Pyramiding Recessive Resistance Genes Enhances Bacterial Leaf Spot Resistance in Peppers by Suppressing In Planta Bacterial Growth

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript by Mousami Poudel et al presented an nice study showing that pyramiding multiple recessive resistance genes enhances bacterial leaf spot resistance. The author showed that three different Xanthomonas species showed lower growth rate in the triple mutant ECW568 compared to the major single mutant ECW5. While the experiment is well conducted and the conclusion is supported, this work lack mechanistic insights and some other necessary investigation.

1, Regarding the mechanism, the previous study showed that the Bs5 enhances resistance by transcriptonal upregulation of PTI pathway genes. Does the pyramiding of Bs5, 6 and 8 further enhances expression of these genes? It is recommended to compare some PTI marker gene expression between ECW, ECW5, ECW568 by RT-qPCR.

2, Resistance and plant growth are usually antagonistic to each other. Are there any growth and yeild differences among ECW, ECW5 and ECW568?

3, Line 62, BLS should be fully spelled in its first appearance.

Author Response

Reviewer Comments:

Reviewer 1

Regarding the mechanism, the previous study showed that the Bs5 enhances resistance by transcriptional upregulation of PTI pathway genes. Does the pyramiding of Bs5, 6 and 8 further enhances expression of these genes? It is recommended to compare some PTI marker gene expression between ECW, ECW5, ECW568 by RT-qPCR.

Author Response: We appreciate the reviewer’s insightful suggestion. Investigating whether pyramiding bs5, bs6, and bs8 leads to enhanced expression of PTI-associated genes is a compelling direction for future work. However, this specific question falls outside the current scope of our study, which focused on phenotypic resistance outcomes. We have not yet conducted RT-qPCR analyses to assess PTI marker gene expression in the resistant genotypes, but we agree that such data would strengthen the mechanistic understanding and plan to explore this in subsequent research.

Resistance and plant growth are usually antagonistic to each other. Are there any growth and yield differences among ECW, ECW5 and ECW568?

Author Response: Field evaluations of bs5 and bs6 genotypes have shown no significant reduction in growth or yield compared to the ECW background, indicating that these resistance loci do not appear to impose a major fitness cost under field conditions (Vallejos et al. 2010). However, we currently lack agronomic performance data for the bs8 genotype, and its potential impact on plant vigor remains to be evaluated.

Line 62, BLS should be fully spelled in its first appearance.

Author Response: This has been corrected.

Reviewer 2

Remove unneeded bold.

Author Response: Additional bolded areas have been removed.

Is there no problem with colonies from other phyllosphere bacteria?

Author Response: Infiltrating a very high concentration of the strain of interest directly into the tomato leaf helps to bypass most phyllosphere competitors. The plants are also grown in a controlled, clean environment to limit growth of unwanted pathogens. We have experienced minimal issues with this experimental protocol with regards to competitors.

Line 373: Add ref for PTI.

Author Response: Reference added.

Reviewer 3

Can authors consider a Grouped Bar Chart to present the data point clearer to readers for Figure 1, 3, and 5? Some points are overlayed, which causes low visibility due to the color code.

Author Response: While the Grouped Bar Chart is an effective way to present data of this sort in many cases, we believe that it will be less effective due to the amount of data being shared. When we attempted to make a Grouped Bar Chart of this data, we needed 32 bars per bacterial strain to demonstrate all 8 genotypes across days 0, 3, 6, and 9. We therefore believe that the line graph is the most effective graphic for this scenario, as it is also the most standard form of presenting bacterial populations data over time.

Is there a better way to organize Table 1, which should be posted before Figure 2, due to the main text? And is it a screenshot posted as Table 1? If so, please correct it accordingly as the way shown in Table 2 that shows the proper format and alignment, such as the consistent 5 decimal places. The same for Table 3.

Author Response: We have reorganized Table 1 (now Table 2) from lowest to highest values to demonstrate the most effective treatments by this metric. We have also moved the Table location, and reformatted other tables according to these suggestions.

As for Figure 2, the representative leaves showed the visual disease symptoms, could authors indicate the specific spots by adding the arrows or dashed circles to point out the symptom area if applicable. The same for Figures 4 and 6.

Author Response: We have now marked the inoculation area for all the leaves to see the affected area more clearly.

Figure 6 showed the representative leaves of more genotypes than Figures 2 and 4, including ECW56 and ECW68. Why does the author post different data set? Please explain. And is it possible to post the data set for the representation of all three Xanthomonas species?

Author Response: Figure 6 represents data from X. perforans, which is an emerging pathogen on pepper. We were therefore more interested in the behavior of these genotypes against X. perforans, and so included more representative leaves.

Line 272 mentioned “ranging from 28-35 18-28 C incubation’, which may confuse the reader. Line 298 mentioned “at 6 dpi”, whereas Line 320 mentioned “resistant genotypes at 0, 3, 7 days post-inoculation,” and Figure 5 does not present the data points at 6 dpi.

Author Response: Line 272 has been edited for clarity. Line 320 has been corrected to 0, 3, and 9 dpi and line 298 also has been changed to “at 9 dpi”. Figure 5 does not present the data points at 6 dpi unlike Figures 1 and 3 due to issues with the data from Day 6. A previous iteration of the Xp experiment was conducted on Days 0, 3, and 7, and had very similar results as the data from Figure 5, and so we feel confident in the durability of our data. The 0, 3, and 7 experiment was not included in this manuscript due to the difference in sampled days, which occurred due to a hurricane closing campus on the days we needed to sample.

Format consistency using either “Figure” or “Fig.”

Author Response: These have been edited for consistency.

Is there a reference to be introduced in the main text from Ln 373 to Ln 376.

Author Response: Reference was unintentionally left out and has been added.

Please also check the resolution/quality of each Figure after posting in the main text.

Author Response: Figures have been re-uploaded in a different version to increase resolution and quality.

Reviewer 2 Report

Comments and Suggestions for Authors

The paper is of strong interest because it reveals the temperature sensitivity of the basic trait of resistance to pathogens.  Climate change means most common varieties of plants are now subjected to different environmental pressures, including higher temperatures.

Well prepared paper-  images of leaves are valuable. 

There are a few format problems noted with sticky notes.

Comments for author File: Comments.pdf

Author Response

Reviewer Comments:

Reviewer 1

Regarding the mechanism, the previous study showed that the Bs5 enhances resistance by transcriptional upregulation of PTI pathway genes. Does the pyramiding of Bs5, 6 and 8 further enhances expression of these genes? It is recommended to compare some PTI marker gene expression between ECW, ECW5, ECW568 by RT-qPCR.

Author Response: We appreciate the reviewer’s insightful suggestion. Investigating whether pyramiding bs5, bs6, and bs8 leads to enhanced expression of PTI-associated genes is a compelling direction for future work. However, this specific question falls outside the current scope of our study, which focused on phenotypic resistance outcomes. We have not yet conducted RT-qPCR analyses to assess PTI marker gene expression in the resistant genotypes, but we agree that such data would strengthen the mechanistic understanding and plan to explore this in subsequent research.

Resistance and plant growth are usually antagonistic to each other. Are there any growth and yield differences among ECW, ECW5 and ECW568?

Author Response: Field evaluations of bs5 and bs6 genotypes have shown no significant reduction in growth or yield compared to the ECW background, indicating that these resistance loci do not appear to impose a major fitness cost under field conditions (Vallejos et al. 2010). However, we currently lack agronomic performance data for the bs8 genotype, and its potential impact on plant vigor remains to be evaluated.

Line 62, BLS should be fully spelled in its first appearance.

Author Response: This has been corrected.

Reviewer 2

Remove unneeded bold.

Author Response: Additional bolded areas have been removed.

Is there no problem with colonies from other phyllosphere bacteria?

Author Response: Infiltrating a very high concentration of the strain of interest directly into the tomato leaf helps to bypass most phyllosphere competitors. The plants are also grown in a controlled, clean environment to limit growth of unwanted pathogens. We have experienced minimal issues with this experimental protocol with regards to competitors.

Line 373: Add ref for PTI.

Author Response: Reference added.

Reviewer 3

Can authors consider a Grouped Bar Chart to present the data point clearer to readers for Figure 1, 3, and 5? Some points are overlayed, which causes low visibility due to the color code.

Author Response: While the Grouped Bar Chart is an effective way to present data of this sort in many cases, we believe that it will be less effective due to the amount of data being shared. When we attempted to make a Grouped Bar Chart of this data, we needed 32 bars per bacterial strain to demonstrate all 8 genotypes across days 0, 3, 6, and 9. We therefore believe that the line graph is the most effective graphic for this scenario, as it is also the most standard form of presenting bacterial populations data over time.

Is there a better way to organize Table 1, which should be posted before Figure 2, due to the main text? And is it a screenshot posted as Table 1? If so, please correct it accordingly as the way shown in Table 2 that shows the proper format and alignment, such as the consistent 5 decimal places. The same for Table 3.

Author Response: We have reorganized Table 1 (now Table 2) from lowest to highest values to demonstrate the most effective treatments by this metric. We have also moved the Table location, and reformatted other tables according to these suggestions.

As for Figure 2, the representative leaves showed the visual disease symptoms, could authors indicate the specific spots by adding the arrows or dashed circles to point out the symptom area if applicable. The same for Figures 4 and 6.

Author Response: We have now marked the inoculation area for all the leaves to see the affected area more clearly.

Figure 6 showed the representative leaves of more genotypes than Figures 2 and 4, including ECW56 and ECW68. Why does the author post different data set? Please explain. And is it possible to post the data set for the representation of all three Xanthomonas species?

Author Response: Figure 6 represents data from X. perforans, which is an emerging pathogen on pepper. We were therefore more interested in the behavior of these genotypes against X. perforans, and so included more representative leaves.

Line 272 mentioned “ranging from 28-35 18-28 C incubation’, which may confuse the reader. Line 298 mentioned “at 6 dpi”, whereas Line 320 mentioned “resistant genotypes at 0, 3, 7 days post-inoculation,” and Figure 5 does not present the data points at 6 dpi.

Author Response: Line 272 has been edited for clarity. Line 320 has been corrected to 0, 3, and 9 dpi and line 298 also has been changed to “at 9 dpi”. Figure 5 does not present the data points at 6 dpi unlike Figures 1 and 3 due to issues with the data from Day 6. A previous iteration of the Xp experiment was conducted on Days 0, 3, and 7, and had very similar results as the data from Figure 5, and so we feel confident in the durability of our data. The 0, 3, and 7 experiment was not included in this manuscript due to the difference in sampled days, which occurred due to a hurricane closing campus on the days we needed to sample.

Format consistency using either “Figure” or “Fig.”

Author Response: These have been edited for consistency.

Is there a reference to be introduced in the main text from Ln 373 to Ln 376.

Author Response: Reference was unintentionally left out and has been added.

Please also check the resolution/quality of each Figure after posting in the main text.

Author Response: Figures have been re-uploaded in a different version to increase resolution and quality.

Reviewer 3 Report

Comments and Suggestions for Authors

Please check the attached file~

Comments for author File: Comments.pdf

Author Response

Reviewer Comments:

Reviewer 1

Regarding the mechanism, the previous study showed that the Bs5 enhances resistance by transcriptional upregulation of PTI pathway genes. Does the pyramiding of Bs5, 6 and 8 further enhances expression of these genes? It is recommended to compare some PTI marker gene expression between ECW, ECW5, ECW568 by RT-qPCR.

Author Response: We appreciate the reviewer’s insightful suggestion. Investigating whether pyramiding bs5, bs6, and bs8 leads to enhanced expression of PTI-associated genes is a compelling direction for future work. However, this specific question falls outside the current scope of our study, which focused on phenotypic resistance outcomes. We have not yet conducted RT-qPCR analyses to assess PTI marker gene expression in the resistant genotypes, but we agree that such data would strengthen the mechanistic understanding and plan to explore this in subsequent research.

Resistance and plant growth are usually antagonistic to each other. Are there any growth and yield differences among ECW, ECW5 and ECW568?

Author Response: Field evaluations of bs5 and bs6 genotypes have shown no significant reduction in growth or yield compared to the ECW background, indicating that these resistance loci do not appear to impose a major fitness cost under field conditions (Vallejos et al. 2010). However, we currently lack agronomic performance data for the bs8 genotype, and its potential impact on plant vigor remains to be evaluated.

Line 62, BLS should be fully spelled in its first appearance.

Author Response: This has been corrected.

Reviewer 2

Remove unneeded bold.

Author Response: Additional bolded areas have been removed.

Is there no problem with colonies from other phyllosphere bacteria?

Author Response: Infiltrating a very high concentration of the strain of interest directly into the tomato leaf helps to bypass most phyllosphere competitors. The plants are also grown in a controlled, clean environment to limit growth of unwanted pathogens. We have experienced minimal issues with this experimental protocol with regards to competitors.

Line 373: Add ref for PTI.

Author Response: Reference added.

Reviewer 3

Can authors consider a Grouped Bar Chart to present the data point clearer to readers for Figure 1, 3, and 5? Some points are overlayed, which causes low visibility due to the color code.

Author Response: While the Grouped Bar Chart is an effective way to present data of this sort in many cases, we believe that it will be less effective due to the amount of data being shared. When we attempted to make a Grouped Bar Chart of this data, we needed 32 bars per bacterial strain to demonstrate all 8 genotypes across days 0, 3, 6, and 9. We therefore believe that the line graph is the most effective graphic for this scenario, as it is also the most standard form of presenting bacterial populations data over time.

Is there a better way to organize Table 1, which should be posted before Figure 2, due to the main text? And is it a screenshot posted as Table 1? If so, please correct it accordingly as the way shown in Table 2 that shows the proper format and alignment, such as the consistent 5 decimal places. The same for Table 3.

Author Response: We have reorganized Table 1 (now Table 2) from lowest to highest values to demonstrate the most effective treatments by this metric. We have also moved the Table location, and reformatted other tables according to these suggestions.

As for Figure 2, the representative leaves showed the visual disease symptoms, could authors indicate the specific spots by adding the arrows or dashed circles to point out the symptom area if applicable. The same for Figures 4 and 6.

Author Response: We have now marked the inoculation area for all the leaves to see the affected area more clearly.

Figure 6 showed the representative leaves of more genotypes than Figures 2 and 4, including ECW56 and ECW68. Why does the author post different data set? Please explain. And is it possible to post the data set for the representation of all three Xanthomonas species?

Author Response: Figure 6 represents data from X. perforans, which is an emerging pathogen on pepper. We were therefore more interested in the behavior of these genotypes against X. perforans, and so included more representative leaves.

Line 272 mentioned “ranging from 28-35 18-28 C incubation’, which may confuse the reader. Line 298 mentioned “at 6 dpi”, whereas Line 320 mentioned “resistant genotypes at 0, 3, 7 days post-inoculation,” and Figure 5 does not present the data points at 6 dpi.

Author Response: Line 272 has been edited for clarity. Line 320 has been corrected to 0, 3, and 9 dpi and line 298 also has been changed to “at 9 dpi”. Figure 5 does not present the data points at 6 dpi unlike Figures 1 and 3 due to issues with the data from Day 6. A previous iteration of the Xp experiment was conducted on Days 0, 3, and 7, and had very similar results as the data from Figure 5, and so we feel confident in the durability of our data. The 0, 3, and 7 experiment was not included in this manuscript due to the difference in sampled days, which occurred due to a hurricane closing campus on the days we needed to sample.

Format consistency using either “Figure” or “Fig.”

Author Response: These have been edited for consistency.

Is there a reference to be introduced in the main text from Ln 373 to Ln 376.

Author Response: Reference was unintentionally left out and has been added.

Please also check the resolution/quality of each Figure after posting in the main text.

Author Response: Figures have been re-uploaded in a different version to increase resolution and quality.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors addressed reviewers concern's very well.