Small Cationic Cysteine-Rich Defensin-Derived Antifungal Peptide Controls White Mold in Soybean

Round 1

Reviewer 1 Report

In this study, the GMA4CG_V6 peptide was used to prevent soybean from S. sclerotiorum. The peptide of GMA4CG_V6 has great antifungal activity against S. sclerotiorum in vitro and vivo. The paper is well organized and written yet the current manuscript suffers from some problems:

Major revision required:

Abstract: The abstract should explain the specific research results.

Discussion: The section of discussion should focus on comparing with other research results and highlighting the innovative points of this study.

Minor revision required:

Introduction: Line 61-66: the results of this study should not appear in the section of introduction. The specific research content should be reflected in this part.

Material and methods:

Line 73: the name of strain is not italicized. Please checked throughout the full text.

Line 76: Specify the specific cultivation temperature

Author Response

Reviewer 1

In this study, the GMA4CG_V6 peptide was used to prevent soybean from S. sclerotiorum. The peptide of GMA4CG_V6 has great antifungal activity against S. sclerotiorum in vitro and vivo. The paper is well organized and written yet the current manuscript suffers from some problems:

Major revision required:

Abstract: The abstract should explain the specific research results.

Addressed.

Discussion: The section of discussion should focus on comparing with other research results and highlighting the innovative points of this study.

We have provided this comparison where possible and highlighted the innovative points of this study (see Conclusions).

Minor revision required:

Introduction: Line 61-66: the results of this study should not appear in the section of introduction. The specific research content should be reflected in this part.

We have deleted these lines from Introduction and moved them to discussion.

Material and methods:

Line 73: the name of strain is not italicized. Please checked throughout the full text.

Done

Line 76: Specify the specific cultivation temperature

Done

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript jof-2428210 entitled "Small cationic cysteine-rich defensin-derived antifungal peptide controls white mold in soybean" by Djami-Tchatchou et al. describes the antifungal activity and mode of action of a synthetic peptide variant GMA4CG_V6. This peptide variant was designed based on the parent peptide GMA4CG that comprises the gamma-core motif of the MtDef4 defensin.

The efficacy of the peptide variant against Sclerotinia sclerotiorum was investigated in detached soybean leaves, pods and stems and it was tested in soybean plants. The results indicate that GMA4CG_6 can reduce sclerotia production and white mold disease symptoms without being phytotoxic. It promises high potential for the development as a bio-fungicide and application in the agricultural sector.

This study is an important contribution to the joint effort to identify new antimicrobial biomolecules that can overcome the rapid development of resistance in phytopathogens. Therefore, this study addresses an important topic that fosters the development of peptide-based antimicrobial drugs based on rationally designed synthetic peptides.

The paper is well prepared and concisely written. The experiments were conducted using state-of-the-art methodology and the data were analyzed applying the appropriate statistics.

The data are well presented, and the authors propose a model of the mode of action of GMA4CG_V6 against S. sclerotiorum based on the obtained results of the study.

However, there are some ambiguities and issues that the authors need to clarify or revise before this study can be accepted for publication.

Main issues:

1. The authors conducted the experiment to study the expression of S. sclerotiorum genes related to sclerotia production in cultures grown in liquid medium. It would make more sense to grow the S. sclerotiorum on PDA plates and analyze the expression of genes in response to the antifungal peptide in surface cultures which more closely resemble the fungal growth in nature. Does S. sclerotiorum produce sclerotia under submers conditions at all? The results obtained from gene expression analyses might significantly differ between fungal cultures grown under submers and surface conditions as genes involved in the regulation of differentiation are differently regulated.

2. The results indicate a very strong antifungal efficacy when the peptide was applied together with the inoculum on plant leaves, pods or stems. But what about the curative potential of GMA4CG_V6? The different parts of the plants should be infected first to establish the infection before the peptide is applied. How effective is the peptide against an established infection?

3. In few experiments, two other peptide variants, GMA4CG_V6_lin and GMA4CG_V6_Ala3 were used for comparison of the efficacy of GMA4CG_V6 to inhibit sclerortia production and gene expression analysis. Peptide GMA4CG_V6_Ala3 exhibited a significantly less antifungal efficacy than its parent peptide. It is an ideal control peptide to be used in all other experiments as well. Furthermore, the result obtained with this peptide in the gene expression analysis raises important questions. The expression of the analyzed genes (cna1, pac1, pka2, smk1) is significantly induced in S. sclerotiorum under GMA4CG_V6_Ala3 treatment, the expression is even significantly higher than in the untreated controls. This result remains undiscussed.

An important experiment would be to investigate what effect GMA4CG_V6_Ala3 has on the infectivity of S. sclerotiorum in plants in the presence of this peptide. Does this peptide variant increase the infectivity of the white mold? This peptide should be included in all experiments of this study.

4. The authors investigated the membrane permeabilization activity of the antifungal peptide and uptake of sublethal concentrations of GMA4CG_V6 in fungal hyphae. They captured the localization of TMR-labelled peptide only after 2-5 min exposure, whereas the membrane permeabilization was visualized with SYTOX Green in hyphae after 30 min of incubation with the peptide. Does this indicate that membrane permeabilization is a secondary effect and might be induced AFTER the peptide was taken up into the fungal cell?

Why was this experiment conducted with sub-lethal concentrations of the peptide?

For investigating the mode of action and membrane activity, the effective concentration of the peptide should be used, and preferentially live cell imaging applied to unravel how the peptide interacts with the cell and affects the fungal membrane right upon exposure of the cell to the compound. Did the peptide retain its antifungal efficacy after TMR-labelling or did the labelling reduce the peptide activity? Were the EC50 and the MIC determined for TMR-labelled peptide in S. sclerotiorum?

Since the peptide showed effects against sclerotia, the localization of the peptide should also be shown in these reproductive organs of the fungus.

Further issues:

Materials and Methods:

1. Explain how to prepare ground mycelium

2. What kind of device is used to spray the peptide solution in volumes as small as 1-2 ml on plants?

3. Define abbreviation EC50

4. Define the volume of the "drop" that is applied to treat parts of the plants (line 208)

Results:

1. Figure 1(b) should not be part of a figure but be a separate table

2. The statistical significance is not indicated in Figure 2

3. Improve Figure 3, the label on top of panel (a) cannot be read

4. Define what "mock" means (inoculation with buffer?)

5. Figure 4: The arrows in panel (f) are hardly visible. Improve. In the legend it is stated for panel (c) that "groups indicated with different letters are significantly different according to ANOVA with Tukey' post hoc test", but no groups labelled with letters are shown in the graph in this bar diagram.

Author Response

Response to Reviewer 2

The manuscript jof-2428210 entitled "Small cationic cysteine-rich defensin-derived antifungal peptide controls white mold in soybean" by Djami-Tchatchou et al. describes the antifungal activity and mode of action of a synthetic peptide variant GMA4CG_V6. This peptide variant was designed based on the parent peptide GMA4CG that comprises the gamma-core motif of the MtDef4 defensin.

The efficacy of the peptide variant against Sclerotinia sclerotiorum was investigated in detached soybean leaves, pods and stems and it was tested in soybean plants. The results indicate that GMA4CG_6 can reduce sclerotia production and white mold disease symptoms without being phytotoxic. It promises high potential for the development as a bio-fungicide and application in the agricultural sector.

 This study is an important contribution to the joint effort to identify new antimicrobial biomolecules that can overcome the rapid development of resistance in phytopathogens. Therefore, this study addresses an important topic that fosters the development of peptide-based antimicrobial drugs based on rationally designed synthetic peptides.

 The paper is well prepared and concisely written. The experiments were conducted using state-of-the-art methodology and the data were analyzed applying the appropriate statistics.

The data are well presented, and the authors propose a model of the mode of action of GMA4CG_V6 against S. sclerotiorum based on the obtained results of the study.

We greatly appreciate the positive comments made by the reviewer. We have addressed in detail specific comments made by the reviewer below. 

However, there are some ambiguities and issues that the authors need to clarify or revise before this study can be accepted for publication.

Main issues:

1. The authors conducted the experiment to study the expression of S. sclerotiorum genes related to sclerotia production in cultures grown in liquid medium. It would make more sense to grow the S. sclerotiorum on PDA plates and analyze the expression of genes in response to the antifungal peptide in surface cultures which more closely resemble the fungal growth in nature. Does S. sclerotiorum produce sclerotia under submers conditions at all? The results obtained from gene expression analyses might significantly differ between fungal cultures grown under submers and surface conditions as genes involved in the regulation of differentiation are differently regulated.

sclerotiorum 555 can produce sclerotia at the surface of liquid SFM cultures (see picture below). As such, we think that looking at the expression of sclerotia-inducing genes from fungi grown in liquid culture is appropriate, although we acknowledge that surface cultures on PDA would have been a more natural mimic of S. sclerotiorum growth in nature. We chose the liquid culture approach because of the ease of collecting tissue and because the suspended mycelia received a more even exposure to peptides than would have occurred when spraying the peptide on plates.

Red arrow points to a sclerotia that developed when S. sclerotiorum 555 was grown in liquid SFM for 3 weeks.

2. The results indicate a very strong antifungal efficacy when the peptide was applied together with the inoculum on plant leaves, pods or stems. But what about the curative potential of GMA4CG_V6? The different parts of the plants should be infected first to establish the infection before the peptide is applied. How effective is the peptide against an established infection?

We have conducted an experiment to test the curative antifungal activity of GMA4CG_V6. We have been able to determine that this peptide does reduce white mold symptoms curatively when spray-applied on soybean plants 24 h post-infection. These results are now reported in the manuscript (Figure 4).

3. In few experiments, two other peptide variants, GMA4CG_V6_lin and GMA4CG_V6_Ala3 were used for comparison of the efficacy of GMA4CG_V6 to inhibit sclerortia production and gene expression analysis. Peptide GMA4CG_V6_Ala3 exhibited a significantly less antifungal efficacy than its parent peptide. It is an ideal control peptide to be used in all other experiments as well. Furthermore, the result obtained with this peptide in the gene expression analysis raises important questions. The expression of the analyzed genes (cna1, pac1, pka2, smk1) is significantly induced in S. sclerotiorum under GMA4CG_V6_Ala3 treatment, the expression is even significantly higher than in the untreated controls. This result remains undiscussed.

An important experiment would be to investigate what effect GMA4CG_V6_Ala3 has on the infectivity of S. sclerotiorum in plants in the presence of this peptide. Does this peptide variant increase the infectivity of the white mold? This peptide should be included in all experiments of this study.

The reviewer is raising an important question of whether GMA4CG_V6_Ala3 variant might actually increase infectivity of the pathogen. As part of an independent study, we will be conducting a more detailed analysis of all GMA4CG_V6 alanine scanning mutants as well as single amino acid substitution mutants for their potential influence on the infectivity of S. sclerotiorum on soybean plants in future.

4. The authors investigated the membrane permeabilization activity of the antifungal peptide and uptake of sublethal concentrations of GMA4CG_V6 in fungal hyphae. They captured the localization of TMR-labelled peptide only after 2-5 min exposure, whereas the membrane permeabilization was visualized with SYTOX Green in hyphae after 30 min of incubation with the peptide. Does this indicate that membrane permeabilization is a secondary effect and might be induced AFTER the peptide was taken up into the fungal cell?

Why was this experiment conducted with sub-lethal concentrations of the peptide?

We have conducted confocal microscopy study examining the internalization of the TMR-labeled GMA4CG_V6 in fungal hypha. We have confirmed that this peptide gains entry into fungal hypha almost immediately. However, SYTOX green uptake study revealed that this dye is taken up very slowly and the signal is clearly discernible only after several minutes. Our results clearly reveal that membrane permeabilization is a secondary effect. We have revised the text to state this conclusion accordingly.

The sub-lethal concentration of the peptide was used to prevent the peptide from killing the fungal cells since the uptake of the peptide is almost instantaneous and can cause rapid cell death. We have also determined that no uptake of the peptide is observed at a low concentration of 6 µM (Figure S2).

For investigating the mode of action and membrane activity, the effective concentration of the peptide should be used, and preferentially live cell imaging applied to unravel how the peptide interacts with the cell and affects the fungal membrane right upon exposure of the cell to the compound. Did the peptide retain its antifungal efficacy after TMR-labelling or did the labelling reduce the peptide activity? Were the EC50 and the MIC determined for TMR-labelled peptide in S. sclerotiorum?

Since the peptide showed effects against sclerotia, the localization of the peptide should also be shown in these reproductive organs of the fungus.

We appreciate this suggestion made by the reviewer. However, the localization of the peptide in the reproductive organs will be determined in a future study.

Further issues:

Materials and Methods:

1. Explain how to prepare ground mycelium

Done (inserted in the Materials and Methods)

2. What kind of device is used to spray the peptide solution in volumes as small as 1-2 ml on plants?

10mL portable refill bulk atomizer spray (inserted in the text)

3. Define abbreviation EC50

The half-maximal effective concentration (inserted in the text)

4. Define the volume of the "drop" that is applied to treat parts of the plants (line 208)

Done

Results:

1. Figure 1(b) should not be part of a figure but be a separate table

Done as Table S3

2. The statistical significance is not indicated in Figure 2

Done

3. Improve Figure 3, the label on top of panel (a) cannot be read

Done

4. Define what "mock" means (inoculation with buffer?)

Inoculation with water and PDB. We have added an explanation in the legends of Figure 3 and 4 to clarify.

5. Figure 4 now Figure 5: The arrows in panel (f) are hardly visible. Improve. In the legend it is stated for panel (c) that "groups indicated with different letters are significantly different according to ANOVA with Tukey' post hoc test", but no groups labelled with letters are shown in the graph in this bar diagram.

Done.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The authors have answered most of my criticisms and suggestions to my satisfaction and revised the manuscript accordingly.

However, some points that I have raised under "Further issues" were insufficiently addressed.

(1) Statistics:

The indication of significant differences of data between controls and samples was unfortunately not carried out consistently and needs to be corrected.

Any highlighting of data in diagrams using an asterisk to indicate a significant difference between the data of samples compared to that of the control must be explained accordingly in the legend. It must be stated which data were compared (sample-control) and which statistical test was applied to analyze the data (Tukey's post hoc test or Student's t-test). The significant difference between the data must be indicated with a defined P-value.

Figure 2: In response to my criticism, the authors have inserted an asterisk in Figure 2 panel d, but did not explain the significant difference of this data in the Figure legend. - Please correct.

Figure 5 (former Figure 4): In the Figure legend, there is no indication of significant differences for data marked with asterisks in panel (c). The definition should be the difference between the samples compared to the control. Reword last sentence in legend of Figure 5 "....*indicates significant differences between data of the control and the treated samples with P<0.05." Indicate which statistical test was applied to evaluate the data presented in the different bar diagrams presented in Figure 5. - Please correct

(2) I asked to define what "mock" means. The authors stated in their reply that they have added an explanation in the legends of Figure 3 and 4 to clarify. I cannot find any declaration in the respective legends.- Please correct.

Author Response

Figure legends

Figure 1. In vitro and semi-in planta antifungal activities of GMA4CG_V6 against S. sclerotiorum 555 on soybean leaves. (a) Representative pictures showing the antifungal activity of GMA4CG_V6 against S. sclerotiorum 555 in SFM media.  (b) Inhibition of fungal growth at different concentrations of GMA4CG_V6. Each data point represents the average % growth from three replications and error bars represent the standard errors of the means between replicates. (c) Representative pictures (under white light and with CropReporter) showing the antifungal activity of GMA4CG_V6 against S. sclerotiorum 555 on detached soybean leaves. (d) Relative disease lesion area following GMA4CG_V6 application on soybean leaf surface at different concentrations. Each data point represents the average lesion size from five leaves relative to control (no peptide, 0 µM), with error bars representing the standard errors of the means between replicates. Results were analyzed using ANOVA, followed by a Tukey’s post hoc test. * Indicates significant differences between data of the control and the treated samples with P<0.05.This experiment was repeated three times with similar results.

Figure 2. Semi-in planta antifungal activity of GMA4CG_V6 against S. sclerotiorum 555 on soybean pods and stems. (a) Soybean pods detachment assay showing the effect of GMA4CG_V6 on disease development at 3 dpi. Representative pictures (under white light and with CropReporter). (b) Relative disease lesion area at 3dpi following the application of GMA4CG_V6 on the pods. (c) Soybean stems detachment assay showing the effect of GMA4CG_V6 on disease development at 3 dpi. (d) Relative lesion size following the application of GMA4CG_V6 on the stems surface at 3 dpi. Antifungal activities at 7dpi (e, f). For panel (b) and (d) each data point represents the average lesion area/size of five samples relative to control (no peptide, 0 µM), with error bars representing the standard errors of the means between replicates. Results were analyzed using ANOVA, followed by a Tukey’s post hoc test. * Indicates significant differences between data of the control and the treated samples with P<0.05. This experiment was repeated three times with similar results.

Figure 3. In planta preventative antifungal activity of GMA4CG_V6 against S. sclerotiorum 555

In-pot assay showing the antifungal activity of GMA4CG_V6 against S. sclerotiorum 555 on tobacco leaves sprayed with S. sclerotiorum 555 ground mycelia. (b) Representative pictures (CropReporter) showing the antifungal activity of GMA4CG_V6 against S. sclerotiorum 555 on tobacco plants. (c) In-pot assay showing the antifungal activity of GMA4CG_V6 against S. sclerotiorum 555 on soybean leaves sprayed with S. sclerotiorum 555 ground mycelia. (d) Representative pictures (CropReporter) showing the antifungal activity of GMA4CG_V6 against S. sclerotiorum soybean leaves. The mock samples were sprayed only with the 1x SFM without the fungus and the peptide. 

Figure 4. In planta curative antifungal activity of GMA4CG_V6 against S. sclerotiorum 555

A suspension of ground mycelium at optical density (OD) 0.5 was used to spray soybean leaves followed by the spray of GMA4CG_V6. The mock samples were sprayed only with the 1x SFM without the fungus and the peptide. (a) Representative pictures of in pots assay showing the curative antifungal activity of GMA4CG_V6 against S. sclerotiorum 555 at 48h post-infection (b) Representative pictures (under white light and with CropReporter based on the value of potential photosynthetic efficiency Fv/Fm) taken 48h post-infection showing the curative antifungal activity of GMA4CG_V6. (Arrow) for treated leaf.

Figure 5. Effect of GMA4CG_V6  on sclerotia production by S. sclerotiorum 555

Representative pictures showing the effect of various GMA4CG_V6 variants against sclerotia production in vitro after 14-day treatment. (b) Picture showing the total number of sclerotia produced 30 days following peptide treatment. Six plates were used for each treatment. (c) Average number of sclerotia per plate. Each data point represents the average of six plates, with error bars representing the standard errors of the means between replicates. Results were analyzed using ANOVA, followed by a Tukey’s post hoc test. * Indicates significant differences between data of the control and the treated samples with P<0.05.  (d) Representative pictures showing the antifungal activity of GMA4CG_V6 against sclerotia production on soybean pods fourteen days post-treatment. (e) Average number of sclerotia per pod. Each data point represents the average of five pods, with error bars representing the standard errors of the means between replicates. Results were analyzed using ANOVA, followed by a Tukey’s post hoc test. * Indicates significant differences between data of the control and the treated samples with P<0.05. (f) Confocal microscopy images showing the inhibition of sclerotia development by GMA4CG_V6 after two days of growth in vitro. Arrows showing the early development of sclerotia—growth of hyphal tips and dichotomous branching. (g) Effect of different GMA4CG_V6 variants on the expression level of genes involved in sclerotia production in S. sclerotiorum 555. Results were analyzed using ANOVA, followed by a Tukey’s post hoc test. * Indicates significant differences between data of the control and the treated samples with P<0.05.

These changes were made in the new version of the manuscript

Round 3

Reviewer 2 Report

The  manuscript can now be accepted for publication.