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  1. Author Response

    Reviewer #3 (Public Review ):

    Rhodes et al. explored novel signalling peptides by searching genes encoding small proteins having signal peptide, which are transcriptionally induced upon biotic elicitor treatments in Arabidopsis thaliana. They found that small potentially secreted proteins, designate as CTNIPs based on the conserved sequence motif, are transcriptionally induced upon 7 different elicitors. In A. thaliana, 5 CTINPs are encoded in the genome, and CTNIP4 is strongly induced upon the elicitor treatments. Chemically synthesized signal peptide-deleted CTNIP proteins except for CTNIP5 show the activities to induce Ca2+ influx and MAP kinases phosphorylation in A. thaliana, which are the hallmarks of elicitor-induced immune signalling. The authors found that CTNIP4 can induce ROS burst in a BAK1-dependent manner in A. thaliana, suggesting that CTNIP4 receptor uses BAK1 as a co-receptor for CTNIP4-induced signalling. Moreover, they show that the C-terminal 23 amino acids of CTINP4 is sufficient to induce the responses, and the conserved 2 Cys residues in this C-terminal region is required for the activity. Based on these findings, the authors further explored a CTNIP receptor by identifying proteins that interact with BAK1 upon CTNIP4 treatment using an IP-MS approach. This approach identified HSL3, which is a leucine-rich repeat receptor-like kinase (LRR-RLK), as a receptor candidate. The authors elegantly demonstrate that HSL3 is a receptor of CTNIPs in A. thaliana by taking complementary biochemical and genetic approaches. They provide some evidences that CTNIP4-HSL3 pathway regulates root growth of A. thaliana. Lastly, the authors proposed that the HSL3-CTNIP signalling module is evolutionarily ancient, which appeared before the divergence of angiosperm species.

    The conclusions of this paper regarding the CTNIP-HSL3 pair in A. thaliana are well supported by data. The identification of the CTNIP-HSL3 pair is very significant in the area of plant research.

    Thank you for the positive comments.

    Weaknesses of this paper would be

    1. There is no evidence provided that CTNIPs are actually secreted from plant cells. And, mature forms of CTNIPs are not examined. And, thus, there is space for discussion whether CTNIPs function as secreted peptide hormones. However, generally speaking, addressing these are rather challenging.

    Thank you for your comments, as mentioned previously, we agree that these are important considerations and limitations of the manuscript. We now discuss this further within the manuscript.

    1. The CTNIPs in A. thaliana are initially screened and identified based on the inducibility by biotic elicitors. However, contributions of the CTNIP-HSL3 module to disease resistance are not examined.

    In the current manuscript, we are reporting the initial identification of the HSL3-CTNIP signalling module and its phylogeny. Further work is now required to elucidate its physiological role(s). Under our conditions, we were unable to observe any phenotype upon spray-infection with P. syringae pv tomato DC3000 ΔAvrPto/ΔAvrPto, and have now included this data for information (Figure3-figure supplement 5). It remains to be established whether the HSL3-CTNIP signalling module contributes to resistance under different conditions or to different pathogens, or plays a role in the regulation of plant growth upon microbial perception. These are also now discussed in the text.

    1. The authors have performed a phylogenetic analysis using full-length sequences of the receptor kinases. However, in order to discuss co-evolution of ligand-receptor pairs, it would be more appropriate to use ectodomains of the receptor kinases for the purpose. Actually, the phylogenetic tree in this paper is different form the trees in the published study (Furumizu et al. doi:10.1093/PLCELL/KOAB173), which used ectodomains for the analysis. Conclusions in this paper can be drawn differently. Based on Furumizu et al., HSL3-related LRR-RLKs in monocots are diversified and less related to HSL3 homologs in dicots. This raise a question whether HSL3 homologs in monocots are HSL3 orthologs to draw the conclusion that the HSL3-CTNIP module is conserved and diversified among angiosperms. It is favourable to test and show that CTNIP-HSL3 combinations from monocots also function as the functional module, for instance, using the Nicotiana benthamiana system. Related, testing one pair each for A. thaliana and M. truncatula is not sufficient to deliver the conclusion related to a co-evolution of ligand-receptor specificity because A. thaliana has 5 CTNIPs and Medicago truncatula has 7 HSL3 homologs and 5 CTNIPs, and thus different combinations may still function.

    Thank you for your suggestions. We have included additional phylogenetic analyses based upon the full-length, ectodomain and the kinase domain.

    We agree that further work is required to establish HSL3 homologs are functional orthologs and have now stated this explicitly within the text. Going forward it would be interesting to test this in the N. benthamiana and native systems.

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  2. Evaluation Summary:

    Beginning with transcriptome data, Rhodes et al. identify a new family of peptides with signalling function called CTNIP in the model plant Arabidopsis thaliana. They use an elegant biochemical capture approach to pinpoint the SERK-dependent LRR receptor kinase HSL3 as the only receptor for these peptides. They provide convincing genetic and biochemical evidence that HSL3 binds CTNIP and that CTNIP perception triggers HSL3-dependent cytoplasmic calcium influx, ROS production and transcriptional changes. Furthermore, they provide initial evidence that the CTNIP-HSL3 module may participate in regulating root growth.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    Using transcriptome data reporting responses to biotic stress Rhodes et al identified a new family of small peptides, called CTNIPs, which elicit cytoplasmic Ca2+-influx and MAP-kinase phosphorylation in a bak1 dependent manner. Based on the requirement of BAK1, they searched for BAK1-interactors in the presence of CTNIP using a combination of BAK1-pulldown with proteomics and identified the orphan receptor-like kinase HSL3. They use a combination of biochemical binding assays, protein modelling and genetics (based on root growth responses to the peptides) to confirm that HSL3 is indeed the receptor of CTNIPs. Thus, the study provides a novel small peptide ligand-receptor pair, with a likely function in the regulation of directional root growth. The work is thoroughly conducted and includes several orthogonal approaches to show that HSL3 is the receptor for CTNIP peptides. The manuscript is well and clearly written.

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  4. Reviewer #2 (Public Review):

    Rhodes et al., use an elegant biochemical capture approach to identify the SERK-dependent LRR receptor kinase HSL3 as the only receptor for the newly identified CTNIP peptides in Arabidopsis, and in other species. This idea is based on their finding that synthetic CTNIP peptides trigger cytoplasmic calcium influx and ROS production in wild-type but not bak1-5 mutant Arabidopsis plants. Using the BAK1 co-receptor and the synthetic peptide they identify HSL3 as a major BAK1 signaling complex component and then elegantly demonstrate in vivo and in vitro that CTNIPs are direct ligands targeting the HSL3 extracellular domain. HSL3, despite its name, is thus no receptor for IDA/IDL peptides but for a new family of peptides with possible roles in stress responses and in root development. While most experiments are performed to a high technical standard and are well documented, the bioinformatic characterization of the CTNIP peptide family could be described in more detail. In addition it remains unclear if the peptide fragment used in this study corresponds to the mature signaling peptide processed in vivo. It also remains to be clarified if the two invariant cysteine residues in CTNIPs are involved in intra- or intermolecular disulfide bonds, and if the peptides could be post-translationally modified.

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  5. Reviewer #3 (Public Review):

    Rhodes et al. explored novel signalling peptides by searching genes encoding small proteins having signal peptide, which are transcriptionally induced upon biotic elicitor treatments in Arabidopsis thaliana. They found that small potentially secreted proteins, designate as CTNIPs based on the conserved sequence motif, are transcriptionally induced upon 7 different elicitors. In A. thaliana, 5 CTINPs are encoded in the genome, and CTNIP4 is strongly induced upon the elicitor treatments. Chemically synthesized signal peptide-deleted CTNIP proteins except for CTNIP5 show the activities to induce Ca2+ influx and MAP kinases phosphorylation in A. thaliana, which are the hallmarks of elicitor-induced immune signalling. The authors found that CTNIP4 can induce ROS burst in a BAK1-dependent manner in A. thaliana, suggesting that CTNIP4 receptor uses BAK1 as a co-receptor for CTNIP4-induced signalling. Moreover, they show that the C-terminal 23 amino acids of CTINP4 is sufficient to induce the responses, and the conserved 2 Cys residues in this C-terminal region is required for the activity. Based on these findings, the authors further explored a CTNIP receptor by identifying proteins that interact with BAK1 upon CTNIP4 treatment using an IP-MS approach. This approach identified HSL3, which is a leucine-rich repeat receptor-like kinase (LRR-RLK), as a receptor candidate. The authors elegantly demonstrate that HSL3 is a receptor of CTNIPs in A. thaliana by taking complementary biochemical and genetic approaches. They provide some evidences that CTNIP4-HSL3 pathway regulates root growth of A. thaliana. Lastly, the authors proposed that the HSL3-CTNIP signalling module is evolutionarily ancient, which appeared before the divergence of angiosperm species.

    The conclusions of this paper regarding the CTNIP-HSL3 pair in A. thaliana are well supported by data. The identification of the CTNIP-HSL3 pair is very significant in the area of plant research.

    Weaknesses of this paper would be

    1. There is no evidence provided that CTNIPs are actually secreted from plant cells. And, mature forms of CTNIPs are not examined. And, thus, there is space for discussion whether CTNIPs function as secreted peptide hormones. However, generally speaking, addressing these are rather challenging.
    2. The CTNIPs in A. thaliana are initially screened and identified based on the inducibility by biotic elicitors. However, contributions of the CTNIP-HSL3 module to disease resistance are not examined.
    3. The authors have performed a phylogenetic analysis using full-length sequences of the receptor kinases. However, in order to discuss co-evolution of ligand-receptor pairs, it would be more appropriate to use ectodomains of the receptor kinases for the purpose. Actually, the phylogenetic tree in this paper is different form the trees in the published study (Furumizu et al. doi:10.1093/PLCELL/KOAB173), which used ectodomains for the analysis. Conclusions in this paper can be drawn differently. Based on Furumizu et al., HSL3-related LRR-RLKs in monocots are diversified and less related to HSL3 homologs in dicots. This raise a question whether HSL3 homologs in monocots are HSL3 orthologs to draw the conclusion that the HSL3-CTNIP module is conserved and diversified among angiosperms. It is favourable to test and show that CTNIP-HSL3 combinations from monocots also function as the functional module, for instance, using the Nicotiana benthamiana system. Related, testing one pair each for A. thaliana and M. truncatula is not sufficient to deliver the conclusion related to a co-evolution of ligand-receptor specificity because A. thaliana has 5 CTNIPs and Medicago truncatula has 7 HSL3 homologs and 5 CTNIPs, and thus different combinations may still function.
    Was this evaluation helpful?