Dear Adam R. Bentham, Juan Carlos De la Concepcion and Sophien Kamoun,  Thank you very for your comments on the pre-print version of our manuscript deposited in bioRxiv \cite{barley}. Your critical comments were fully taken into account to prepare the final version of the manuscript, which will be published in the Focus Issue 'Activation, Regulation, and Evolution of MTI and ETI' of Molecular Plant-Microbe Interactions  \cite{m6d7mx}.  Our response to your comments is summarised below.  I'd like to thank again for investing your time to thoroughly evaluate our study.Sincerely,Takaki  Maekawa Response to BIORXIV comments Reviewers Adam R. Bentham and Juan Carlos De la Concepcion, Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich, UK. Sophien Kamoun. The Sainsbury Laboratory, Norwich Research Park, Norwich, UK.   Findings and comments (1)  Overall comments The lack of functional analyses with pathogens and/or effectors and the fact that the study does not identify a differential phenotype between the reported subfamilies, do not support the statements about functional diversification.  We thank the reviewer for the critical reading of our manuscript and acknowledge the lack of recognition specificities of subfamily 2 members of MLA proteins. However, several lines of evidence strongly support specialised functions of each MLA subfamily: i) known MLA receptor variants that confer Bgh resistance belong exclusively to one subfamily, ii) no Bgh resistance activity was detected for at least three subfamily 2 members (MLA16-1, MLA18-1, and MLA25-1), iii) maintenance of subfamily 2 members with positive selection signature in wild barley populations, iv) undetectable negative impact on agronomic performance of an allele encoding a subfamily 2 member of MLA (FT394) in cultivated barley rejects the hypothesis that introgression of this subfamily in domesticated barley has been subject to counterselection. Therefore, taken together, these data do support the idea that MLA immune receptor family diversification is linked to functional specialization. We think that the evolutionary diversification of a potent NLR family in wild populations, which is mainly explained by the CC signalling domain (Fig. 1), is without precedence and provides future opportunities to identify pathogens in wild barley recognized by the subfamily 2 members. CC domain differences are proposed to drive the diversification of the MLA receptor. However, the phylogeny of the conserved NB domain does not fully recapitulate this diversification. In our phylogenetic analysis, NB domain sequences belonging to the same subfamily tend to cluster together. The manuscript would benefit from a more thorough functional assessment of a range of CC domains. Only one CC domain from the new subclade, which is also the closest homolog to Sr50, has been assayed. Testing more members across the newly identified subfamily will help to draw a general conclusion about the new subclade. Thank you very much for this suggestion. It is indeed important to test if CC domains of SF2 generally trigger cell death or trigger immune responses without cell death. However, such experiments would lie beyond the scope of this paper.  The manuscript proposes a possible diversification in signalling capabilities, however, the chimera experiments of (Jordan 2011) are not consistent with this hypothesis and suggest conserved signalling capacities. For the chimera experiment, as we discussed (Line 425 in the original version), determining whether the known Bgh population is able to overcome (suppress) signalling mediated by the CC domains of subfamily 2, requires re-examination with native Mla gene expression levels and not overexpression. We admit that the term “overcome” might be misleading with respect to the currently available dataset, and that it is more accurate to state that signalling mediated by the CC domains of subfamily 2 is not effective against Bgh populations in cultivated barley varieties. Probably, the required signaling pathway is not effective in subfamily 2 proteins in cultivated barley varieties. Please also note that the invariant CC domain (1–46 amino acids) representing subfamily 1 interacts with WRKYs, while the same region in the CC domains of two subfamily 2 members (MLA18-1 and MLA25-1) does not (Jordan 2011). Irrespective of the biological relevance of interaction with WRKYs, these data imply that the CC domains of the two subfamilies behave differently at the molecular level. This is consistent with the idea that the two Mla subfamilies have functionally diverged from each other.  (2)  Comments on structural predictions The 21st residue of the MLA family CC domains is generally occupied by an aspartate or a glutamate whereas it is a glycine in Sr33, and it is suggested in the manuscript that this may account for the reported differences in the structures—Sr33 was described as a four-helix bundle by (Casey 2016), and MLA10 as a helix-loop-helix in an obligate dimer by (Maekawa 2011). The (Casey 2016) paper has shown that the CC domains of Sr33, MLA10 and Rx all maintain the same oligomeric state and four-helix bundle fold in solution, and this is supported by biophysical analyses of recombinantly produced protein. As such, the current debate on the CC domain structure is not centred around differences in their tertiary structures. What is unknown is whether the MLA10 CC domain dimeric helix-loop-helix structure represents an alternative quaternary conformation, for example a post-activation conformation. To this end, the manuscript does not address the “alternative activation state hypothesis” (discussed by (El Kasmi 2016)), rather the text implies that the tertiary structures of Sr33 and MLA10 are different. To support the statement that Sr33 and MLA10 CC domains maintain different tertiary structures, the authors applied secondary structure prediction with PSIPRED and protein stability modelling with the STRUM web-server. However, published biochemical and biophysical data demonstrating the structural similarity of the Sr33 and MLA10 proteins in solution are not fully considered.  Secondary structure predictions of the MLA10 and Sr33 CC domains were stated to be performed with the first 40 amino acids “for simplicity”. This is problematic as secondary structure prediction using PSIPRED can vary depending on the length of the sequence submitted. Indeed, when the first 160 amino acids of MLA10 and Sr33 are submitted to PSIPRED, the observed differences in the "looped vs helical" regions of the first 40 residues of Sr33 and MLA10 reported in this manuscript are no longer apparent. Considering that the expression of the 1-160 region of the CC domains (or equivalent) triggered cell death in planta (Figure 4a), we believe this region to be more appropriate for secondary structure predictions. It was hypothesized that the presence of the glycine at the 21st residue in Sr33 is the determinant of the “structural differences between MLA10 and Sr33”, and subsequently used the STRUM server (structure-based prediction of protein stability changes upon single-point mutation), to predict whether reciprocal mutations of polymorphisms between MLA10 and Sr33 (Sr33 V20T and Sr33 G21E; MLA10 T20V and MLA10 E21G) would be sufficient to destabilise the MLA10 and Sr33 structures, respectively. The results of STRUM suggest the MLA10 T20V and MLA10 E21G would likely destabilise the MLA10 structure, however the reciprocal mutations in Sr33 would have no effect. There are several questions that this analysis raises listed below. What was the structure used to model the destabilisation? The original MLA10 structure (3QFL) could be compared to the four-helix bundle fold of the Rx and Sr33 CC domains. The small-angle X-ray scattering (SAXS) data for MLA10 CC domain published by (Casey 2016) indicates the MLA10 CC domain also likely forms a four-helix bundle in solution, therefore a better approach to this experiment would be to generate a homology model of MLA10 based on the Sr33 CC domain four-helix bundle and then assess the effects of the mutants on protein stability using STRUM. Additionally, it would be ideal if all the approaches taken to the predictions in the manuscript could be detailed in the materials and methods.To simulate protein stability, it would be more appropriate to use the entire functional region of the protein instead of a non-functional shorter fragment, as it is likely that the missing residues could contribute to stability of the protein. Unfortunately, there is no structure of the active CC domain of MLA10 (a minimum of 142 residues) and as STRUM requires a structure to predict destabilisation caused by point mutations, it is not possible to determine the destabilising effect of the Sr33 V20T and Sr33 G21E mutants in a functional CC domain, even via homology modelling. Consequently, any stability prediction using either the current MLA10 or Sr33 CC domain structures are essentially not directly relevant to the minimal functional domain. Biochemical and biophysical analyses would be the more robust approaches than predictions of protein stability. These experiments are possible given that MLA10 CC domain can be purified in quantities sufficient for structural studies as in (Maekawa 2011). For example, stability of MLA10 and Sr33 CC domain mutants could be analysed by circular dichroism (CD), 2D NMR, or by ThermoFluor. These experiments would be much more conclusive than prediction servers, and these data would significantly benefit the manuscript.Finally, the MLA10 T20V and MLA10 E21G mutants appear to not have any effects on the cell death phenotype nor on the accumulation of these proteins in planta (Fig. 5). These observations are inconsistent with the hypothesis that the mutations destabilize the proteins. Further discussion of these observations would be beneficial.Thank you very much for making these detailed point-to-point comments and suggestions. As structure prediction software programs might be subject to false errors even when using a template, we used only a short sequence without template to examine the local stability of the fragment carrying the unique amino acid. Actually, we do see a difference in the accumulation of proteins between wild-type MLA10 and MLA10 (T20V E21G) in planta (Fig. 5C), which was reproducible in three independent experiments. But such a difference was not seen for Sr33. In the current experimental setting, the level of the mutant protein is higher than that of the wild type. This result seemingly contradicts the structural prediction: however, we think that this is due to the weakened cell death activity of the mutant (see below), although we did not detect a marked difference between the two variants. The current experimental setup is probably not suitable for detection of such a small difference. The lack of difference in protein accumulation between wild-type Sr33 and the Sr33 mutant could be due to their weak cell death activity even with the mutation that might potentiate the Sr33-mediated cell death in our setting, in which both proteins accumulate close to maximal levels. Based on your critical concerns regarding this section and because the relevance of this section for the rest of the paper is only minor, we have removed the data regarding structural prediction. (In general, a rapid and strong cell death response prevents the accumulation of proteins in the Agrobacterium-based transient expression system. This appears to be true for the other experiment (Fig, 4)). (3)  Other comments Figure S1 is a great positive control for the bioinformatics pipeline. It is not clear why the second clade is described as a SUB-family of MLA given that ALL known MLA are in the other clade. The second clade is better described as MLA-like or MLA sister clade. The term “subfamily” was first introduced in (Jordan 2011) for Mla sequences found in cultivated barley varieties. We used the same nomenclature for our study to avoid unnecessary confusion in the field.   Some plants carry two (or even three) members of MLA. In these cases, do they belong to the same or a different subclade? It was not clear in the text and is worth commenting as this situation complicates allelic analyses. Yes, an accession can carry up to three copies, which can be different subfamily members.  These data were shown in Table S1. In the revised version of the manuscript, we have added few sentences to mention this finding. Line 127: as there is not structural data available and to avoid confusion, the text should state “predicted to be located…” This has been amended.Lines 142-143: The statement that RGH1/MLA family has been driven by subfamily-specific functionalization to distinct pathogens is highly speculative.  Is there evidence for a second pathogen? Is possible that this subfamily detects uncharacterized powdery mildew strains. This contradicts lines 410-412 "Whether subfamily 2 NLRs confer disease resistance to avirulence genes present in yet uncharacterized Bgh populations or other pathogens remains to be tested". The distinct pathogens that we refer to might include uncharacterized Bgh populations. Thus, we do not think that our statements are contradictory. Previous pathotyping studies of Bgh field isolates with different barley varieties carrying different powdery mildew R genes suggested that the Central European pathogen population can be considered as a single epidemiological unit (Limpert 1987). Thus, even if an uncharacterized Bgh population that is recognized by subfamily 2 Mla exists, then it would be unlikely to be present in central Europe.  Line 257: “Bootstrap not very high”. Perhaps include the bootstrap number in brackets. This has been indicated.Line 384. It is unclear how they can conclude from RNAseq data only that "in wild barley Rgh1/Mla has undergone frequent gene duplication (Table S1)".  We admit that this is an overstatement. We have modified the text as follows:in wild barley Rgh1/Mla sequences can be duplicated/triplicated Could these sequences be allelic? If what is meant is whether SF1 and SF2 members are allelic to each other, then we cannot answer this question. As mentioned in the text, based on the latest genome assembly of the cultivar ‘Morex’ (Mascher, et al. 2017), Rgh1/Mla family members can be encoded at distant locations in the barley genome.  FT352-1, for example, belongs to subfamily 2, while its LRR sequence is clustered with LRR sequences of subfamily 1 (Fig. 1 and 2). This gene is considered to be a product of recombination between two subfamilies, suggesting that the parental genes are allelic or close enough for recombination. To answer this question, we ultimately require genomic sequences containing Rgh1/Mla family members, which is beyond the scope of this paper. Lines 450-456. They cite (Shen 2007) as evidence that MLA-CC functions by binding WRKY transcription factors and derepressing them. Our understanding is that this model was drawn from an experiment with the inappropriate avirulence effector.  We are aware that the inappropriate avirulence effector was used for the FLIM-FRET data in (Shen 2007). However, this does not affect the finding that a subset of WRKYs interact with the MLA CC domain and that these transcription factors act as repressors of PAMP-triggered basal defense and in MLA-specified resistance as originally shown by both WRKY gene silencing and overexpression experiments. Repetition of the FLIM-FRET experiment with the appropriate avirulence effector is ongoing by the respective authors. Thus, the differential interaction capacity of CC domains of subfamily 1 and 2 with WRKYs implies different molecular functions of the two CC domain types. Therefore, we still mention the data shown in (Shen 2007) as well as (Jordan 2011) and have added a sentence in the Discussion section as follows. The different interaction capacities of the CC domains of subfamily 1 and 2 with WRKYs implies diversified molecular function of the two CC domain haplotypes.Figures 4 and 5: the loading control would be easier to distinguish when showing the band corresponding to RuBisCO (55KDa).  RuBisCO loading is now shown in Figure 4. (Figure 5 has been removed from the manuscript.) Please also note that we have rescanned the original membrane and adjusted the color contrast for better visualization.