Expanding the Toolkit of Fluorescent Biosensors for Studying Mitogen Activated Protein Kinases in Plants

Round 1

Reviewer 1 Report

Presented manuscript provides logistic and technological view how measurement and readout of MAPK activation in plant living tissues can be utilized using fluorescence-based genetically-encoded biosensors. As such, by the scientific content, formal layout, structural organization and mode of writing fits well to the scope of Special Issue of the International Journal of Molecular Science, related to Plant Mitogen Activated Protein Kinases.

Authors follow up their own publication originally presenting only one currently used MAPK biosensor in plants. Importantly, they describe herein an approach providing tools for designing, developing and practical testing of new plant MAPK biosensors. To this aim, they describe and practically show characterization of construct bearing MAPK docking domains for in vitro FRET-based assay, and creation of a translocation-based kinase biosensor. In both presented approaches, by activity profiles in response to constitutively active MAPK variants effectively expressed as recombinant proteins using the EKAREV plant MAPK docking domain trap, and by a site directed mutagenesis utilizing “phospho-dead” variant and “phosphomimetic” variant constructs, the functionality in plant system has been documented. As a proof-of-concept, authors show assays with translocation biosensors as a type of kinase reporters changing subcellular localization in response to phosphorylation of a bNLS/NES module present within the biosensor Kinase Localization Reporter (KLR) using MAPK activity-stimulating agents. Transient reductions of the nuclear fluorescence of KLR-MKP1 and KLR-AP2C1 in response to chitin, flg22 and NaCl is convincing to show that the actual activity of MAPKs was reported in vivo.

As authors clearly stated in the manuscript, outcome of the translocation biosensor, in particular the KLR-MKP1, displaying different responses to chitin, flg22, and NaCl, might reflect differential activation of MAPK pathway(s) upon different stimuli. The selectivity and positive/negative roles of particular MAPKs in particular signalling pathways makes the point. The question is: Could this be physiologically tracked using presented biosensors? This point is partially addressed in Results (lines 442-445), but it is addressed more extensively in the Discussion. Authors clearly state that one of the potential applications of presented plant MAPK docking domain trap approach would be the development of fluorescent biosensors that specifically report the activation of a single MAPK isoform. I appreciate this part a lot, as it helps to understand the complexity of MAPK-based signalling, subcellular topology of the response and versatility of available biosensors. Overall, the manuscript is well written, logically organized and satisfactory documented by experimental data. This study is worth to publish with high potential of broad interest for the IJMS readership.

 

My comments to the technical part of the manuscript:

-In Fig. 3, Fig. 6, Suppl. Fig. 2, it is stated that samples were treated with 200 mg/ml chitin. In Materials and Methods is specified that for chitin treatment, 200 mg of chitin was grounded in a 1.5 ml Eppendorf and the chitin powder was resuspended in 5 ml of pure water prior to use. Please, crosscheck it because these two information (200 mg/ml and 200 mg in 5 ml) are inconsistent. The correct working concentration of chitin used for treatments should be provided.

-In all Figures and Supplementary Figures where the water mock control treatment was used, “H2O” should be replaced by “H₂O”.

-In the legend to the Suppl. Fig. 7 is written that cotyledons were incubated in „200 l“ water (should be „200 µl“). In all Figure legends time points are indicated in „min“, but in Suppl. Fig. 7 this is indicated as ’ (0’, 22’ and 58’). This should be unified as well.

Author Response

1. In Fig. 3, Fig. 6, Suppl. Fig. 2, it is stated that samples were treated with 200 mg/ml chitin. In Materials and Methods is specified that for chitin treatment, 200 mg of chitin was grounded in a 1.5 ml Eppendorf and the chitin powder was resuspended in 5 ml of pure water prior to use. Please, crosscheck it because these two information (200 mg/ml and 200 mg in 5 ml) are inconsistent. The correct working concentration of chitin used for treatments should be provided.

 

Authors response:  We thank the reviewer for catching this error. We have updated all mentions of chitin to display the final working concentration of “40 mg/ml”, including the videos.

 

2. In all Figures and Supplementary Figures where the water mock control treatment was used, “H2O” should be replaced by “H₂O”.

 

Authors response:  We have changed all instances of “H2O” to “H₂O”.

 

3. In the legend to the Suppl. Fig. 7 is written that cotyledons were incubated in „200 l“ water (should be „200 µl“). In all Figure legends time points are indicated in „min“, but in Suppl. Fig. 7 this is indicated as ’ (0’, 22’ and 58’). This should be unified as well.

    

Authors response:  We fixed Suppl. Fig. 7 legend such that the units are correct. We have corrected Suppl. Fig. 7 so that all instances of ‘ are now ‘min’.

Reviewer 2 Report

The submitted manuscripit by Seitz and Kryspan presents two complimentary tools (FRET biosensor and transloction biosensor) dedicated to study MAPK signalling in Arabidopsis. The part of paper regarding FREt biosensor is based on Authors' and their coworkers previous work with SOMA domain. Herein, Authors mainly focused on  EKAREV and compare result with biosensor with SOMA domain. My question is regarding FRET calculation - why values for Turq are so high in Table 1 , as they are lower in Figures (f.example for EKAREV in Fig 1B) MAybe I just missed something, so please explain.

I also think that instead of describing how the FRET system works Authors should present Figure similiar to figure 1a) in Ref 44

Minor comments

A. page 2 line 60 should be spectrofluorometer instead of  spectrophotometer

B. Why in caption of Figure 1 is written "excitation with 435 nm" whereas in experimental (line 625) is written 420 nm. 

Author Response

1. My question is regarding FRET calculation - why values for Turq are so high in Table 1 , as they are lower in Figures (f.example for EKAREV in Fig 1B) Maybe I just missed something, so please explain.

Authors response:  We thank the reviewer for catching this error.  The y-axis for the in vitro FRET assay graphs were labelled incorrectly. These errors have been fixed and should now reflect the values from Table 1.

 

2. I also think that instead of describing how the FRET system works Authors should present Figure similiar to figure 1a) in Ref 44

Authors response:  We thank the reviewer for this helpful suggestion.  A cartoon diagram of a FRET biosensors similar to Figure 1 from (Zaman et al., 2019) has been added to Figure 1 as Figure 1, panel B. Other panels within Figure 1 have been edited to reflect new ordering. Figure 1 Legend and the manuscript section describing Figure 1 have also been edited to reflect this change.

 

Minor comments

1. Page 2 line 60 should be spectrofluorometer instead of  spectrophotometer

Authors response:  We have changed the instance of “spectrophotometer” to    “spectrofluorometer”.

 

2. Why in caption of Figure 1 is written "excitation with 435 nm" whereas in experimental (line 625) is written 420 nm. 

    Authors response: We thank the reviewer for finding this inconsistency.  We have corrected the Figure 1 legend “excitation with 435 nm” to read the correct value of excitation with “420 nm”

Reviewer 3 Report

In the work “Expanding the toolkit of fluorescent biosensors for studying mitogen activated protein kinases in plants” the authors report development of two tools, namely in vitro assay for screening and characterization of MAPK docking domains and a translocation-based kinase biosensor. The first tool is based on previously published FRET biosensor EKAREV; the authors used this biosensor to create MAPK docking domain trap, which can be used for identifying the specificity of MAPK docking domains and as FRET biosensor in vivo.

The second tool is the translocation-based kinase biosensor, developed on the basis of nKTR used for imaging MAPK activity in C.elegans. The authors created a new plant-based biosensor, named KLR and demonstrated phosphorylation-dependent translocation of the biosensor from the nucleus to the cytoplasm. By adding MAPK docking domains from two known MAPK substrates to KLR, the authors showed specific translocation of this biosensor caused by treatments known to induce MAPK activity.

In general, the work is an important proof of principal for the described new tools and an important step forward in developing specific tools for monitoring MAPK activity in plants allowing the better understanding the mechanisms of action for these kinases. The study is a solid and careful investigation with all necessary controls, which provides new tools for plant science.

However, the manuscript requires minor editing as follows:

1. The panels of Figure 4 do not correspond to the text on page 11, which describes the Figure 4, for example in the text Figure 4B should be instead Figure 4E. Similarly in the text Figure 4D should be Figure 4F and so on. Please check carefully the panels for Figure 4 and their mention in the text.
2. The same applies to the Figure 5.
3. Page 15, line 494: Figure 6B should be Figure 6E.
4. Page 16, line 581: EKAEV should be EKAREV

Author Response

1. The panels of Figure 4 do not correspond to the text on page 11, which describes the Figure 4, for example in the text Figure 4B should be instead Figure 4E. Similarly in the text Figure 4D should be Figure 4F and so on. Please check carefully the panels for Figure 4 and their mention in the text.

Authors response: We thank the reviewer for finding this inconsistencies.  All mismatches between Figure panels and mentions in text have been corrected.

 

2. The same applies to the Figure 5.

Authors response: All mismatches between Figure panels and mentions in text have been corrected.

 

3. Page 15, line 494: Figure 6B should be Figure 6E.

Authors response: All mismatches between Figure panels and mentions in text have been corrected.

 

4. Page 16, line 581: EKAEV should be EKAREV

Authors response: We have changed the instance of EKAEV to EKAREV