The leucine-rich repeats in allelic barley MLA immune receptors define specificity towards sequence-unrelated powdery mildew avirulence effectors with a predicted common RNase-like fold
Fig 2
Mla6 and AVRa6 co-expression in barley protoplasts and N. benthamiana causes a specific cell death response.
(A) Barley cv. Golden Promise protoplasts were transfected with pIPKb002 vectors containing cDNAs of Mla6 or Mla1 and either an empty vector (EV), AVRa6, AVRa6-V2, AVRa1, AVRa1-V1, or AVRa1-V2 variants lacking their respective signal peptides together with a pUBI:Luciferase construct. The LUC activity relative to the EV sample was measured as a proxy for cell death 16 h post transfection. Box plot diagrams show median of the relative LUC activity of six independent transfections, which are represented by dots, while the box shows the interquartile range. Significant differences between samples were analyzed using non-parametric Kruskal-Wallis (KW) analysis followed by a Dunn’s test. Calculated KW p-values are as follows: Mla6: p = 0.007146; Mla1: p = 0.0007392. Samples labeled with identical letters did not differ significantly (p < 0.05) in the Dunn’s test for the corresponding Mla variant. (B) cDNAs of clade 1 AVRa6 family members BLGH_00698, BLGH_00697, BLGH_00700, AVRa6, and AVRa1 variants were expressed without their respective signal peptides and stop codons and with a C-terminal mYFP fusion under the control of a 35S promotor in N. benthamiana. The effectors were co-expressed with Mla1 and Mla6 cDNAs fused C-terminally with a 4xmyc tag under the control of a 35S promotor. Cell death was scored five days post infiltration and Figures show a representative of at least 15 co-transformations. (C) Protein levels of AVRA1-mYFP, AVRA6-mYFP, AVRA6-V2-mYFP, BLGH_00698-mYFP, BLGH_00697-mYFP and BLGH_00700-mYFP. Samples for total protein extraction were harvested two days post infiltration. mYFP fusion proteins were enriched by an GFP-Trap. Proteins were separated using 10% or 12% polyacrylamide gels and proteins were detected using α-GFP and α-myc western blotting (WB). IP = immunoprecipitation. CBB = Coomassie Brilliant Blue.