Variation in bradyrhizobial NopP effector determines symbiotic incompatibility with Rj2-soybeans via effector-triggered immunity

Genotype-specific incompatibility in legume–rhizobium symbiosis has been suggested to be controlled by effector-triggered immunity underlying pathogenic host-bacteria interactions. However, the rhizobial determinant interacting with the host resistance protein (e.g., Rj2) and the molecular mechanism of symbiotic incompatibility remain unclear. Using natural mutants of Bradyrhizobium diazoefficiens USDA 122, we identified a type III-secretory protein NopP as the determinant of symbiotic incompatibility with Rj2-soybean. The analysis of nopP mutations and variants in a culture collection reveal that three amino acid residues (R60, R67, and H173) in NopP are required for Rj2-mediated incompatibility. Complementation of rj2-soybean by the Rj2 allele confers the incompatibility induced by USDA 122-type NopP. In response to incompatible strains, Rj2-soybean plants activate defense marker gene PR-2 and suppress infection thread number at 2 days after inoculation. These results suggest that Rj2-soybeans monitor the specific variants of NopP and reject bradyrhizobial infection via effector-triggered immunity mediated by Rj2 protein.


1.
The authors interpret their results as suggesting that certain residues in the NopP protein are critical for Rj2 recognition. However, their results would also be consistent with the fact that these amino acid changes could disrupt secretion of the NopP protein from the bacterium to the plant. Ideally, it would be good to actually see movement of the protein into the plant, which is now readily possible using methods pioneered in plant pathogens. However, short of this, the authors need to at least show that the various forms of NopP do not vary with regard to the ability of the bacterium to deliver the protein to the culture supernatant.

2.
The authors interpret their results as indicative of a plant immune response being triggered via NopP-Rj2 recognition. However, they present no data in the paper that such a defense response does occur and, most importantly, is dependent on NopP-Rj2 recognition. These data are necessary if the authors on going to continue to claim a plant pathogen-type model for their results.
More specific comments: p. 6, top: It would be nice to at least see more microscopy on these USDA122 spontaneous mutants. The varying fixation phenotypes suggests that the mutants may be blocked at differing steps in the symbiosis, which would be useful information to better understand how Rj2 restriction is mediated. Note that, although R genes are well studied, to date there has been no clear mechanism attributed to how R genes actually confer resistance. Perhaps this rhizobial system might provide better information.
p. 10, bottom: There appears to be an apparent inconsistency in the results that needs some mention in the paper. Specifically, work with USDA122 indicated that NopP residues R60 and H173 were the most critical, with R67 also playing a lesser role. The authors used these data to suggest that these were critical residues for Rj2 recognition. However, in their studies of NopP in the ST3, ST5, and ST6 strains that obtain an opposite result, NopP strains having the R60 and H173 residues nodulated normally, while those with R67 did not nodulate. The authors cannot have it both ways. Either R60 and H173 are critical for Rj2 recognition or they are not. Some explanation is required.
p. 11, top: I find the results mentioned here interesting. I would ask that the authors compare the NopP lineage to that obtained by comparing the nifHDK and nodABC genes. The latter differ from one another and also do not follow the rRNA phylogeny. It would be interesting to see if nopP reflects co-evolution with either the nitrogen fixation or nodulation genes. (see also discussion on p. 15). The location of NopP within the nif cluster suggests co-evolution with the nif genes, which seems counter-intuitive if the primary role of NopP is to regulate the nodulation process and not nitrogen fixation. Hence, I would again underline the need for the authors to better define the infection blockage imposed by Rj2 in relation to NopP-determined specificity. One cannot rely solely on the published results of others.
p. 14, line 300: The suggestion that the R and H residues in NopP are the sites of phosphorylation is a bit too much speculation, especially considering that relatively easy experiments could be done to look at this. p. 14, line 308: Y2H is notorious for both false positives and false negatives and, hence, mentioning these data seems irrelevant.
p. 15, bottom: What we now know about R genes-effector interactions and the associated complexity far exceeds the older 'zigzag' model. Hence, I suggest that authors cite a more recent review paper that more adequately describes the extant knowledge in this area. In the many references have wrong lowercase or capital letter or italic.
Reviewer #3 (Remarks to the Author): In this manuscript by Sugawara and colleagues, the authors identified NopP as an avirulence protein that is necessary for USDA122 to trigger Rj2-dependent incompatibility. Comparisons between NopP alleles suggested that compatibility and incompatibility is due to variation in one three amino acids.
This work is amazing! I am exceptionally enthusiastic about this manuscript because the work is very impactful. It draws parallels between mechanisms of plant-pathogen and plant-mutualist interactions and will definitely influence the thinking in the field. The data suggest that plant immunity may not distinguish beneficial from detrimental symbiont; NopP likely dampens PTI. The data further suggest that plant immunity has key nodes that are susceptible to attack by microbeassociated effector proteins. I also think it is possible that if Rj2 is a guard, that NopP and a pathogen effector protein likely modify in the same manner, the same guardee. That incompatibility and compatibility differ by few amino acids in NopP is novel; in plant-pathogen interactions, it is typically a presence/absence polymorphism in type III effector genes. Last, the results are wholly consistent with the conclusion that type III effector genes of rhizobia exhibit mutualistic co-evolution with host defenses.
The work is simple, yet brilliant. The experiments were logical and comprehensive and the results are convincing. The manuscript is tight and easy to follow. The authors smartly relied on spontaneous mutants and the easily visualized gain of nodulation phenotype to identify causative mutations in NopP. They engineered strains and also relied on natural variation in nopP alleles to test their hypothesis. Sugawara and colleagues confirmed the gene-for-gene relationship between nopP and Rj2. Last, the authors even cleared up a previous study and showed that NopP has beneficial effects in mung bean.
However, my review is not without some minor comments. I felt the writing needs to be improved in three areas. First, in key places, there needs to be more attention to precision in language. Second, there are just enough errors and awkwardly phrased sentences to be noticeable. Third, the impact could be elevated if the authors were to discuss the broader implications of their work. I am a little worried that the discussion focuses too much on the specific NopP-Rj2 system and does not adequately relate the work to the larger implications of immunity-effector interactions in plantmicrobe interactions. The impact of the work may not be sufficiently recognized by members of the wider field. Below are some, but not all, cases: Precision: Line 128: secretion is not sufficient to call it an effector. It is safer to say that NopP is secreted in a T3SS-dependent manner.
Line 135: did not test nitrogen fixation (strike these words out).
Line 138: change "induces" to "is necessary for" Line 237: this is not clear to me. I am guessing that the authors sequenced the nopP alleles of the Is-1 mutants to ask if there were second site mutations in nopP? Errors/awkward:

Line 18
Line 32 Line 43: escape PTI, not the patterns.
I am very supportive of this manuscript. I loved it.

Dear Reviewers,
We are grateful for your critical comments and useful suggestions that have helped us to improve our paper. As indicated in the responses that follow, we have taken all these comments and suggestions into account in preparing the revised version of our paper. All the points revised according to the reviewer comments were highlighted as red letters in the revised manuscript.
Reviewer #1 (Remarks to the Author): Comment: The soybean Rj2 gene encodes an NBS-LRR protein that confers resistance to nodulation by specific rhizobial strains. The Rj2 function is dependent on the bacterial type III secretion system; as such, Rj2 presumably recognizes a bacterial effector to trigger immune responses but such effectors have not been identified. This manuscript describes the identification and validation of NopP as the cognized effector of Rj2. The manuscript is generally well written but still needs significant improvements. The data presented are solid and strongly support the conclusion. However, in my opinion, the novelty of the research does not warranty its publication in Nature Communications. It is more appropriate for specialized journals such as Molecular Plant-Microbe Interactions (MPMI). Response: Although genetic loci of soybeans restricting nodulation with specific rhizobial strains were found as Rj2, Rj3 and Rj4 in 1960's, the molecular mechanism of this genotype-specific incompatibility have been not fully elucidated so far. In particular, Rj2-mediated incompatibility has been strongly expected to reveal the crucial role of plant immunity of rhizobium-legume symbioses (Hayashi et al. 2012, Gourion et al. 2014, because Rj2 gene encodes a typical R protein to counter the virulence functions of pathogen effector (Yang et al. 2010). As compared to our understanding of plant pathogen interactions, much remains to be elucidated for roles of effector-triggered immunity (ETI) in symbiosis. The present work clearly shows that the variations of NopP effector protein secreted by the incompatible strains determine Rj2-mediated incompatibility via ETI. According to additional experiments recommended by reviewers, we obtained two key results: By incompatible strains, Rj2-soybean plant activated a defense gene, PR-2, and suppressed infection thread number, 2 days after inoculation. By the solid and data, we are able to demonstrate the framework that Rj2-soybeans monitor the specific variants of NopP and reject bradyrhizobial infection via ETI mediated by the host Rj2 protein. We believe that our results could provide important contributions towards our understandings for plant immunity between symbiosis and pathogenesis. These are reasons that we submit our manuscript to Nature Communications. I believe that the paper could ultimately be a very nice publication in Nature Communications but is currently lacking some critical pieces of additional evidence, which I have listed below and then expand upon in some of the specific comments below.

Comment 1:
The authors interpret their results as suggesting that certain residues in the NopP protein are critical for Rj2 recognition. However, their results would also be consistent with the fact that these amino acid changes could disrupt secretion of the NopP protein from the bacterium to the plant. Ideally, it would be good to actually see movement of the protein into the plant, which is now readily possible using methods pioneered in plant pathogens. However, short of this, the authors need to at least show that the various forms of NopP do not vary with regard to the ability of the bacterium to deliver the protein to the culture supernatant. Response: Thank you for this valuable comment. We agree with your suggestion. According to your suggestion, we examined NopP secretion by USDA 110 and 110nopP 122 (USDA110 strain having USDA122-type nopP) to the culture by western blotting. The results have shown that both strains secreted NopP into the culture supernatants. Thus, we suggested that substitutions of NopP on the amino acid residues at positions 60, 67, 173, and 271 does not affect the ability of the bacterium to deliver the protein. We have included this data in Figure 4a, and re-written the sentence in the revised manuscript. In addition, we are now trying to construct a USDA122 mutant, which is able to secrete CyaA-fused NopP. We would like to examine the movement of CyaA-NopP into the roots of Hardee by measurement of Adenylate cyclase activity.
Comment 2. The authors interpret their results as indicative of a plant immune response being triggered via NopP-Rj2 recognition. However, they present no data in the paper that such a defense response does occur and, most importantly, is dependent on NopP-Rj2 recognition. These data are necessary if the authors on going to continue to claim a plant pathogen-type model for their results. Response: Based on the reviewer comment, we examined the expressions of defense marker genes (PR-1, PR-2 and PR-5) in Hardee (Rj2) inoculated with USDA 122 and 122nopP 110 . The expression of PR-2 was significantly induced 2 days after inoculation with USDA 122 as compared with 122nopP 110 and uninoculated control. Together with the results of Rj2/rj2 complementation analyses ( Figure 5), our data suggested that NopP/Rj2-dependent incompatibility occurs via host defense response. We have added the procedure, results and discussions to the revised manuscript.

More specific comments:
Comment 3: p. 6, top: It would be nice to at least see more microscopy on these USDA122 spontaneous mutants. The varying fixation phenotypes suggests that the mutants may be blocked at differing steps in the symbiosis, which would be useful information to better understand how Rj2 restriction is mediated. Note that, although R genes are well studied, to date there has been no clear mechanism attributed to how R genes actually confer resistance. Perhaps this rhizobial system might provide better information. Response: We agree with the reviewer comment. We also think that this rhizobial system may provide mechanistic understanding of R gene. However, the lacking of nitrogen fixation activity was caused by spontaneous mutation (deletion) of nif genes (Supp. Fig. 2 to 5). Therefore, we believe that Fix-mutants are not blocked at different stages in terms of nodulation processes.
Comment 4: p. 10, bottom: There appears to be an apparent inconsistency in the results that needs some mention in the paper. Specifically, work with USDA122 indicated that NopP residues R60 and H173 were the most critical, with R67 also playing a lesser role. The authors used these data to suggest that these were critical residues for Rj2 recognition. However, in their studies of NopP in the ST3, ST5, and ST6 strains that obtain an opposite result, NopP strains having the R60 and H173 residues nodulated normally, while those with R67 did not nodulate. The authors cannot have it both ways. Either R60 and H173 are critical for Rj2 recognition or they are not. Some explanation is required. microbe-associated effector proteins. I also think it is possible that if Rj2 is a guard, that NopP and a pathogen effector protein likely modify in the same manner, the same guardee. That incompatibility and compatibility differ by few amino acids in NopP is novel; in plant-pathogen interactions, it is typically a presence/absence polymorphism in type III effector genes. Last, the results are wholly consistent with the conclusion that type III effector genes of rhizobia exhibit mutualistic co-evolution with host defenses. The work is simple, yet brilliant. The experiments were logical and comprehensive and the results are convincing. The manuscript is tight and easy to follow. The authors smartly relied on spontaneous mutants and the easily visualized gain of nodulation phenotype to identify causative mutations in NopP. They engineered strains and also relied on natural variation in nopP alleles to test their hypothesis. Sugawara and colleagues confirmed the gene-for-gene relationship between nopP and Rj2. Last, the authors even cleared up a previous study and showed that NopP has beneficial effects in mung bean. However, my review is not without some minor comments. I felt the writing needs to be improved in three areas. First, in key places, there needs to be more attention to precision in language. Second, there are just enough errors and awkwardly phrased sentences to be noticeable. Third, the impact could be elevated if the authors were to discuss the broader implications of their work. I am a little worried that the discussion focuses too much on the specific NopP-Rj2 system and does not adequately relate the work to the larger implications of immunity-effector interactions in plant-microbe interactions. The impact of the work may not be sufficiently recognized by members of the wider field. Below are some, but not all, cases: Response: Thank you for your positive evaluations. According to the first and second comments, we edited precision in English language, errors and awkwardly sentences throughout text as possible as we can. As for the third comments, we obtained additional key results that were required by other reviewers: By incompatible strains, Rj2-soybean plant activated a defense gene, PR-2, and suppressed infection thread number, 2 days after inoculation (Fig. 3d, Fig. 6, Suppl. Fig. 8), providing genetic and morphological evidence for defense reaction via ETI. Thus, we changed tile including ETI from "Variation of three amino acid residues in bradyrhizobial NopP effector determines symbiotic incompatibility with Rj2-soybeans" to "Variation in bradyrhizobial NopP determines symbiotic incompatibility with Rj2-soybeans via effector-triggered immunity" for attracting broad readers. In addition, we added and edited new descriptions in the discussion section to emphasize the impact of our work and the implications of immunity-effector interactions in plant-microbe interactions.