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 7
The LRR domain of MLA10 accounts for the specific interaction with AVRA10 in planta and in yeast.
(A) Nicotiana benthamiana leaves were transformed transiently with vectors containing cDNAs of Mla10-cLUC, Mla10Lrr22-cLUC or Mla22Lrr10-cLUC together with vectors containing cDNAs of AVRa10-nLUC or AVRa10-V/ AVRa22-V nLUC lacking signal peptides (SPs), under the control of a 35S promoter. LUC activity was determined forty hours after transfection. The experiment was performed on at least three independent days with two to four replicates (independent set of plants) each day. Significant differences between AVRa10-nLUC or AVRa10-V/ AVRa22-V nLUC were analyzed using one-way Kruskal-Wallis (KW) analysis. Calculated KW p-values are as follows: Mla10: p = 0.0001491; Mla22Lrr10: p = 0.009035; Mla10Lrr22: p = 0.8079. Samples labeled with different letters differed significantly (p < 0.05). (B) Protein levels of MLA10-cLUC, MLA22LRR10-cLUC and MLA10LRR22-cLUC in N. benthamiana leaf extracts. Proteins were separated on a 8% SDS-PAGE gel and a detected using anti-LUC western blot (WB). (C) Yeast was co-transformed with cDNAs of N-terminal LexABD-fused MLA and N-terminal B42AD-fused AVRA variants. Growth on media lacking Leucine indicates association of respective proteins fused to AD (activation domain) and BD (Binding domain). (D) Protein levels of LexABD-MLA and B42AD-AVRA variants. Proteins were precipitated using an ammonium-acetate buffer and dissolved in a urea-SDS sample buffer before separation on a 10% or 12% polyacrylamide gel and detection by either α-LexA or α-HA WB.