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. 2015 Mar 17;10(3):e0120214.
doi: 10.1371/journal.pone.0120214. eCollection 2015.

Refined requirements for protein regions important for activity of the TALE AvrBs3

Tom Schreiber  1 , Anika Sorgatz  1 , Felix List  2 , Doreen Blüher  1 , Sabine Thieme  1 , Matthias Wilmanns  3 , Ulla Bonas  1
Affiliations

Refined requirements for protein regions important for activity of the TALE AvrBs3

Tom Schreiber et al. PLoS One. .

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. AvrBs3 complex formation interferes with DNA binding.
(A) AvrBs3 dimerizes via disulfide bonds. 0.5 μg purified His6::AvrBs3, treated with different DTT concentrations for 1 h at room temperature (RT) or with 10 mM DTT overnight at 8°C (10*), was separated by a non-reducing SDS-polyacrylamide gel and analyzed by immunoblot with an α-His antibody. a, multimeric His6::AvrBs3; b, monomeric His6::AvrBs3. (B) AvrBs3 binds to DNA as a monomer. Electromobility shift assay (EMSA) using 333 fmol biotin-labeled 36-bp DNA-fragments derived from the UPA20 promoter (WT) as a probe. The DNA was incubated with untreated or DTT-treated His6::AvrBs3 (+, 10 mM DTT overnight). Unlabeled WT and mutant ubm2 fragments (m, [8]) were used as competitor DNA. The experiments were repeated at least once with similar results.
Fig 2
Fig 2. Cysteines in AvrBs3 repeats are required for target gene induction.
(A) Constructs used for Agrobacterium-mediated T-DNA delivery into leaves of N. benthamiana. The effector construct allows expression of 4× c-Myc-tagged AvrBs3 derivatives under control of the 35S promoter. The reporter construct contains the 19-bp effector binding element (EBE) of AvrBs3 in front of the tomato Bs4-minimal promoter driving the uidA (GUS) reporter gene [47]. LB, left border; RB, right border. (B) GUS activity was determined in N. benthamiana leaves three days after Agrobacterium-mediated co-delivery of the reporter construct with silencing inhibitor p19 and effector constructs encoding AvrBs3 or derivatives. GFP served as negative, 35S:uidA as positive control. Gene-inducing activities of the AvrBs3 derivatives were determined relative to the GUS activity induced by the WT AvrBs3 protein (set to 100%). Grey box, T3S signal; black boxes, NLSs; black arrow, AD. White ovals represent WT repeats, grey and black ovals denote repeats with C30S and C30A substitutions, respectively. The C-terminal region of AvrBs3 containing C912S and C963S substitutions is indicated in grey. Errors bars indicate standard deviations (SD). Asterisks indicate statistically significant differences to GUS activity induced by WT AvrBs3 (t-test; *, P < 0.05; **, P < 0.01; ***, P < 0.001). The experiment was repeated once, and four times using the UPA20 box instead of EBE AvrBs3, with similar results. Columns on the right-hand side summarize the results of the HR induction assays in leaves of resistant pepper (ECW-30R) and Bs3-transgenic N. benthamiana plants after Agrobacterium-mediated delivery (“T-DNA”), and in pepper ECW-30R plants after inoculation of Xcv expressing AvrBs3 and a subset of cysteine mutants, respectively. HR development was monitored three to five dpi. +, HR three dpi; (+), delayed/partial HR five dpi;-, no HR five dpi; n.a., not analyzed. The experiments were repeated three times.
Fig 3
Fig 3. The AvrBs3 cysteine mutant is monomeric and binds specifically to DNA in vitro.
(A) 0.5 μg purified His6::AvrBs3ΔN152 and His6::AvrBs3ΔN152(C30S)Rep, respectively, were treated with different concentrations of DTT for 1 h at RT or with 10 mM DTT overnight at 8°C (10*). Samples were separated by a non-reducing SDS-polyacrylamide gel and analyzed by immunoblot with an α-His antibody. a, multimeric His6::AvrBs3ΔN152; b, monomeric His6::AvrBs3ΔN152. (B) EMSA. 333 fmol biotin-labeled 36-bp DNA-fragments derived from the UPA20 promoter were incubated with reduced His6::AvrBs3ΔN152 and His6::AvrBs3ΔN152(C30S)Rep (+, incubation with 10 mM DTT overnight). Unlabeled WT and mutant ubm2 DNA fragments (m, [8]) were used as competitor DNA. The experiments were repeated at least twice with similar results.
Fig 4
Fig 4. AvrBs3 cysteine mutants do not compete with AvrBs3 for DNA binding in planta.
(A) T-DNA constructs used. Two different effector constructs, A and B, allow expression of (A) GFP- or (B) 4× c-Myc tagged AvrBs3 and derivatives under control of the 35S promoter. The reporter construct contains the 19-bp UPA20 UPA box in front of the tomato Bs4 minimal promoter driving a promoterless uidA reporter gene [48]. LB, left border; RB, right border. (B) GUS activity was determined in N. benthamiana leaves three days after Agrobacterium-mediated co-delivery of the reporter construct with the avrBs3- or gfp construct (effector construct A) and effector construct B encoding one of the indicated proteins. Values are displayed relative to the GUS activity induced by GFP and WT AvrBs3. Error bars indicate SD. 35S:uidA served as control. Asterisks indicate statistically significant differences to the GUS activity induced by GFP and WT AvrBs3 (t-test, P < 0.05). The experiment was repeated twice with similar results.
Fig 5
Fig 5. In planta activity of AvrBs3 N- and C-terminal deletion mutants.
AvrBs3 and deletion derivatives were expressed in N. benthamiana leaves as 4× c-Myc fusions under control of the 35S promoter by Agrobacterium-mediated transformation. (A) N-terminal deletion constructs are schematically shown on the left and are named by the number of deleted amino acids. (B) Names of C-terminal deletion constructs indicate the number of amino acids remaining in the C-terminal part of AvrBs3. To compensate for the loss of NLSs and AD in CTR mutants the SV40 NLS and the AvrBs3 AD were added to the new C-termini. GUS activities were determined in N. benthamiana leaves three days after Agrobacterium-mediated co-delivery of the reporter construct (UPA20 box-minimal pBs4 driving a promoterless uidA [48]), the silencing inhibitor p19, and avrBs3, avrBs3-derivatives or gfp (negative control). GUS activity induced by WT AvrBs3 was set to 100%. NTR box, T3S signal; CTR boxes, AvrBs3 NLSs; box with asterisk, SV40 NLS; arrow, AD. Error bars indicate SD. Asterisks indicate statistically significant differences to the GUS activity induced by WT AvrBs3 (t-test, P < 0.05). The right column shows the HR induction by AvrBs3 and deletion derivatives in leaves of resistant pepper (ECW-30R) and Bs3-transgenic N. benthamiana plants three days after Agrobacterium-mediated delivery of the avrBs3 or derivative constructs. The experiments were repeated three times with similar results.
Fig 6
Fig 6. Analysis of the imperfect leucine zipper motif in AvrBs3.
(A) Sequence comparison between the leucine zipper-like motif in the CTR of AvrBs3 and a subset of homologs using ClustalW. Identical amino acids (white letters on black background) and similar amino acids (white letters on grey background) were shaded using Boxshade. Leucines corresponding to the proposed leucine repeats were labelled with a black asterisk, leucines that do not correspond to leucine repeats with a grey asterisk and aa of the basic region with a black dot. (B) AvrBs3 was mutated in the basic region (AvrBs3-LZm1) and in leucines of the leucine-rich region (AvrBs3-LZm2). Leucines are given in black, the basic regions are boxed. (C) UPA20 box activation by AvrBs3 and AvrBs3 mutant derivatives shown in B. GUS activities were determined in leaves of N. benthamiana three days after Agrobacterium-mediated co-delivery of the reporter construct (UPA20 box-minimal pBs4 driving a promoterless uidA [48]), together with silencing inhibitor p19 and gfp, avrBs3 or avrBs3 derivatives. GUS activities are given relative to the GUS activity induced by WT AvrBs3. Error bars indicate SD. Asterisks indicate statistically significant differences as compared to WT AvrBs3 (t-test, P < 0.05). (D) HR induction by AvrBs3 and mutant derivatives in Bs3 ECW-30R plants. Xcv 85–10 containing pGGX1 (empty vector, ev) or pGGX1 driving expression of avrBs3, avrBs3-LZm1 and avrBs3-LZm2, respectively, were inoculated into leaves of Bs3 pepper plants (ECW-30R). Leaves were harvested three dpi and bleached with ethanol to better visualize the HR. (E) Bs3 gene induction by AvrBs3 and mutant derivatives in leaves of pepper ECW-30R. RT-PCR analysis 10 h after inoculation of the Xcv strains described in D. EF1α was used as control for equal cDNA amounts. RT-PCR was repeated once, the other experiments at least twice with similar results.
Fig 7
Fig 7. AvrBs3 NTR and CTR contribute to DNA binding to different extents.
Fluorescence polarization titrations of fluorescein-labeled dsDNA by AvrBs3 and deletion mutants. Increasing concentrations of purified His6-tagged AvrBs3 (open circles), AvrBs3ΔN152 (closed circles) and AvrBs3ΔN152-C16 (CTR deleted, except for the first 16 aa downstream of the repeats; open squares) were incubated under reducing conditions with fluorescein-labeled 36-bp DNA fragments derived from the UPA20 promoter. Fluorescence polarization intensities were normalized and plotted as function of protein concentration. The results are representative of three independent measurements. Dissociation constants (KD app) were determined by curve fitting with a one-site saturation and nonspecific binding model using Kaleidagraph 4.0 (Synergy Software). The KD app for AvrBs3 is 23.2 ± 1.6 nM, and KD values of 99.2 ± 7.5 nM and 282.2 ± 18.1 nM were calculated for the truncated derivatives AvrBs3ΔN152 and AvrBs3ΔN151-C16, respectively.
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