrBs3 region of 17.5 34-amino acid repeats that functions as a DNA-binding domain mediating recognition in a “one-repeat-to-one base pair” manner. Although AvrBs3 forms homodimers in the plant cell cytosol prior to nuclear import, it binds DNA as a monomer. Here, we show that complex formation of AvrBs3 proteins negatively affects their DNA-binding affinity in vitro. The conserved cysteine residues at position 30 of each repeat facilitate AvrBs3 complexes via disulfide bonds in vitro but are also required for the gene-inducing activity of the AvrBs3 monomer, i.e., activation of plant gene promoters. Our data suggest that the latter is due to a contribution to protein plasticity and that cysteine substitutions to alanine or serine result in a different DNA-binding mode. In addition, our studies revealed that extended parts of both the N-terminal and C-terminal regions of AvrBs3 contribute to DNA binding and, hence, gene-inducing activity in planta.

The Xanthomonas effector XopL uncovers the role of microtubules in stromule extension and dynamics in Nicotiana benthamiana

“…(). MAP4 was amplified from pCAMBIA3000 :: eGFP : MAP4 described in Mathur and Chua (), and cloned into pGGA2 (Schreiber et al ., ), or pGGA12 (generation of plasmid as described for pGGA11 ), fusing it to an N‐terminal eGFP or mOrange2 tag, respectively. pUBN‐Dest :: TUA5 ( TUA5 = CDS of TUBULIN ALPHA5 from A . thaliana ); first used in plants by Gutierrez et al .…”

“…(i) pGGA2::MAP4 and pGGA12::MAP4 (MAP4 = CDS of microtubule-associated protein 4 from Mus musculus); first use of MAP4 in plants was described by Marc et al (1998). MAP4 was amplified from pCAMBIA3000::eGFP:MAP4 described in Mathur and Chua (2000), and cloned into pGGA2 (Schreiber et al, 2015), or pGGA12 (generation of plasmid as described for pGGA11), fusing it to an N-terminal eGFP or mOrange2 tag, respectively. (ii) pUBN-Dest::TUA5 (TUA5 = CDS of TUBULIN ALPHA5 from A. thaliana); first used in plants by Gutierrez et al (2009).…”

Non-host Resistance Induced by the Xanthomonas Effector XopQ Is Widespread within the Genus Nicotiana and Functionally Depends on EDS1

“…Fragments were cloned into pUC57 or pJET1.2/blunt (Thermo Fisher Scientific), respectively, and then by Bsa I cut-ligation (Engler et al, 2008) into the expression vectors pBRM (Szczesny et al, 2010b) or pGGX1 for Xcv , and pGGA1 (Schulze et al, 2012), pGGA2 (Schreiber et al, 2015) and pGGA7, respectively, for Agrobacterium -mediated expression in planta . The binary vector pGGA7 contains the backbone of pBGWFS7 (Karimi et al, 2002), the chloramphenicol resistance- ccd B selection cassette from pGWB2 (Nakagawa et al, 2007), and allows in planta expression of genes 3′-translationally fused to 4 × c-Myc under the control of the cauliflower mosaic virus 35S promoter.…”

Behind the lines–actions of bacterial type III effector proteins in plant cells

“…Notably, however, DNA binding of TAL effectors to EBEs is not only determined by the RVDs but also by N-terminal non-canonical repeats as well as C-terminal protein regions and can be affected by epigenetic modifications of the DNA (Boch, Bonas and Lahaye 2014; Schreiber et al. 2015). …”