Journal: The EMBO Journal

Article Title: The barley MLA13-AVR A13 heterodimer reveals principles for immunoreceptor recognition of RNase-like powdery mildew effectors

doi: 10.1038/s44318-025-00373-9

Figure Lengend Snippet: ( A ) SEC profile of the N-terminal, GST-tagged MLA13 K98E/K100E in complex with C-terminal, Twin-Strep-HA-tagged AVR A13 -1 sample purified by a two-step affinity purification (Appendix Figure ). Elution of the heterodimer (~155 kDa) is indicated by the peak at ~15.5 mL. Inset CBB-stained, SDS-PAGE gel presents fractions highlighted with alternating shades in the SEC profile. Samples run on a 12% gel. ( B ) Representative negative staining TEM image of the peak elution volume (~15.5 mL) from ( A ) diluted fivefold. Scale bar represents 100 nm. ( C ) Three orientations of the Coulomb potential map (above), atomic model (middle) and domain architecture (below) of the heterodimer. Arrows indicate the approximate locations of the MLA13 K98E/K100E substitutions introduced to impede in planta cell death. The workflow of cryo-EM data processing is presented as Fig. . .

Article Snippet: AVR a13 -1 (codon altered for expression in N. benthamiana ) , GeneArt (Thermo Fisher Scientific Inc.) , Not applicable.

Techniques: Purification, Affinity Purification, Staining, SDS Page, Negative Staining, Cryo-EM Sample Prep

Journal: The EMBO Journal

Article Title: The barley MLA13-AVR A13 heterodimer reveals principles for immunoreceptor recognition of RNase-like powdery mildew effectors

doi: 10.1038/s44318-025-00373-9

Figure Lengend Snippet: Elution volumes of the Sr35 resistosome, Sr50 resistosome and MLA13 K98E/K100E -AVR A13 -1 heterodimer are shown with a dotted line when purified using the same method (Appendix Figs. , and main Fig. , respectively). ( A ) MLA13 K98E/K100E -2S-HA expressed and purified alone. ( B ) MLA13 K98E/K100E/D502V -2S-HA expressed and purified alone. ( C ) MLA13 K98E/K100E -GST expressed with AVR A13 -2S-HA. ( D ) MLA13 K98E/K100E expressed with AVR A13 -2S-HA. ( E ) MLA13 L11E/L15E expressed with AVR A13 -2S-HA. All samples were purified from 100 g of leaf tissue and with a single-step affinity purification via the Twin-Strep-tag® (2S-HA), followed by SEC. Fractions from the SEC profiles are numbered and presented on the accompanying CBB-stained, 10 or 12% SDS-PAGE gels. .

Article Snippet: AVR a13 -1 (codon altered for expression in N. benthamiana ) , GeneArt (Thermo Fisher Scientific Inc.) , Not applicable.

Techniques: Purification, Affinity Purification, Strep-tag, Staining, SDS Page

Journal: The EMBO Journal

Article Title: The barley MLA13-AVR A13 heterodimer reveals principles for immunoreceptor recognition of RNase-like powdery mildew effectors

doi: 10.1038/s44318-025-00373-9

Figure Lengend Snippet: ( A ) Structural alignment of the CC domains of ZAR1–RKS1 (light blue; PDB: 6J5W), ZAR1–RKS1-PBL2 UMP (blue; PDB: 6J5V) and ZAR1 resistosome (dark blue; PDB: 6J5T) to the CC domain of the MLA13 K98E/K100E –AVR A13 -1 heterodimer (beige). ( B ) Structural alignment of ZAR1–RKS1 (light blue; PDB: 6J5W), ZAR1–RKS1-PBL2 UMP (blue; PDB: 6J5V) and ZAR1 resistosome (dark blue; PDB: 6J5T) to the MLA13 K98E/K100E –AVR A13 -1 heterodimer. Only the MLA13 NBD and LRR, AVR A13 -1 and NBDs of ZAR1 are shown. The red–yellow–red traces illustrate the major mode of conformational heterogeneity observed for the MLA13 NBD (average position shown in pink). ( C ) Top five models for the MLA13 K98E/K100E –AVR A13 -1 complex as predicted by AlphaFold 3 using five independent runs with different seeds. All five models were aligned to the MLA13 experimental atomic model and predicted MLA13 models are coloured by their RMSD deviation to the experimental model. Predicted parts of MLA13 not present in the experimental model and AVR A13 -1 model are coloured grey. The experimentally observed Coulomb potential map is shown in transparent grey. For all models, the position of the NBD does not align with the experimental model. In three predicted models the fourth helix of the CC bundle is too far elongated. ( D ) AlphaFold 3 predicts multiple orientations of AVR A13 -1 (coloured rainbow) that are all incorrectly rotated compared to the experimentally observed position (pink). The MLA13–AVR A13 -1 experimental Coulomb density map and model are shown in transparent grey and grey, respectively. For an overview of AlphaFold3 quality scores see Appendix Fig. .

Article Snippet: AVR a13 -1 (codon altered for expression in N. benthamiana ) , GeneArt (Thermo Fisher Scientific Inc.) , Not applicable.

Techniques:

Journal: The EMBO Journal

Article Title: The barley MLA13-AVR A13 heterodimer reveals principles for immunoreceptor recognition of RNase-like powdery mildew effectors

doi: 10.1038/s44318-025-00373-9

Figure Lengend Snippet: ( A ) Cryo-EM-derived atomic model of AVR A13 -1 with side chains shown for residues predicted to contribute to the interface with MLA13 and experimentally tested for loss of MLA13-mediated cell death. ( B ) Co-expression of MLA13 with AVR A13 -1 substitution mutants in barley protoplasts. Luminescence is normalised to EV + MLA13 ( = 1). High relative luminescence suggests low cell death response. Six data points represent two technical replicates performed with three independently prepared protoplast samples. Treatments labelled with different letters differ significantly ( P < 0.05) according to the Dunn’s test ( P values are included as Source Data for Fig. 3). In the box plot, the top, middle, and bottom horizontal lines of the box correspond to the upper quartile, the median, and the lower quartile, respectively. The whiskers extend to the smallest and largest data points within 1.5 times the interquartile range from Q1 and Q3. Any points outside this range are plotted as dots and considered outliers. ( C ) Agrobacterium -mediated co-expression of MLA13 with AVR A13 -1 interface substitution mutants in leaves of N. benthamiana . Three independent replicates were performed with two Agrobacterium transformations and plant batches (Source Data for Fig. 3). ( D ) Western blot analysis of AVR A13 -1 substitution mutants. Samples were run on a 10% gel. .

Article Snippet: AVR a13 -1 (codon altered for expression in N. benthamiana ) , GeneArt (Thermo Fisher Scientific Inc.) , Not applicable.

Techniques: Cryo-EM Sample Prep, Derivative Assay, Expressing, Western Blot

Journal: The EMBO Journal

Article Title: The barley MLA13-AVR A13 heterodimer reveals principles for immunoreceptor recognition of RNase-like powdery mildew effectors

doi: 10.1038/s44318-025-00373-9

Figure Lengend Snippet: ( A ) The MLA13–AVR A13 -1 interface exhibiting MLA13 residues that were experimentally shown to contribute to AVR A13 -1-mediated cell death. ( B ) Co-expression of AVR A13 -1 with MLA13 substitution mutants in barley protoplasts. Each MLA13 variant was normalised to its own autoactivity; luminescence is normalised to EV + MLA13 variant (= 1). High relative luminescence suggests low cell death response. The four data points represent two technical replicates performed with two independently prepared protoplast samples. Treatments labelled with different letters differ significantly ( P < 0.05) according to the Dunn’s test ( P values are included as Source Data for Fig. 4). In the box plot, the top, middle, and bottom horizontal lines of the box correspond to the upper quartile, the median, and the lower quartile, respectively. The whiskers extend to the smallest and largest data points within 1.5 times the interquartile range from Q1 and Q3. Any points outside this range are plotted as dots and considered outliers. ( C ) Agrobacterium -mediated co-expression of AVR A13 -1 with MLA13 substitution mutants believed to contribute to MLA13 interface and cell death response in leaves of N. benthamiana . Three independent replicates were performed with two Agrobacterium transformations and plant batches (Source Data for Fig. 4). ( D ) Western blot analysis of MLA13 substitution mutants. Samples were run on a 12% gel. .

Article Snippet: AVR a13 -1 (codon altered for expression in N. benthamiana ) , GeneArt (Thermo Fisher Scientific Inc.) , Not applicable.

Techniques: Expressing, Variant Assay, Western Blot

Journal: The EMBO Journal

Article Title: The barley MLA13-AVR A13 heterodimer reveals principles for immunoreceptor recognition of RNase-like powdery mildew effectors

doi: 10.1038/s44318-025-00373-9

Figure Lengend Snippet: ( A ) Co-expression of MLA7 and MLA7 L902S with AVR A7 and AVR A13 variants in barley protoplasts. Luminescence is normalised to EV + MLA7 ( = 1) or EV + MLA7 L902S ( = 1). High relative luminescence suggests low cell death response. The six data points represent two technical replicates performed with three independently prepared protoplast samples. Treatments labelled with an asterisk differ significantly ( P < 0.05) according to the Welch two-sample t test. The P values for AVR a22 , AVR a7 -1 , AVR a7 -2 , AVR a13 -1 and AVR a13 -V2 are 0.682, 0.059, 0.386, 0.002 and 0.010, respectively. In the box plot, the top, middle, and bottom horizontal lines of the box correspond to the upper quartile, the median, and the lower quartile, respectively. The whiskers extend to the smallest and largest data points within 1.5 times the interquartile range from Q1 and Q3. Any points outside this range are plotted as dots and considered outliers. ( B ) Agrobacterium -mediated co-expression of MLA7 and MLA7 L902S with AVR A7 and AVR A13 variants in N. benthamiana leaves. Three independent replicates were performed (Source Data for Fig. 5). ( C ) Western blot analysis of the effector variants tested in ( B ). ( D ) Western blot analysis of MLA7 and MLA7 L902S . Samples were run on a 12% SDS-PAGE gel. .

Article Snippet: AVR a13 -1 (codon altered for expression in N. benthamiana ) , GeneArt (Thermo Fisher Scientific Inc.) , Not applicable.

Techniques: Expressing, Western Blot, SDS Page

Journal: The EMBO Journal

Article Title: The barley MLA13-AVR A13 heterodimer reveals principles for immunoreceptor recognition of RNase-like powdery mildew effectors

doi: 10.1038/s44318-025-00373-9

Figure Lengend Snippet: ( A ) Structural alignment of AVR A13 -1 (dark goldenrod colour) and crystal structure of AVR A7 -1 (burgundy colour; PDB: 8OXL). The basal loops of AVR A13 -1 are coloured in red. ( B ) Sequence alignment of AVR A13 and AVR A7 variants. Alignment performed using MUSCLE and visualised using ESPript 3.0.

Article Snippet: AVR a13 -1 (codon altered for expression in N. benthamiana ) , GeneArt (Thermo Fisher Scientific Inc.) , Not applicable.

Techniques: Sequencing