<i>Sl</i>FLS2 co-immunoprecipitates with <i>Sl</i>SERK3A and <i>Sl</i>SERK3B.

<p><i>Nicotiana benthamiana</i> leaves transiently expressing <i>Sl</i>SERK3A-HA or <i>Sl</i>SERK3B-HA constructs and <i>Sl</i>FLS2-GFP were elicited (+) or not (−) with 100 nM flg22 for 5 min. Total proteins (input) were subjected to reciprocal immunoprecipitation and immunoblotting. Immunoprecipitation with anti-GFP Protein A agarose beads (upper panel) or anti-HA (middle panel). This experiment was repeated once with similar results. WB: Western blot.</p

<i>Sl</i>SERK3A and <i>Sl</i>SERK3B are active protein kinases.

<p>Auto-phosphorylation and <i>trans</i>-phosphorylation of MBP were tested <i>in vitro</i> using freshly expressed and purified GST-tagged fusion proteins corresponding to the cytoplasmic domain of both <i>Sl</i>SERK3A and <i>Sl</i>SERK3B and their respective kinase dead mutants, <i>Sl</i>SERK3A* CD (D418N) and <i>Sl</i>SERK3B* CD (D420N). Proteins were fractionated on 12% SDS-PAGE. Coomassie blue stained and dried gel, lower panel; radiolabeled bands were revealed by autoradiography, upper panel. This experiment was repeated twice.</p

<i>SlSERK3A</i> and <i>SlSERK3B</i> co-silenced plants are compromised in cell death control and BR sensitivity.

<p>(<b>A</b>) Transcript levels of VIGS-silenced genes were evaluated using qRT-PCR. Tomato cv. Moneymaker plants treated with TRV empty vector (TRV), TRV-<i>SlSERK3A</i>, TRV-<i>SlSERK3B</i> or TRV-<i>SlSERK3AB</i> were evaluated. Expression was normalized against <i>UBI3</i>. Two independent samples were analyzed per construct. Values are average ± SE of three technical replicates. *<i>P</i><0.05 significant difference from TRV (two-sample <i>t</i>-test). Experiment was repeated three times with similar results. (<b>B</b>) Phenotype of individually silenced <i>SlSERK3A</i>, <i>SlSERK3B</i> and co-silenced plants. (<b>C</b>) Cell death lesions in <i>SlSERK3A</i> and <i>SlSERK3B</i> co-silenced tomato leaflets. (<b>D</b>) Defense and senescence-related <i>SlPRIa</i>, <i>SlPR2</i>, <i>SlPR5</i>, and <i>SlACS2</i> gene regulation in <i>SlSERK3A or SlSERK3B</i> silenced and co-silenced plants. Transcript levels were evaluated using qRT-PCR normalized against <i>SlUBI3</i>. Values are average ± SE (n = 3). * indicates significance difference from TRV at <i>P</i><0.05 (two-sample t-test). Experiment was repeated twice with similar results. (<b>E</b>) Aniline blue-stained tomato leaf discs. Callose accumulation was detected near the edges of leaf patches showing cell-death in co-silenced <i>SlSERK3A</i> and <i>SlSERK3B</i> plants and TRV control. Leaves treated with 1 µM flg22 for 24 h were used as control. (<b>F</b>) Leaflets of tomato plants silenced for <i>SlSERK3A</i>, <i>SlSERK3B</i> or co-silenced and TRV control were treated with 10 µM BL for 12 h for <i>SlCPD</i> expression evaluation. Transcript levels were evaluated using qRT-PCR normalized against <i>SlUBI3</i>. Values are average ± SE (n = 3). *<i>P</i><0.05 and **<i>P</i><0.01 indicate significant difference from the respective –BL control (two-sample <i>t</i>-test). This experiment was repeated twice with similar results.</p

<i>SlSERK3A</i> and <i>SlSERK3B</i> partially complemented the Arabidopsis <i>bak1-4</i> null mutant.

<p>(<b>A</b>) <i>SlSERK3A and SlSERK3B</i> transcript levels in transgenic <i>bak1-4</i> plants expressing pBAK1-<i>SlSERK3A (bak1-4 SlSERK3A)</i> or pBAK1-<i>SlSERK3B (bak1-4 SlSERK3B)</i> were evaluated using qRT-PCR. Values are average ± SE (n = 3) normalized relative to <i>AtActin</i> and calibrated to expression of <i>BAK1</i> in Col-0. *<i>P</i><0.05 and ** <i>P</i><0.001 significant difference from Col-0 (two-sample <i>t</i>-test). (<b>B</b>) Relative root growth of 9-day-old seedlings grown on medium with or without 1 nM BL. Root length is presented relative to untreated control for each genotype. Values are average ± SE (n = 50). Values were arcsine transformed for statistical analysis. Letters above the graphs denote significance difference at <i>P</i><0.01 (ANOVA Tukey HSD test). This experiment was repeated twice with similar results. (<b>C</b>) Leaf discs were floated on water overnight. ROS burst was measured using a luminol-based assay within 25 min after 1 µM flg22 treatment. Values are average ± SE (n = 8). (<b>D</b>) A photo of representative short-day grown 4.5-week-old Arabidopsis plants of the indicated genotypes.</p

<i>Sl</i>SERK3A and <i>Sl</i>SERK3B co-localize with BAK1 at the plasma membrane (PM).

<p><i>Agrobacterium</i>-mediated transient expression of <i>Sl</i>SERK3A-GFP or <i>Sl</i>SERK3A-GFP with BAK1-mCherry in <i>Nicotiana benthamiana</i> leaves. Localization of PM-associated BAK1-mCherry was compared with that of <i>Sl</i>SERK3A and <i>Sl</i>SERK3B (merged). Differential interference contrast (DIC) image. Leaf epidermal cells were imaged by confocal microscopy 72 h after infiltration with <i>Agrobacterium</i>. Bar = 20 µm.</p

The Tomato Leucine-Rich Repeat Receptor-Like Kinases <i>SlSERK3A</i> and <i>SlSERK3B</i> Have Overlapping Functions in Bacterial and Nematode Innate Immunity

<div><p>The Somatic Embryogenesis Receptor Kinase 3 (SERK3)/Brassinosteroid (BR) Insensitive 1-Associated Kinase 1 (BAK1) is required for pattern-triggered immunity (PTI) in <i>Arabidopsis thaliana</i> and <i>Nicotiana benthamiana</i>. Tomato (<i>Solanum lycopersicum</i>) has three <i>Sl</i>SERK members. Two of them exhibit particularly high levels of sequence similarity to <i>At</i>SERK3 and, therefore, were named <i>Sl</i>SERK3A and <i>Sl</i>SERK3B. To characterize a role for <i>SlSERK3A</i> and <i>SlSERK3B</i> in defense, we suppressed each gene individually or co-silenced both using virus-induced gene silencing (VIGS) in the tomato cv. Moneymaker. Co-silencing <i>SlSERK3A</i> and <i>SlSERK3B</i> resulted in spontaneous necrotic lesions and reduced sensitivity to exogenous BR treatment. Silencing either <i>SlSERK3A</i> or <i>SlSERK3B</i> resulted in enhanced susceptibility to root knot-nematode and to non-pathogenic <i>Pseudomonas syringae</i> pv. <i>tomato (Pst)</i> DC3000 <i>hrcC</i> indicating that both <i>SlSERK3</i>s are positive regulators of defense. Interestingly, silencing <i>SlSERK3B</i>, but not <i>SlSERK3A</i>, resulted in enhanced susceptibility to the pathogenic strain <i>Pst</i> DC3000 indicating distinct roles for these two <i>SlSERK3</i> paralogs. <i>Sl</i>SERK3A and <i>Sl</i>SERK3B are active kinases, localized to the plasma membrane, and interact <i>in vivo</i> with the Flagellin Sensing 2 receptor in a flg22-dependent manner. Complementation of the <i>Atserk3/bak1-4</i> mutant with either <i>SlSERK3A</i> or <i>SlSERK3B</i> partially rescued the mutant phenotype. Thus, <i>SlSERK3A</i> and <i>SlSERK3B</i> are likely to constitute tomato orthologs of <i>BAK1</i>.</p></div

Silencing <i>ACS</i> genes in tomato does not compromise <i>Mi-1</i>-mediated resistance to root-knot nematodes.

<p>Two-week-old tomato plants cvs. Moneymaker (<i>mi/mi</i>) and Motelle (<i>Mi-1/Mi-1</i>) were used in agroinfiltration of tobacco rattle virus (TRV) empty vector, and cv. Motelle was used with TRV containing a portion of <i>Mi-1</i> (TRV-Mi-1) or containing <i>1-aminocyclopropane-1-carboxylic acid synthase</i> (<i>ACS</i>) constructs (TRV-ACSI and TRV-ACSII), which were either individually or simultaneously agroinfiltrated (TRV-ACSI+II). Three weeks after agroinfiltration, plants were infected with 10,000 second-stage juvenile root-knot-nematodes and evaluated for nematodes reproduction 8 weeks later. Dots represent the number of egg masses counted on a single root system (<i>n</i> = 18–25). Two independent experiments were performed with similar results and data from one are presented.</p

Root-knot nematodes (<i>Meloidogyne incognita</i>) induce the expression of ethylene biosynthetic genes in tomato.

<p><i>In vitro</i> grown seedlings of near isogenic tomato cvs. Moneymaker and Motelle were infected with 100–150 second-stage juvenile root-knot-nematodes in sterile conditions. The infected root tips were sampled at 0, 12, 24 and 36 h post infection (hpi). Expression of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase genes (<i>ACO</i>) and ACC synthase genes (<i>ACS</i>) was determined by semi-quantitative RT-PCR using gene-specific primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063281#pone.0063281.s002" target="_blank">Table S1</a>) in two biological replicates with similar results. PCR amplification from a single sample is presented for each time point and genotype. Amplification of the tomato ubiquitin <i>Ubi3</i> gene was used as internal control. PCR cycles are indicated on the right side of the panel.</p

Effect of MCP treatment on ethylene response and resistance to root-knot nematode in tomato roots.

<p>(<b>A</b>) Efficiency of the 1-methylcyclopropene (MCP)-blocking of ethylene (ET) perception was assessed by monitoring the expression of <i>E4</i> after induction by ET. Seven-week-old cv. Moneymaker plants (+MCP/+ET) were pre-treated with MCP, and two plants were treated daily for 18 h with 10 µl/l ET prior to harvest. Root tissues were pooled and frozen. Tissues from untreated plants (−MCP/−ET) or plants only induced by ET (−MCP/+ET) were used as control. Total RNA (25 µg) for each sample was used for RNA blot analysis. The blot was hybridized sequentially with <i>E4</i> and an 18S rDNA probe used to normalize expression. (<b>B</b>, <b>C</b>) Five-week-old tomato plants cvs. Moneymaker and Motelle were treated with MCP (+MCP) or untreated (−MCP) prior root-knot nematode (RKN) infection with 3,000 second-stage juvenile. During the first 2 weeks following RKN infection, the plants (+MCP) were repeatedly treated with MCP every 2 days. RKN reproduction was evaluated 7 weeks after infection as (<b>B</b>) egg masses and (<b>C</b>) egg production. Results are presented relative to the fresh weight (FW) of roots. Error bars indicate standard error of the mean (<i>n</i> = 16), where bars with different letters denote significant difference at <i>P</i><0.05. The bioassay was performed twice with both tomato cultivars tested and twice more with cv. Moneymaker only. In all experiments, results from the same genotypes were similar. Data from one representative experiment are presented.</p

Root-knot nematodes reproduction on tomato is affected by the <i>Nr</i> mutation in compatible host only.

<p>Root-knot nematodes (RKN) reproduction was evaluated on <i>Never ripe (Nr)</i> mutant, wild type tomato cvs. Pearson and VFN, and the <i>Nr</i> introgressed line VFNx<i>Nr</i>. Four-week-old plants were infected with 3,000 second-stage juvenile RKN. (<b>A</b>) Egg masses and (<b>B</b>) eggs production were evaluated 6 weeks after RKN infection. Results are presented relative to the fresh weight (FW) of roots. Error bars indicate standard error of the mean (<i>n</i> = 20–30), where bars with different letters denote significant difference at <i>P</i><0.05. Two independent experiments were performed with similar results and data from one are presented.</p