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

  1. Aaron W. Lawson
  2. Andrea Flores-Ibarra
  3. Yu Cao
  4. Chunpeng An
  5. Ulla Neumann
  6. Monika Gunkel
  7. Isabel M. L. Saur
  8. Jijie Chai
  9. Elmar Behrmann
  10. Paul Schulze-Lefert

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Co-evolution between cereals and pathogenic grass powdery mildew fungi is exemplified by sequence diversification of an allelic series of barley resistance genes encoding Mildew Locus A (MLA) nucleotide-binding leucine-rich repeat (NLR) immunoreceptors with a N-terminal coiled-coil domain (CNLs). Each immunoreceptor recognises a matching, strain-specific powdery mildew effector encoded by an avirulence gene ( AVR a ) . We present here the cryo-EM structure of barley MLA13 in complex with its cognate effector AVR A13 -1. The effector adopts an RNase-like fold when bound to MLA13 in planta , similar to crystal structures of other RNase-like AVR A e ffectors purified from E. coli . AVR A13 -1 interacts via its basal loops with MLA13 C-terminal leucine rich repeats (LRRs) and the central winged helix domain (WHD). Co-expression of structure-guided MLA13 and AVR A13 -1 substitution variants show that the receptor–effector interface plays an essential role in mediating immunity-associated plant cell death. Furthermore, by combining structural information from the MLA13–AVR A13 -1 heterocomplex with sequence alignments of other MLA receptors, we designed a single amino acid substitution in MLA7 that enables expanded effector detection of AVR A13 -1 and the virulent variant AVR A13 -V2. In contrast to the pentameric conformation of previously reported effector-activated CNL resistosomes, MLA13 was purified and resolved as a stable heterodimer from an in planta expression system. Our study suggests that the MLA13–AVR A13 -1 heterodimer might represent a CNL output distinct from CNL resistosomes and highlights opportunities for the development of designer gain-of-function NLRs.

Article activity feed

  1. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/13285226.

    Summary

    This study explores the structural basis of pathogen recognition by the barley immune receptor MLA13 and its interaction with the effector protein AVRA13-1 from the barley powdery mildew fungus Blumeria hordei.

    Lawson et al. determined the structure of the MLA13 receptor bound to AVRA13-1 at a resolution of 3.8 Å, revealing a stable 1:1 heterodimeric complex between plant and pathogen protein. This enabled the identification of key amino acids involved in receptor-effector interactions. The authors conducted mutagenesis experiments to test the functional significance of these interactions using hypersensitive cell death assays in barley leaf protoplasts and N. benthamiana leaves, demonstrating how specific mutations affect immune signaling and cell death. Moreover, they leverage their structural understanding of the NLR-effector interaction interface to bioengineer AVRA13-1 and AVRA13-V2 recognition in MLA7, an MLA allele that normally does not recognise these effectors.

    The manuscript is comprehensive and presents a robust structural and functional framework for understanding the interaction between the barley immune receptor MLA13 and the fungal effector AVRA13-1. The cryo-EM data and subsequent mutagenesis experiments are well-executed, providing significant insights into the immune signaling mechanisms. However, there are several areas where clarity and depth could be improved. Below, we offer suggestions that may enhance the manuscript's readability and scientific rigor. We hope these comments are useful to the authors.

    Title

    We found the use of the term "heterodimer" in the title and throughout the manuscript confusing. Typically, NLR-effector complexes are not referred to as heterodimers. For example, Sr35 and Roq1 resistosomes, which include effectors in them, are referred to as pentamers and tetramers rather than heterodecamers or heterooctamers, respectively. Please clarify the rationale for this terminology.

    Abstract

    Lines 41-43: The statement "Our study suggests that the MLA13–AVRA13-1 heterodimer might represent a CNL output distinct from CNL resistosomes" is not clear. Are the authors implying that MLA13 may trigger cell death without forming a resistosome? This needs clearer articulation. Later on, they entertain the possibility that this is more likely to do with different CNLs potentially having different equilibria between the primed monomer and oligomeric resistosome states. That's a reasonable explanation, but the way this is phrased in the abstract is confusing as it could be intepreted as the manuscript featuring data on resistosome-independent signaling activity for the MLA13- AVRA13-1 complex.

    Results

    Line 136: The authors co-express MLA13 and AVRA13 in N. benthamiana using K98E/K100E mutations in MLA13 to abrogate cell death. It would be beneficial to discuss why mutations in the a1 helix, used previously for Sr35, NRC2, and NRC4, were not employed.

    The authors use the fact that MLA13K98E/K100E/D502V still triggers cell death as evidence for the fact that these mutants are not "generally disrupted in receptor-mediated signalling" but it may well be that the D502V mutant forms an overall more stable resistosome than the effector activated one. This is evidenced in the BN-PAGE experiments in Extended Data Fig. 6, where the D502V mutant of MLA13 can form a stable resistosome but the effector activated variant of MLA13 cannot. In the case of effector activated MLA13, the K98E and K100E may be leading to a de-stabilized resistosome.

    Related to this, can the authors also discuss why the D502V mutant is able to oligomerize in N. benthamiana? Why would potential co-factors be required for the effector activated complex (as mentioned in the discussion) but not for the autoactive mutant?

    The comparison between the MLA13 and Sr35 resistosome formation in TEM using different mutations (K98E/K100E for MLA13 and L11E/L15E for Sr35) needs justification. Although the authors include SEC data and BN-PAGE data for the MLA13L11E/L15E mutant showing that an oligomer does not form, negative stain TEM data for MLA13L11E/L15E purified under the Sr35 conditions would make the comparisons more robust.

    Related to this point, throughout the manuscript we found it hard to understand which variant of MLA13 was being used (which mutation, N or C-terminally tagged, etc.). As so many different variants of MLA13 are used throughout the manuscript, it should be clearer to the reader which version is being used in which experiment.

    Lines 147 and Lines 158: The MLA13L11E/L15E mutation is introduced at this point in the manuscript. A primary citation for where this mutation comes from is missing (Adachi et al. 2019, eLife).

     Line 177-180: The BN-PAGE data is an intriguing result that adds to this story. This is the first instance that an MLA allele has been shown to oligomerize in planta, and as such this is worth emphasizing in a main figure.

    Lines 180-183: "Collectively, this suggests that the heterodimeric MLA13-AVRA13-1 complex might represent an intermediate effector-activated CNL complex and that the equilibrium between heterodimeric and pentameric resistosomes may be differentially regulated among sensor CNLs."

    The main points of the paper are that somehow the MLA-AVRA13 complex has signaling capacities form and exhibits a different equilibrium between the primed intermediate and oligomerized form. This is primarily based on the negative finding that a higher-order resistosome was not detected for mutant variants of MLA13. This negative result could be due to a number of issues which are covered in the discussion, but still a lot of emphasis is being made on "a non-canonical conformation" (lines 321-322) or on "a CNL output distinct from CNL resistosomes" (lines 41-43). In general, we feel like the authors should be more upfront about the limitations of this study throughout the manuscript.

    Line 194: "Three independent MLA13-AVRA13-1 heterocomplex samples were prepared for cryo-EM analysis." Does the cryo-EM structure solved and presented in Fig. 1 correspond to the MLA13K98E/K100E mutant? This is not immediately clear from the way the results are written. Please specify which MLA13 variant was used to determine the structure. This is related to the comment made above.

    Additional comment:

    In a previous manuscript on the activity of AVRA13-V2 as a dominant negative allele that breaks down resistance, the authors showed that this virulent allele escapes MLA13 recognition by substituting a serine for a leucine at the C-terminus, and this resulted in enhanced MLA13 association. It would be nice to add a couple of sentences addressing where this residue locates in reference to the MLA13-AVRA13-1 binding interface. This residue would presumably be located outside of the basal loop of AVRA13-1 that is in contact with MLA13. It would be interesting to discuss how AVRA13-V2 might outcompete AVRA13-1 binding and act as a dominant negative allele based on the structure of the MLA13 heterodimer.

     Figures

    One general comment: As mentioned above, all main and supplementary figures would benefit from more specificity about which NLRs constructs are being used (N or C-term tag, which mutations are introduced, etc.).

    Figure 1: The figure is overall very good. Perhaps a close-up of the interface, highlighting important residues could be added?

    Figure 2: While the message of this figure is important, we found this figure difficult to understand at times. In panel B, the layout comparing the different ZAR1 conformations to MLA13 heterodimer is difficult to understand. Simplifying this comparison, potentially making it pairwise, or providing pairwise RMSD values could improve clarity. What is the overall conclusion? Is the MLA13-AVRA13 completely different to all ZAR1 states characterized to date?

    Regarding panel C, overall AlphaFold model quality metrics are missing which would help the readers interpret the results better. Moreover, for AlphaFold 3, good practice involves running protein complexes multiple times using different seeds each time and selecting higher confidence models from each round. It appears that in this case, the authors only selected models from one modelling round with 1 seed. We encourage the authors try this modelling again with several modelling rounds using several seeds as this may yield better predictions.

    Figure 3: We appreciate that it is obvious that these blots are spliced together from different images. However, we would appreciate a dotted line or a mention of this in the legend. Same comment applies to Figure 4 and Extended Data Figure 1.

    Figure 4: See comment made for Figure 3.

    Figure 5: While the results from the barley protoplasts are very convincing, the positive control used in the N. benthamiana cell death assays (MLA7+AVRA7) is not . The cell death for MLA7+AVRA7-1 looks indistinguishable from the cell death triggered by MLA7+AVRA22 (the negative control). This looks to be the case in most of the leaves shown in Supplementary Figure 4. Could the authors provide quantification or perhaps UV images? While we are convinced by the protoplast results, we feel like the statement "The same co-expression experiments were performed in leaves of N. benthamiana with qualitatively similar results" is not necessarily accurate as it looks like MLA7-AVRA7-1 does not seem to trigger robust cell death in N. benthamiana.

    Discussion

    The discussion is comprehensive but could be made more precise.

    Lines 321-322: The term "non-canonical conformation" should be clarified, especially since similar intermediate forms have been reported for other CC-NLRs like ZAR1. What makes this MLA13-AVRA13 conformation non-canonical?

    Lines 343-345: Clarify whether the authors are referring to three distinct states (effector-dependent, intermediate, and oligomeric) or two states (effector-dependent intermediate and oligomeric). This distinction is crucial for understanding the proposed model. Maybe a comma is missing.

    Lines 345-347: As mentioned above, address the presence of MLA13D502V oligomers in BN-PAGE despite the suggestion that additional components might be missing in N. benthamiana for detectable oligomer formation. Why does effector activation require co-factors but the MLA13D502V mutant does not?

    Discuss the possibility of transient effector binding in planta leading to less stable resistosomes, or the possibility that the L11E/L15E or other CC domain mutations could be de-stabilizing the effector activated form.

    We commend the authors for having a section considering several alternative explanations for the absence of oligomers, such as different extraction conditions or inherent stability differences between MLA13 and other CNL resistosomes. However, this is only mentioned at the end of the discussion. In the abstract, introduction and results sections, a lot of conclusions are being drawn and no mention of these limitations is made. As mentioned above, we feel like the authors should be more cautious about drawing conclusions from negative data.

    Conclusion

    Overall, the manuscript presents significant advances in understanding the structural and functional dynamics of MLA13 and its interaction with AVRA13-1. However, we feel the authors should clarify what their interpretation of the complex is.

    Througout the manuscript, several conclusions drawn from this finding and it is not clear which one the authors are more confident on. From the abstract, it appears the authors are claiming that this MLA13-AVR-A13 complex has resistosome-independent signaling activities, which we feel is a conclusion that is not supported by the data presented and would require further specific testing. For instance, do MLA mutants that don't oligomerize into a resistosome still respond to the AVR? It's plausible that they have purified an intermediate activation stage and that an MLA13 resistosome will be formed under the correct conditions.

    In general, our opinion is that the main advance of this study is a better structural understanding of the MLA13-AVRA13 interaction interface as well as the MLA7 bioengineerin. We think it is premature to draw conclusions on MLA13 activation mechanisms and dynamics at this point based on the absence of a resistosome under the purification conditions used.

    Competing interests

    The authors declare that they have no competing interests.

  2. Version published to 10.1101/2024.07.14.603419v1 on bioRxiv