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].