Soybean RIN4 represents a mechanistic link between plant immune and symbiotic signaling
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eLife Assessment
The manuscript reports fundamental findings supported by convincing data that supports the biological mechanism for optimal nodulation in soybean. The results are of relevance to understanding the signaling pathways (specifically those dependent on RIN4/RPM1-interacting protein 4) underpinning beneficial rhizobia symbiosis, while repressing the immune response.
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Abstract
Abstract
The legume-rhizobium symbiosis represents a unique and beneficial interaction between legumes and nitrogen-fixing soil bacteria, called rhizobia. The initiation and development of this symbiosis is complex and begins with recognition of key molecular signals, produced by the plant and its symbiont, which determine symbiotic compatibility. Current data suggest that the invading symbiont initially triggers plant immune responses that are subsequently suppressed. Hence, there is growing evidence that features of plant immunity may be relevant to symbiotic establishment. RIN4 is a key immune regulator in plants, regulating basal immunity and it is also targeted by pathogen effector proteins that either confer susceptibility or resistance, depending on the presence of the appropriate resistance protein. Surprisingly, we found that RIN4 was rapidly phosphorylated upon rhizobial inoculation of soybean root hairs. RNAi silencing and mutant studies indicate that RIN4 expression is essential for effective nodulation of soybean. RIN4 phosphorylation occurs within a fifteen amino acid motif, which is highly conserved within the Fabales (legumes) and Rosales orders, which comprise species capable of nitrogen-fixing endosymbiosis with rhizobia. RIN4 proteins mutated in this conserved phosphorylation site failed to support efficient soybean nodulation. Phosphorylation of this site is mediated by the symbiotic receptor-like kinase, SymRK, a well-studied member of the symbiotic signaling pathway. The data implicate RIN4 phosphorylation as a key mediator of rhizobial compatibility, interconnecting symbiotic and immune signaling pathways.
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eLife Assessment
The manuscript reports fundamental findings supported by convincing data that supports the biological mechanism for optimal nodulation in soybean. The results are of relevance to understanding the signaling pathways (specifically those dependent on RIN4/RPM1-interacting protein 4) underpinning beneficial rhizobia symbiosis, while repressing the immune response.
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Reviewer #1 (Public review):
The authors set out to illuminate how legumes promote symbiosis with beneficial nitrogen fixing bacteria while maintaining a general defensive posture towards the plethora of potentially pathogenic microbes in their environment. Intriguingly, a protein involved in plant defence signalling, RIN4, is implicated as a type of 'gatekeeper' for the symbiosis, connecting symbiosis signalling with defence signalling. Although questions remain about how exactly RIN4 enables the symbiosis, the work opens an important door to new discoveries in this area.
Strengths:
The study uses a multidisciplinary, state-of-the-art approach to implicate RIN4 in soybean nodulation and symbiosis development. The results support the authors' conclusions.
Weaknesses:
None after thoughtful revision.
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Reviewer #3 (Public review):
Summary:
This manuscript by Toth et al reveals a conserved phosphorylation site within the RIN4 (RPM1-interacting protein 4) R protein that is exclusive to two of the four nodulating clades, Fabales and Rosales. The authors present persuasive genetic and biochemical evidence that phosphorylation at the serine residue 143 of GmRIN4b, located within a 15-aa conserved motif with a core five amino acids 'GRDSP' region, by SymRK, is essential for optimal nodulation in soybean. The experimental design and results are robust, the manuscript's discussion has been satisfactorily updated. Results described here are important to understand how the symbiosis signaling pathway prioritizes associations with beneficial rhizobia, while repressing immunity-related signals.
Strengths:
The manuscript asks an important question …
Reviewer #3 (Public review):
Summary:
This manuscript by Toth et al reveals a conserved phosphorylation site within the RIN4 (RPM1-interacting protein 4) R protein that is exclusive to two of the four nodulating clades, Fabales and Rosales. The authors present persuasive genetic and biochemical evidence that phosphorylation at the serine residue 143 of GmRIN4b, located within a 15-aa conserved motif with a core five amino acids 'GRDSP' region, by SymRK, is essential for optimal nodulation in soybean. The experimental design and results are robust, the manuscript's discussion has been satisfactorily updated. Results described here are important to understand how the symbiosis signaling pathway prioritizes associations with beneficial rhizobia, while repressing immunity-related signals.
Strengths:
The manuscript asks an important question in plant-microbe interaction studies with interesting findings.
Overall, the experiments are detailed, thorough and very well-designed. The findings appear to be robust.
The authors provide results that are not overinterpreted and are instead measured and logical.
Weaknesses:
No major weaknesses.
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Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public Review):
The authors set out to illuminate how legumes promote symbiosis with beneficial nitrogen-fixing bacteria while maintaining a general defensive posture towards the plethora of potentially pathogenic bacteria in their environment. Intriguingly, a protein involved in plant defence signalling, RIN4, is implicated as a type of 'gatekeeper' for symbiosis, connecting symbiosis signalling with defence signalling. Although questions remain about how exactly RIN4 enables symbiosis, the work opens an important door to new discoveries in this area.
Strengths:
The study uses a multidisciplinary, state-of-the-art approach to implicate RIN4 in soybean nodulation and symbiosis development. The results support the …
Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public Review):
The authors set out to illuminate how legumes promote symbiosis with beneficial nitrogen-fixing bacteria while maintaining a general defensive posture towards the plethora of potentially pathogenic bacteria in their environment. Intriguingly, a protein involved in plant defence signalling, RIN4, is implicated as a type of 'gatekeeper' for symbiosis, connecting symbiosis signalling with defence signalling. Although questions remain about how exactly RIN4 enables symbiosis, the work opens an important door to new discoveries in this area.
Strengths:
The study uses a multidisciplinary, state-of-the-art approach to implicate RIN4 in soybean nodulation and symbiosis development. The results support the authors' conclusions.
Weaknesses:
No serious weaknesses, although the manuscript could be improved slightly from technical and communication standpoints.
Reviewer #2 (Public Review):
Summary:
The study by Toth et al. investigates the role of RIN4, a key immune regulator, in the symbiotic nitrogen fixation process between soybean and rhizobium. The authors found that SymRK can interact with and phosphorylate GmRIN4. This phosphorylation occurs within a 15 amino acid motif that is highly conserved in Nfixation clades. Genetic studies indicate that GmRIN4a/b play a role in root nodule symbiosis. Based on their data, the authors suggest that RIN4 may function as a key regulator connecting symbiotic and immune signaling pathways.
Overall, the conclusions of this paper are well supported by the data, although there are a few areas that need clarification.
Strengths:
This study provides important insights by demonstrating that RIN4, a key immune regulator, is also required for symbiotic nitrogen fixation.
The findings suggest that GmRIN4a/b could mediate appropriate responses during infection, whether it is by friendly or hostile organisms.
Weaknesses:
The study did not explore the immune response in the rin4 mutant. Therefore, it remains unknown how GmRIN4a/b distinguishes between friend and foe.
Reviewer #3 (Public Review):
Summary:
This manuscript by Toth et al reveals a conserved phosphorylation site within the RIN4 (RPM1-interacting protein 4) R protein that is exclusive to two of the four nodulating clades, Fabales and Rosales. The authors present persuasive genetic and biochemical evidence that phosphorylation at the serine residue 143 of GmRIN4b, located within a 15-aa conserved motif with a core five amino acids 'GRDSP' region, by SymRK, is essential for optimal nodulation in soybean. While the experimental design and results are robust, the manuscript's discussion fails to clearly articulate the significance of these findings. Results described here are important to understand how the symbiosis signaling pathway prioritizes associations with beneficial rhizobia, while repressing immunity-related signals.
Strengths:
The manuscript asks an important question in plant-microbe interaction studies with interesting findings.
Overall, the experiments are detailed, thorough, and very well-designed. The findings appear to be robust.
The authors provide results that are not overinterpreted and are instead measured and logical.
Weaknesses:
No major weaknesses. However, a well-thought-out discussion integrating all the findings and interpreting them is lacking; in its current form, the discussion lacks 'boldness'. The primary question of the study - how plants differentiate between pathogens and symbionts - is not discussed in light of the findings. The concluding remark, "Taken together, our results indicate that successful development of the root nodule symbiosis requires cross-talk between NF-triggered symbiotic signaling and plant immune signaling mediated by RIN4," though accurate, fails to capture the novelty or significance of the findings, and left me wondering how this adds to what is already known. A clear conclusion, for eg, the phosphorylation of RIN4 isoforms by SYMRK at S143 modulates immune responses during symbiotic interactions with rhizobia, or similar, is needed.
Recommendations for the authors:
Reviewer #1 (Recommendations For The Authors):
I have no major criticism of the work, although it could be improved by addressing the following minor points:
(1) Page 8, Figure 2 legend. Consider changing "proper symbiosis formation" to "normal nodulation" or something that better reflects control of nodule development/number.
We thank you for the suggestion, the legend was changed to “...required for normal nodule formation” (see Page 10, revised manuscript)
(2) Page 9. Cut "newly" from the first sentence of paragraph 2, as S143 phosphorylation was identified previously.
Thank you for the suggestion, we removed “newly” from the sentence.
(3) Page 10, Figure 3. Panels B showing green-fluorescent nodules are unnecessary given the quantitative data presented in the accompanying panel A. This goes for similar supplemental figures later.
We appreciate the comment; regarding Figure 3 (complementing rin4b mutant, we updated the figures according to the other reviewer’s comment) and Suppl Figure 6 (OE phenotype of phospho-mimic/negative mutants), we removed the panels showing the micrographs. At the same time, we did not modify Figure 2 (where micrographs showing transgenic roots carrying the silencing constructs) for the sake of figure completeness. (See Page 10, revised manuscript)
(4) Consider swapping Figure 3 for Supplemental Figure S7, which I think shows more clearly the importance of RIN4 phosphorylation in nodulation.
We appreciate the comment and have swapped the figures according to the reviewer’s suggestion. Legend, figure description, and manuscript text have been updated accordingly. (See page 12 and 38, revised manuscript)
(5) Page 10. Replace "it will be referred to S143..." with "we refer to S143 instead of ....".
We replaced it according to the comment.
(6) Page 11, delete "While" from "While no interactions could be observed...".
We deleted it according to the suggestion.
(7) Page 33, Fig S5. How many biological replicates were performed to produce the data presented in panel C and what do the error bar and asterisk indicate? Check that this information is provided in all figures that show errors and statistical significance.
Thank you for the remark. The experiment was repeated three times, and this note was added to the figure description. All the other figure legends with error bar(s) were checked whether replicates are indicated accordingly.
(8) Page 37, Fig S11, panel B. Are averages of data from the 2 biological and 3 technical replicates shown? Add error bars and tests of significant difference.
Averages of a total of 6 replicates (from 2 biological replicates, each run in triplicates) are shown. We thank the reviewer for pointing out the missing error bars and statistical test, we have updated the figure accordingly.
(9) Fig S12. Why are panels A, C, E, and G presented? The other panels seem to show the same data more clearly- showing the linear relationship between peak area ratio and protein concentration.
We have taken the reviewer’s comment into consideration and revised the figure, removing the calibration curves and showing only four panels. The figure legend has been corrected accordingly. (Please see page 43, revised masnuscript). The original figure (unlike other revised figures) had to be deleted from the revised manuscript,as it caused technical issues when converting the document into pdf.
Reviewer #2 (Recommendations For The Authors):
Some small suggestions:
(1) It's good to include a protein schematic for RIN4 in Figure 1.
We appreciate the reviewer’s suggestion and we have drawn a protein schematic and added it to Figure 1. The figure legend was updated accordingly.
(2) There appears to be incorrect labeling in Figure 2c; please double-check and make the necessary corrections.
With respect, we do not understand the comment about incorrect labeling. Would the reviewer please help us out and give more explanation? In Figure 2C, RIN4a and RIN4b expression was checked in transgenic roots expressing either EV (empty vector) or different silencing constructs targeting RIN4a/b.
Reviewer #3 (Recommendations For The Authors):
I enjoyed the level of detail and precision in experimental design.
A discussion point could be - What does it mean that nodule number but not fixation is affected? Is RIN4 only involved in the entry stage of infection but not in nodules during N-fixation?
Current/Our data suggest that RIN4 does indeed appear to be involved in infection. This hypothesis is supported by the findings that RIN4a/b was found phosphorylated in root hairs but not in root (or it was not detected in the root). The interaction with the early signaling RLKs also suggests that RIN4 is likely involved in the early stage of symbiosis formation.
How would the authors explain their observation "However, the motif is retained in non-nodulating Fabales (such as C. canadensis, N. schottii; SI Appendix, Figure S2) and Rosales species as well." What does this imply about the role in symbiosis that the authors propose?
We appreciate the reviewer’s question. The motif seems to be retained, however, it might be not only the motif but also the protein structure that in case of nodulating plants might be different. We have not investigated the structure of RIN4, how it would look based on certain features/upon interaction with another protein and/or post-translational modification(s). Griesman et al, (2018) showed the absence of certain genes within Fabales in non-nodulating species, we can speculate that these absent genes can’t interact with RIN4 in those species, therefore the lack of downstream signaling could be possible (in spite of the retained motif in non-nodulating species). At this point, there is not enough data or knowledge to further speculate.
qPCR analysis of symbiotic pathway genes showed that both NIN-dependent and NIN-independent branches of the symbiosis signaling pathway were negatively affected in the rin4b mutant. Please derive a conclusion from this.
We appreciate the comment, it also prompted us to correct the following sentence; original: “Since NIN is responsible for induction of NF-YA and ERN1 transcription factors, their reduced expression in rin4b plants was not unexpected (Fig. 5). “As ERN1 expression is independent of NIN (Kawaharada et al, 2017). The following sentences were also deleted as it represented a repetition of a statement above these sentences: “Soybean NF-YA1 homolog responded significantly to rhizobial treatment in rin4b plants, whereas NF-YA3 induction did not show significant induction (Fig. 5).“
We added the following conclusion/hypothesis: “Based on the results of the expression data presented above, it seems that both NIN-dependent and NINindependent branches of the symbiotic signaling pathways are affected in the rin4b mutant background. This indicates that the role of RIN4 protein in the symbiotic pathway can be placed upstream of CYCLOPS, as the CYCLOPS transcription activating complex is responsible (directly or indirectly) for the activation of all TFs tested in our expression analysis (Singh et al, 2014/47, 48).” (Please see Page 16, revised manuscript)
The authors are highly encouraged to write a thoughtful discussion that would accompany the detailed experimental work performed in this manuscript.
We appreciate the comment, and we did some work on the discussion part of the document. (Please see Pages 17-19, revised manuscript)
Some minor suggestions for overall readability are below.
What about immune signaling genes? Given that authors hypothesize that "Absence of AtRIN4 leads to increased PTI responses and, therefore, it might be that GmRIN4b absence also causes enhanced PTI which might have contributed to significantly fewer nodules." Could check marker immune signaling gene expression FLS2 and others.
We appreciate the reviewer’s comment, and while we believe those are very interesting questions/suggestions, answering them is out of the scope of the current manuscript. Partially because it has been shown that several defenseresponsive genes that were described in leaf immune responses could not be confirmed to respond in a similar manner in root (Chuberre et al., 2018). It was also shown that plant immune responses are compartmentalized and specialized in roots (Chuberre et al., 2018). If we were looking at immune-responsive genes, the signal might be diluted because of its specialized and compartmentalized nature. Another reason why these questions cannot be answered as a part of the current manuscript is because finding a suitable immune responsive gene would require rigorous experiments (not only in root, but also in root hair (over a timecourse) which would be a ground work for a separate study (root hair isolation is not a trivial experiment, it requires at least 250-300 seedlings per treatment/per time-point).
Regarding FLS2, it is known in Arabidopsis that its expression is tissue-specific within the root, and it seems that FLS2 expression is restricted to the root vasculature (Wyrsch et al, 2015). In our manuscript, we showed that RIN4a/b is highly expressed in root hairs, as well as RIN4 phosphorylation was detectable in root hair but not in the root; therefore, we do not see the reason to investigate FLS2 expression.
"in our hands only ERN1a could be amplified. One possible explanation for this observation is that primers were designed based on Williams 82 reference genome, while our rin4b mutant was generated in the Bert cultivar background." Is the sequence between the two cultivars and the primers that bind to ERN1b in both cultivars so different? If not, this explanation is not very convincing.
At the time of performing the experiment the genomic sequence of the Bert cultivar (used for generating rin4b edited lines) was not publicly available. In accordance with the reviewer’s comment, we removed the explanation, as it does not seem to be relevant. (See page 16, revised manuscript)
The figures are clear and there is a logical flow. The images of fluorescing nodules in Figure 2,3 panels with nodules are not informative or unbiased .
We appreciate the comment, as for Figure 3 (complementing rin4b mutant), we updated the figures according to the other reviewer’s comment and Suppl. Figure 6 (OE phenotype of phospho-mimic/negative mutants) we removed the panels showing the micrographs. At the same time, we did not modify Figure 2 (where micrographs showing transgenic roots carrying the silencing constructs) for the sake of figure completeness. (See pages 10, 12 and 38, revised manuscript)
What does the exercise in isolation of rin4 mutants in lotus tell us? Is it worth including?
Isolation of the Ljrin4 mutant suggests that RIN4 carries such an importance that the mutant version of it is lethal for the plant (as in Arabidospis, where most of the evidence regarding the role of RIN4 has been described), and an additional piece of evidence that RIN4 is similarly crucial across most land plant species.
Sentence ambiguous. "Co-expression of RIN4a and b with SymRKßΔMLD and NFR1α _resulted in YFP fluorescence detected by Confocal Laser Scanning Microscopy (SI Appendix, Figure S8) suggesting that RIN4a and b proteins closely associate with both RLKs." Were all 4 expressed together?
Thank you for the remark. Not all 4 proteins were co-expressed together. We adjusted the sentence as follows: “Co-expression of RIN4a/ and b with SymRKßΔMLD as well as and NFR1α resulted in YFP fluorescence…” I hope it is phrased in a clearer way. (See page 13, revised manuscript)
Minor spelling errors throughout.. Costume-made (custom made?)
Thank you for noticing. According to the Cambridge online dictionary, it is written with a hyphen, therefore, we added a hyphen and corrected the manuscript accordingly.
CRISPR-cas9 or CRISPR/Cas9? Keep it consistent throughout. CRISPR-cas9 is the latest consensus.
We corrected it to “CRISPR-Cas9” throughout the manuscript.
References are missing for several 'obvious statements' but please include them to reach a broader audience. For example the first 5 sentences of the introduction. Also, statements such as 'Root hairs are the primary entry point for rhizobial infection in most legumes.'.
Thank you for the comment. To make it clearer, we also added reference #1, after the third sentence of the introduction, as well as we added an additional review as reference. This additional review was also cited as the source for the sentence “Root hairs are the primary…” (Please see page 2, revised manuscript)
Can you provide a percent value? Silencing of RIN4a and RIN4b resulted in significantly reduced nodule numbers on soybean transgenic roots in comparison to transgenic roots carrying the empty vector control. Also, this wording suggests it was a double K.D. but from the images, it appears they were individually silenced.
We appreciate the reviewer's comment. We observed a 50-70% reduction in the number of nodules. We adjusted the text according to the reviewer's remark. (See page 9, revised manuscript)
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eLife assessment
The manuscript describes important findings supported by convincing data. The authors present persuasive genetic and biochemical evidence that supports the biological mechanism for optimal nodulation in soybean presented in this study. The results are of relevance to understanding the signaling pathway underpinning beneficial rhizobia symbiosis, while repressing the immune response. With the discussion part strengthened this paper would be of broad interest to plant biologists working on cell signaling and plant-microbe interactions.
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Reviewer #1 (Public Review):
The authors set out to illuminate how legumes promote symbiosis with beneficial nitrogen-fixing bacteria while maintaining a general defensive posture towards the plethora of potentially pathogenic bacteria in their environment. Intriguingly, a protein involved in plant defence signalling, RIN4, is implicated as a type of 'gatekeeper' for symbiosis, connecting symbiosis signalling with defence signalling. Although questions remain about how exactly RIN4 enables symbiosis, the work opens an important door to new discoveries in this area.
Strengths:
The study uses a multidisciplinary, state-of-the-art approach to implicate RIN4 in soybean nodulation and symbiosis development. The results support the authors' conclusions.Weaknesses:
No serious weaknesses, although the manuscript could be improved slightly from …Reviewer #1 (Public Review):
The authors set out to illuminate how legumes promote symbiosis with beneficial nitrogen-fixing bacteria while maintaining a general defensive posture towards the plethora of potentially pathogenic bacteria in their environment. Intriguingly, a protein involved in plant defence signalling, RIN4, is implicated as a type of 'gatekeeper' for symbiosis, connecting symbiosis signalling with defence signalling. Although questions remain about how exactly RIN4 enables symbiosis, the work opens an important door to new discoveries in this area.
Strengths:
The study uses a multidisciplinary, state-of-the-art approach to implicate RIN4 in soybean nodulation and symbiosis development. The results support the authors' conclusions.Weaknesses:
No serious weaknesses, although the manuscript could be improved slightly from technical and communication standpoints. -
Reviewer #2 (Public Review):
Summary:
The study by Toth et al. investigates the role of RIN4, a key immune regulator, in the symbiotic nitrogen fixation process between soybean and rhizobium. The authors found that SymRK can interact with and phosphorylate GmRIN4. This phosphorylation occurs within a 15 amino acid motif that is highly conserved in N-fixation clades. Genetic studies indicate that GmRIN4a/b play a role in root nodule symbiosis. Based on their data, the authors suggest that RIN4 may function as a key regulator connecting symbiotic and immune signaling pathways.Overall, the conclusions of this paper are well supported by the data, although there are a few areas that need clarification.
Strengths:
• This study provides important insights by demonstrating that RIN4, a key immune regulator, is also required for symbiotic …Reviewer #2 (Public Review):
Summary:
The study by Toth et al. investigates the role of RIN4, a key immune regulator, in the symbiotic nitrogen fixation process between soybean and rhizobium. The authors found that SymRK can interact with and phosphorylate GmRIN4. This phosphorylation occurs within a 15 amino acid motif that is highly conserved in N-fixation clades. Genetic studies indicate that GmRIN4a/b play a role in root nodule symbiosis. Based on their data, the authors suggest that RIN4 may function as a key regulator connecting symbiotic and immune signaling pathways.Overall, the conclusions of this paper are well supported by the data, although there are a few areas that need clarification.
Strengths:
• This study provides important insights by demonstrating that RIN4, a key immune regulator, is also required for symbiotic nitrogen fixation.
• The findings suggest that GmRIN4a/b could mediate appropriate responses during infection, whether it is by friendly or hostile organisms.Weaknesses:
• The study did not explore the immune response in the rin4 mutant. Therefore, it remains unknown how GmRIN4a/b distinguishes between friend and foe. -
Reviewer #3 (Public Review):
Summary:
This manuscript by Toth et al reveals a conserved phosphorylation site within the RIN4 (RPM1-interacting protein 4) R protein that is exclusive to two of the four nodulating clades, Fabales and Rosales. The authors present persuasive genetic and biochemical evidence that phosphorylation at the serine residue 143 of GmRIN4b, located within a 15-aa conserved motif with a core five amino acids 'GRDSP' region, by SymRK, is essential for optimal nodulation in soybean. While the experimental design and results are robust, the manuscript's discussion fails to clearly articulate the significance of these findings. Results described here are important to understand how the symbiosis signaling pathway prioritizes associations with beneficial rhizobia, while repressing immunity-related signals.Strengths:
The …Reviewer #3 (Public Review):
Summary:
This manuscript by Toth et al reveals a conserved phosphorylation site within the RIN4 (RPM1-interacting protein 4) R protein that is exclusive to two of the four nodulating clades, Fabales and Rosales. The authors present persuasive genetic and biochemical evidence that phosphorylation at the serine residue 143 of GmRIN4b, located within a 15-aa conserved motif with a core five amino acids 'GRDSP' region, by SymRK, is essential for optimal nodulation in soybean. While the experimental design and results are robust, the manuscript's discussion fails to clearly articulate the significance of these findings. Results described here are important to understand how the symbiosis signaling pathway prioritizes associations with beneficial rhizobia, while repressing immunity-related signals.Strengths:
The manuscript asks an important question in plant-microbe interaction studies with interesting findings.Overall, the experiments are detailed, thorough, and very well-designed. The findings appear to be robust.
The authors provide results that are not overinterpreted and are instead measured and logical.
Weaknesses:
No major weaknesses. However, a well-thought-out discussion integrating all the findings and interpreting them is lacking; in its current form, the discussion lacks 'boldness'. The primary question of the study - how plants differentiate between pathogens and symbionts - is not discussed in light of the findings. The concluding remark, "Taken together, our results indicate that successful development of the root nodule symbiosis requires cross-talk between NF-triggered symbiotic signaling and plant immune signaling mediated by RIN4," though accurate, fails to capture the novelty or significance of the findings, and left me wondering how this adds to what is already known. A clear conclusion, for eg, the phosphorylation of RIN4 isoforms by SYMRK at S143 modulates immune responses during symbiotic interactions with rhizobia, or similar, is needed. -