MtNFP and MtLYK3, or AtCERK1 (co-)production in <i>Nicotiana</i> leaves induces defence-like responses.

<p>A, Kinetics of cell death development in <i>Nicotiana</i>. <i>Agrobacterium</i> transformants carrying either <i>MtNFP-3xFLAG</i> or <i>MtLYK3-3xFLAG</i> construct were co-infiltrated into <i>Nicotiana</i> leaves at five different time points (1–5). Macroscopic observation (left panel) and subsequent Evans blue staining (right panel) are depicted 42 hai (region 1), 39 hai (region 2), 36 hai (region 3), 33 hai (region 4) and 30 hai (region 5). Mock infiltration (region 6) was done concomitantly with the infiltration of region 1. Bar is 1 cm. B, Changes in leaf autofluorescence upon MtNFP and MtLYK3 co-production. Leaf regions co-producing MtNFP-3xFLAG and MtLYK3-3xFLAG fusions were analyzed between 24 and 48 hai (here depicted 36 hai) using a stereoscope. Note the decrease in chlorophyll content, as indicated by the decrease of far-red autofluorescence of chlorophyll (left panel), and enhanced accumulation of blue light-excited autofluorescence (right panel) within the infiltrated region. Bar is 1 cm. C, Accumulation of phenolic compounds. The following fusions were (co-)produced in <i>Nicotiana</i> leaves: MtNFP-3xFLAG (1); MtLYK3-3xFLAG (2); MtNFP-3xFLAG+MtLYK3-3xFLAG (3); MtNFP-3xFLAG+MtLYK3[G334E]-3xFLAG (4); AtCERK1-3xFLAG (5); or AtCERK1[K349]-3xFLAG (6). Macroscopic observations (left panel) and subsequent UV-excited autofluorescence of ethanol/lactophenol-cleared (right panel) leaf regions are depicted 36 hai (except for 5–30 hai). Bars are 1 cm. D, Induction of <i>NbHIN1, NbPR1 basic</i>, <i>NbACRE31</i>, and <i>NbACRE132</i> expression in response to separate production or co-production of: MtNFP-3xFLAG (NFP), MtLYK3-3xFLAG (LYK3), MtLYK3[G334E]<i>-</i>3xFLAG (LYK3[G334E]), and AtCERK1-3xFLAG (CERK1). Leaf samples were collected 24 hai and induction of gene expression was analyzed using qRT-PCR. Histograms represent induction of <i>NbHIN1</i> (white columns), <i>NbPR1 basic</i> (grey columns), <i>NbACRE31</i> (hatched columns), and <i>NbACRE132</i> (black columns) normalized by one reference gene, <i>MtEF1 α</i>. Induction of each gene was normalized to that caused by mock infiltration, and then calculated as % induction relative to the induction observed upon co-production of MtNFP and MtLYK3 fusions. Bars represent standard deviation of the mean. At least two technical replicates from two biological replicates were analyzed.</p

Cell death induction activity of MtNFP-sYFP2 truncated/mutated variants in <i>Nicotiana</i> leaves.

*<p>-see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065055#pone.0065055-Lefebvre1" target="_blank">[22]</a>, PM-plasma membrane, ER-endoplasmic reticulum.</p><p>The designated constructs were expressed alone or co-expressed with <i>MtLYK3-mCherry</i> construct in <i>Nicotiana</i> leaves. Macroscopic symptoms of cell death were scored 48 hai: only infiltrations that resulted in confluent death of (nearly) the entire infiltrated region were scored and are presented as a fraction of total infiltrations performed. # - In the 3 remaining leaf regions, co-expression of <i>MtNFP</i>[S67A]-<i>sYFP2</i> and <i>MtLYK3-mCherry</i> constructs resulted in increased staining with Evans blue in the entire infiltrated region.</p

Cell death induction upon (co-)expression of various RLK-encoding genes in <i>Nicotiana</i> leaves.

<p># − unless stated differently: with <i>-3xFLAG</i> (*) untagged (**), or <i>−YFP</i>_N (***) tagged construct.</p><p>Indicated constructs were expressed alone or co-expressed with either <i>MtNFP</i> or <i>MtLYK3</i> in <i>Nicotiana</i> leaves, and the infiltrated regions were marked. Macroscopic symptoms of cell death were scored 48 hai: only infiltrations that resulted in confluent death of (nearly) the entire infiltrated region were scored and are presented as a fraction of total infiltrations performed.</p

Co-production of MtNFP and MtLYK3 induces cell death in <i>Nicotiana</i> leaves.

<p>A, The following <i>MtNFP</i> and <i>MtLYK3</i> constructs were expressed alone or co-expressed in <i>Nicotiana</i> leaves: mock infiltration (1); <i>MtNFP</i> untagged+<i>MtLYK3</i> untagged (2); <i>MtNFP-sYFP2</i>+<i>MtLYK3-sYFP2</i> (3); <i>MtLYK3-sYFP2</i> (4); <i>MtLYK3</i> untagged (5); <i>MtNFP-sYFP2</i> (6); <i>MtNFP</i> untagged (7). Macroscopic observation (left panel) and subsequent Evans blue staining (right panel) are depicted 48 hai. Bar is 1 cm. B, <i>MtDMI2-sYFP2</i> construct was expressed alone or co-expressed with either <i>MtNFP-mCherry</i> or <i>MtLYK3-mCherry</i> construct in <i>Nicotiana</i> leaves. Macroscopic observations (left panel) and subsequent Evans blue stainings (right panel) are depicted 48 hai. Bars are 1 cm.</p

Cell death upon MtNFP and MtLYK3 co-production in <i>Nicotiana</i> leaves does not require <i>Sm</i>NF.

<p><i>Agrobacterium</i> transformants carrying either <i>MtNFP-3xFLAG</i> or <i>MtLYK3-3xFLAG</i> construct were co-infiltrated into <i>Nicotiana</i> leaves at a final concentration: OD₆₀₀ [<i>MtNFP</i>] = 0.25 and OD₆₀₀ [<i>MtLYK3</i>] = 0.4 (1); OD₆₀₀ [<i>MtNFP</i>] = 0.15 and OD₆₀₀ [<i>MtLYK3</i>] = 0.25 (2). Twelve hai parts of the transformed regions were syringe-infiltrated with 10⁻⁷mM <i>Sm</i>NF (circled in red) or DMSO diluted to the same concentration (circled in white). Macroscopic observation (left panel) and Evans blue staining (right panel) are depicted 33 hai. Bar is 1 cm.</p

Lanthanum chloride delays the cell death development upon MtNFP and MtLYK3, or AtCERK1 (co-)production.

<p><i>Agrobacterium</i> transformants carrying the following constructs were (co-)infiltrated at a final concentration: OD₆₀₀ [<i>MtNFP-3xFLAG</i>] = 0.125 and OD₆₀₀ [<i>MtLYK3-3xFLAG</i>] = 0.2 (A, B); OD₆₀₀ [<i>AtCERK1-3xFLAG</i>] = 0.2 (C, D). Twelve hai parts of the infiltrated regions were syringe-infiltrated with 5 mM lanthanum chloride (circled in red) or water (circled in white). Macroscopic observations (left panel) and subsequent Evans blue stainings (right panel) are depicted 42 hai for leaf regions co-producing MtNFP and MtLYK3 fusions (A, B), and 33 hai for leaf regions producing AtCERK1 fusion (C, D). Cell death development was scored 42 hai (A, B) or 33 hai (C, D): only infiltrations that showed the lack of tissue collapse and no compromised membrane permeability in the lanthanum chloride- or water-treated region were scored and are presented (right panel) as a fraction of total infiltrations performed. Bars are 1 cm.</p

Cell death induction activity of MtLYK3-sYFP2 mutated variants in <i>Nicotiana</i> leaves.

*<p>-see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065055#pone.0065055-KlausHeisen1" target="_blank">[20]</a>, except for the T480A (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065055#pone.0065055.s002" target="_blank">Fig. S2</a>), **-see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065055#pone.0065055-KlausHeisen1" target="_blank">[20]</a>, except for the P87S <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065055#pone.0065055-Smit1" target="_blank">[12]</a>, K464A and T480A (this study; number of plants nodulated/number of plants tested).</p><p>The designated constructs were expressed alone or co-expressed with <i>MtNFP-mCherry</i> construct in <i>Nicotiana</i> leaves. Macroscopic symptoms of cell death were scored 48 hai: only infiltrations that resulted in confluent death of (nearly) the entire infiltrated region were scored and are presented as a fraction of total infiltrations performed. # - despite the lack of pronounced macroscopic symptoms, the co-expression of <i>MtLYK3</i>[K464A]-<i>sYFP2</i> and <i>MtNFP-mCherry</i> constructs resulted in increased staining with Evans blue in the entire infiltrated region.</p

Production of AtCERK1 in <i>Nicotiana</i> leaves induces cell death that requires AtCERK1 kinase activity.

<p><i>AtCERK1-sYFP2</i> (A) and <i>AtCERK1</i>[K349E]-<i>sYFP2</i> (B) constructs were expressed in <i>Nicotiana</i> leaves. Macroscopic observations (left panel) and subsequent Evans blue stainings (right panel) are depicted 36 hai. Macroscopic symptoms of cell death were scored 36 hai: only infiltrations that resulted in confluent death of (nearly) the entire infiltrated region were scored and are presented (right panel) as a fraction of total infiltrations performed. Bars are 1 cm.</p

MLA10 CC-NB or full-length trigger cell death signaling in the cytoplasm.

<p>(<b>A</b>) Analysis of cell death inducing activity of MLA10 CC-NB fusion proteins. MLA10 CC-NB fusion proteins, CC-NB-YFP, CC-NB-YFP-NES and CC-NB-YFP-NLS, were expressed in <i>N. benthamiana</i> leaves by Agro-infiltration; confocal images were taken at ∼22 hpi (upper panel) and cell-death triggered by each fusion protein was scored by trypan blue staining at ∼48 hpi (lower panel). Scale bar is 50 µm. (<b>B</b>) Analysis of cell death phenotype upon expression of MLA10 CC-NB-YFP-GR or co-expression of MLA10 CC-NB-YFP-NLS and CC-NB-YFP-GR fusions before and after Dex treatment. The indicated fusion(s) were expressed in <i>N. benthamiana</i> leaves by Agro-infiltration. Buffer with/or without Dex was sprayed onto <i>N. benthamiana</i> leafs 20 hpi before the confocal images were taken (upper panel). The cell-death phenotype of each fusion protein was scored by trypan blue staining at ∼48 hrs post treatment with/or without Dex (lower panel). GR: Steroid binding domain of the mammalian glucocorticoid receptor. Scale bar is 50 µm. (<b>C</b>) Analysis of cell death activity of GR fusions of MLA10 WT and autoactive variants. Indicated MLA10-GR fusion, and the C-terminal YFP-GR fusions of MLA10 WT and two autoactive mutant variants were expressed in <i>N. benthamiana</i> leaves by Agro-infiltration. The cell-death phenotype was revealed by trypan blue staining at ∼24 hrs post infiltration without Dex induction.</p

Manipulation of MLA10 subcellular localization and the relevance to MLA10 cell death signaling and disease resistance.

<p>(<b>A</b>) Confocal images of barley leaf epidermal cells expressing MLA10 fusion proteins. Indicated fusion proteins were expressed in barley leaf epidermal cells upon biolistic delivery, the confocal images were taken at 36 hrs post bombardment. Upper panel: A representative barley cell coexpressing MLA10-YFP-NLS and a nucleus marker CFP-WRKY2 (2D <i>z</i> plane). Lower panel: a cell expressing MLA10-YFP-nls alone (2D <i>z</i> plane). NLS: nuclear localization signal; nls: mutated nuclear localization signal. Arrowheads mark the nucleus and scale bar is 50 µm. (<b>B</b>) Analyses of disease resistance activity of MLA10 fusions or mutant variants. Relative single cell resistance/susceptibility is shown by fungal haustorium index upon biolistic delivery of plasmids expressing indicated protein and a GUS reporter into the barley leaf epidermal cells of a susceptible barley line (Golden promise). Bombarded leaves were inoculated with <i>B. graminis</i> fungal spores expressing AVR_A10, and the fungal haustorium index was microscopically scored at 36 hrs post spore inoculation. Histogram bar represents average of three independent experiments and error bar represents SD. In the case of MLA10(F99E) expression, the number of cells expressing GUS reporter were extremely low. (<b>C</b>) Analysis of cell death triggering activity of MLA10 fusion proteins. Indicated MLA10 fusion proteins were expressed in <i>N. benthamiana</i> leaves by Agro-infiltration, and cell-death triggered by each fusion protein was scored by trypan blue staining at 40 hpi. NES: nuclear exclusion signal; nes: mutated nuclear exclusion signal. (<b>D</b>) Protein expression of indicated MLA10 fusions. Proteins were extracted at 23 hpi and MLA was detected by immunoblotting using an anti-MLA27 monoclonal antibody. Asterisk indicates non-specific signals. (<b>E</b>) Quantification of cell-death inducing activity of MLA10 fusion proteins. Upon expression of indicated MLA10 fusion proteins by Agro-infiltration in <i>N. benthamiana</i>, ion leakage was measured each hour from 23 to 34 hpi. Error bars (SE) were calculated from three replicates per time point and construct. Experiment was done at least twice with similar result. Letters (a–c) represent groups with significant differences [<i>p</i><0.05, Tukey's honest significant difference (HSD) test].</p