Cysteines in AvrBs3 repeats are required for target gene induction.

<p>(<b>A</b>) Constructs used for <i>Agrobacterium</i>-mediated T-DNA delivery into leaves of <i>N</i>. <i>benthamiana</i>. The effector construct allows expression of 4× c-Myc-tagged AvrBs3 derivatives under control of the <i>35S</i> promoter. The reporter construct contains the 19-bp effector binding element (EBE) of AvrBs3 in front of the tomato <i>Bs4</i>-minimal promoter driving the <i>uidA</i> (GUS) reporter gene [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120214#pone.0120214.ref047" target="_blank">47</a>]. LB, left border; RB, right border. (<b>B</b>) GUS activity was determined in <i>N</i>. <i>benthamiana</i> leaves three days after <i>Agrobacterium</i>-mediated co-delivery of the reporter construct with silencing inhibitor <i>p19</i> and effector constructs encoding AvrBs3 or derivatives. GFP served as negative, <i>35S</i>:<i>uidA</i> 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 (<i>t</i>-test; *, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001). The experiment was repeated once, and four times using the <i>UPA20</i> box instead of <i>EBE</i>_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 <i>Bs3</i>-transgenic <i>N</i>. <i>benthamiana</i> plants after <i>Agrobacterium</i>-mediated delivery (“T-DNA”), and in pepper ECW-30R plants after inoculation of <i>Xcv</i> 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.</p

AvrBs3 NTR and CTR contribute to DNA binding to different extents.

<p>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 <i>UPA20</i> promoter. Fluorescence polarization intensities were normalized and plotted as function of protein concentration. The results are representative of three independent measurements. Dissociation constants (K_D^app) were determined by curve fitting with a one-site saturation and nonspecific binding model using Kaleidagraph 4.0 (Synergy Software). The K_D^app for AvrBs3 is 23.2 ± 1.6 nM, and K_D values of 99.2 ± 7.5 nM and 282.2 ± 18.1 nM were calculated for the truncated derivatives AvrBs3ΔN152 and AvrBs3ΔN151-C16, respectively.</p

AvrBs3 cysteine mutants do not compete with AvrBs3 for DNA binding <i>in planta</i>.

<p>(<b>A</b>) 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 <i>35S</i> promoter. The reporter construct contains the 19-bp <i>UPA20 UPA</i> box in front of the tomato <i>Bs4</i> minimal promoter driving a promoterless <i>uidA</i> reporter gene [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120214#pone.0120214.ref048" target="_blank">48</a>]. LB, left border; RB, right border. (<b>B</b>) GUS activity was determined in <i>N</i>. <i>benthamiana</i> leaves three days after <i>Agrobacterium</i>-mediated co-delivery of the reporter construct with the <i>avrBs3</i>- or <i>gfp</i> 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. <i>35S</i>:<i>uidA</i> served as control. Asterisks indicate statistically significant differences to the GUS activity induced by GFP and WT AvrBs3 (<i>t</i>-test, <i>P</i> < 0.05). The experiment was repeated twice with similar results.</p

Expression pattern of traditional reference genes in healthy and <i>Xcv</i>-infected tomato plants.

<p>Leaves of <i>S</i>. <i>lycopersicum</i> cv. MM plants were untreated or infiltrated with 10 mM MgCl₂ (mock) and 85–10Δ<i>hrcN</i>, respectively. To analyze mRNA accumulation of selected genes leaf material was harvested at 0, 4, 8 and 16 hours post infiltration (hpi). Relative RNA levels of housekeeping genes traditionally employed as references [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136499#pone.0136499.ref030" target="_blank">30</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136499#pone.0136499.ref061" target="_blank">61</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136499#pone.0136499.ref064" target="_blank">64</a>] were determined by microarray hybridization analysis. Expression values of analyzed plant samples are plotted separately, i.e., three biological replicates per time-point per infiltration. For each gene, expression values were obtained as the mean of the intensities of all probes representing the respective gene. Blue curves represent housekeeping genes that were also analyzed in this study together with novel reference genes. In addition, the most stably expressed gene in the microarray experiment is shown (red curve). <i>ACT</i> (<i>actin</i>), TC194780a; <i>EF-1α</i> (<i>elongation factor 1α</i>), SGN-U212845; <i>GAPDH</i> (<i>glyceraldehyde 3-phosphate dehydrogenase</i>), TC198136a; <i>UBI</i> (<i>ubiquitin</i>), TC193502a; <i>TBP</i> (<i>TATA binding protein</i>), SGN-U329249; <i>RPL8</i> (<i>ribosomal protein L8</i>), X64562; <i>TUA (α-tubulin</i>), AC122540; <i>CYP</i> (<i>cyclophilin</i>), AK326854; <i>TAF6</i> (<i>TFIID subunit 6</i>), Solyc10g006100.2.1.</p

<i>In planta</i> activity of AvrBs3 N- and C-terminal deletion mutants.

<p>AvrBs3 and deletion derivatives were expressed in <i>N</i>. <i>benthamiana</i> leaves as 4× c-Myc fusions under control of the <i>35S</i> promoter by <i>Agrobacterium</i>-mediated transformation. (<b>A</b>) N-terminal deletion constructs are schematically shown on the left and are named by the number of deleted amino acids. (<b>B</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 <i>N</i>. <i>benthamiana</i> leaves three days after <i>Agrobacterium</i>-mediated co-delivery of the reporter construct (<i>UPA20</i> box-minimal p<i>Bs4</i> driving a promoterless <i>uidA</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120214#pone.0120214.ref048" target="_blank">48</a>]), the silencing inhibitor <i>p19</i>, and <i>avrBs3</i>, <i>avrBs3</i>-derivatives or <i>gfp</i> (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 (<i>t</i>-test, <i>P</i> < 0.05). The right column shows the HR induction by AvrBs3 and deletion derivatives in leaves of resistant pepper (ECW-30R) and <i>Bs3</i>-transgenic <i>N</i>. <i>benthamiana</i> plants three days after <i>Agrobacterium</i>-mediated delivery of the <i>avrBs3</i> or derivative constructs. The experiments were repeated three times with similar results.</p

The AvrBs3 cysteine mutant is monomeric and binds specifically to DNA <i>in vitro</i>.

<p>(<b>A</b>) 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>B</b>) EMSA. 333 fmol biotin-labeled 36-bp DNA-fragments derived from the <i>UPA20</i> 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, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120214#pone.0120214.ref008" target="_blank">8</a>]) were used as competitor DNA. The experiments were repeated at least twice with similar results.</p

Analysis of the imperfect leucine zipper motif in AvrBs3.

<p>(<b>A</b>) 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>(B)</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. <b>(C)</b><i>UPA20</i> box activation by AvrBs3 and AvrBs3 mutant derivatives shown in B. GUS activities were determined in leaves of <i>N</i>. <i>benthamiana</i> three days after <i>Agrobacterium</i>-mediated co-delivery of the reporter construct (<i>UPA20</i> box-minimal p<i>Bs4</i> driving a promoterless <i>uidA</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120214#pone.0120214.ref048" target="_blank">48</a>]), together with silencing inhibitor <i>p19</i> and <i>gfp</i>, <i>avrBs3</i> or <i>avrBs3</i> 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 (<i>t</i>-test, <i>P</i> < 0.05). <b>(D)</b> HR induction by AvrBs3 and mutant derivatives in <i>Bs3</i> ECW-30R plants. <i>Xcv</i> 85–10 containing pGGX1 (empty vector, ev) or pGGX1 driving expression of <i>avrBs3</i>, <i>avrBs3-</i>LZm1 and <i>avrBs3-</i>LZm2, respectively, were inoculated into leaves of <i>Bs3</i> pepper plants (ECW-30R). Leaves were harvested three dpi and bleached with ethanol to better visualize the HR. <b>(E)</b><i>Bs3</i> gene induction by AvrBs3 and mutant derivatives in leaves of pepper ECW-30R. RT-PCR analysis 10 h after inoculation of the <i>Xcv</i> strains described in D. <i>EF1α</i> was used as control for equal cDNA amounts. RT-PCR was repeated once, the other experiments at least twice with similar results.</p

AvrBs3 complex formation interferes with DNA binding.

<p>(<b>A</b>) 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>B</b>) AvrBs3 binds to DNA as a monomer. Electromobility shift assay (EMSA) using 333 fmol biotin-labeled 36-bp DNA-fragments derived from the <i>UPA20</i> 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, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120214#pone.0120214.ref008" target="_blank">8</a>]) were used as competitor DNA. The experiments were repeated at least once with similar results.</p

Expression stability of candidate reference genes in <i>Xcv</i>-infected and mock-treated pepper plants evaluated by NormFinder.

<p>Ranking of <i>C</i>. <i>annuum</i> reference genes based on expression stability calculated by NormFinder (n = 48). New reference gene candidates are indicated in bold. The cut-off value for reliable reference genes is indicated by a dashed line. Sample groups were defined based on (a) treatment [MgCl₂, <i>Xcv</i> 85–10, 85–10Δ<i>hrcN</i> and 85-10(p<i>avrBs3</i>)] and (b) time-points of harvesting (0, 6, 10 and 24 hpi).</p

Expression stability of candidate reference genes in <i>Xcv</i>-infected and mock-treated tomato plants evaluated by geNorm.

<p>Tomato reference genes were ranked based on expression stability calculated by geNorm. New reference gene candidates are indicated in bold. <i>M</i> values represent the average expression stability of each gene (n = 48). The cut-off value for reliable reference genes is indicated by a dashed line.</p