A dual function of the IDA peptide in regulating cell separation and modulating plant immunity at the molecular level

Reviewer #1 (Public Review):

The paper titled 'A dual function of the IDA peptide in regulating cell separation and modulating plant immunity at the molecular level' by Olsson Lalun et al., 2023 aims to understand how IDA-HAE/HSL2 signalling modulates immunity, a pathway that has previously been implicated in development. This is a timely question to address as conflicting reports exist within the field. IDL6/7 have previously been shown to negatively regulate immune signalling, disease resistance and stress responses in leaf tissue, however IDA has been shown to positively regulate immunity through the shedding of infected tissues. Moreover, recently the related receptor NUT/HSL3 has been shown to positively regulate immune signalling and disease resistance. This work has the potential to bring clarity to this field, however the manuscript requires some additional work to address these questions. This is especially the case as it contracts some previous work with IDL peptides which are perceived by the same receptor complexes.

Can IDA induce pathogen resistance? Does the infiltration of IDA into leaf tissue enhance or reduce pathogen growth? Previously it has been shown that IDL6 makes plants more susceptible. Is this also true for IDA? Currently cytoplasmic calcium influx and apoplastic ROS as overinterpreted as immune responses - these can also be induced by many developmental cue e.g. CLE40 induced calcium transients. Whilst gene expression is more specific is also true that treatment with synthetic peptides, which are recognised by LRR-RKs, can induce immune gene expression, especially in the short term, even when that is not there in vivo function e.g. doi.org/10.15252/embj.2019103894.

This paper shows that receptors other than hae/hsl2 are genetically required to induce defense gene expression, it would have been interesting to see what phenotype would be associated with higher order mutants of closely related haesa/haesa-like receptors. Indeed recently HSL1 has been shown to function as a receptor for IDA/IDL peptides. Could the triple mutant suppress all response? Could the different receptors have distinct outputs? For example for FRK1 gene expression the hae hsl2 mutant has an enhanced response. Could defence gene expression be primarily mediated by HSL1 with subfunctionalisation within this clade?

One striking finding of the study is the strong additive interaction between IDA and flg22 treatment on gene expression. Do the authors also see this for co-treatment of different peptides with flg22, or is this unique function of IDA? Is this receptor dependent (HAE/HSL1/HSL2)?

It is interesting how tissue specific calcium responses are in response to IDA and flg22, suggesting the cellular distribution of their cognate receptors. However, one striking observation made by the authors as well, is that the expression of promoter seems to be broader than the calcium response. Indicating that additional factors are required for the observed calcium response. Could diffusion of the peptide be a contributing factor, or are only some cells competent to induce a calcium response?
It is interesting that the authors look for floral abscission phenotypes in cngc and rbohd/f mutants to conclude for genetic requirement of these in floral abscission. Do the authors have a hypothesis for why they failed to see a phenotype for the rbohd/f mutant as was published previously? Do you think there might be additional players redundantly mediating these processes?

Can you observe callose deposition in the cotyledons of the 35S::HAE line? Are the receptors expressed in native cotyledons? This is the only phenotype tested in the cotyledons.

Are flg22-induced calcium responses affected in hae hsl2?

Reviewer #2 (Public Review):

Lalun and co-authors investigate the signalling outputs triggered by the perception of IDA, a plant peptide regulating organs abscission. The authors observed that IDA perception leads to a transient influx of Ca2+, to the production of reactive oxygen species in the apoplast, and to an increase accumulation of transcripts which are also responsive to an immunogenic epitope of bacterial flagellin, flg22. The authors show that IDA is transcriptionally upregulated in response to several biotic and abiotic stimuli. Finally, based on the similarities in the molecular responses triggered by IDA and elicitors (such as flg22) the authors proposed that IDA has a dual function in modulating abscission and immunity. The manuscript is rather descriptive and provide little information regarding IDA signalling per se. A potential functional link between IDA signalling and immune signalling remains speculative.

Reviewer #3 (Public Review):

Previously, it has been shown the essential role of IDA peptide and HAESA receptor families in driving various cell separation processes such as abscission of flowers as a natural developmental process, of leaves as a defense mechanism when plants are under pathogenic attack or at the lateral root emergence and root tip cell sloughing. In this work, Olsson et al. show for the first time the possible role of IDA peptide in triggering plant innate immunity after the cell separation process occurred. Such an event has been previously proposed to take place in order to seal open remaining tissue after cell separation to avoid creating an entry point for opportunistic pathogens. The elegant experiments in this work demonstrate that IDA peptide is triggering the defense-associated marker genes together with immune specific responses including release of ROS and intracellular CA2+. Thus, the work highlights an intriguing direct link between endogenous cell wall remodeling and plant immunity. Moreover, the upregulation of IDA in response to abiotic and especially biotic stimuli are providing a valuable indication for potential involvement of HAE/IDA signalling in other processes than plant development.

Strengths:
The various methods and different approaches chosen by the authors consolidates the additional new role for a hormone-peptide such as IDA. The involvement of IDA in triggering of the immunity complex process represents a further step in understanding what happens after cell separation occurs. The Ca2+ and ROS imaging and measurements together with using the haehsl2 and haehsl2 p35S::HAE-YFP genotypes provide a robust quantification of defense responses activation. While Ca2+ and ROS can be detected after applying the IDA treatment after the occurrence of cell separation it is adequately shown that the enzymes responsible for ROS production, RBOHD and RBOHF, are not implicated in the floral abscission.
Furthermore, IDA production is triggered by biotic and abiotic factors such as flg22, a bacterial elicitor, fungi, mannitol or salt, while the mature IDA is activating the production of FRK1, MYB51 and PEP3, genes known for being part of plant defense process.

Weaknesses:
Even though there is shown a clear involvement of IDA in activating the after-cell separation immune system, the use of p35S:HAE-YFP line represent a weak point in the scientific demonstration. The mentioned line is driving the HAE receptor by a constitutive promoter, capable of loading the plant with HAE protein without discriminating on a specific tissue. Since it is known that IDA family consist of more members distributed in various tissues, it is very difficult to fully differentiate the effects of HAE present ubiquitously.
The co-localization of HAE/HSL2 and FLS2 receptors is a valuable point to address since in the present work, the marker lines presented do not get activated in the same cell types of the root tissues which renders the idea of nanodomains co-localization (as hypothetically written in the discussion) rather unlikely.