Functional Characterization of CaSpr2 in Jasmonate-Dependent Induced Defense Against Western Flower Thrips in Capsicum annuum

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents a well-designed and comprehensive study investigating the role of CaSpr2 in jasmonic acid–mediated defense responses against the western flower thrips in pepper. This manuscript is technically sound, well-written, and the data are properly analyzed and interpreted. Several minor revisions are suggested to improve the manuscript's clarity and impact prior to final publication.

1. The experimental descriptions are overly general, lacking specific details such as mixing ratios, inoculation conditions, and culture temperatures.

2.The description of statistical analysis and data visualization requires improvement. The text currently notes the use of Student’s t-test and GraphPad Prism software but omits the significance threshold (e.g., p < 0.05) and the format for data presentation (e.g., mean ± SE).

3. The significance test results in Tables 1 and 2 should be indicated using asterisks (*) rather than letters.

4. We recommend retain only the essential context in Discussion, thereby allowing greater emphasis on the novelty and applied significance of the present findings.

Comments for author File: Comments.pdf

Author Response

Comments 1: The experimental descriptions are overly general, lacking specific details such as mixing ratios, inoculation conditions, and culture temperatures.

Response 1: Thank you for pointing this out. We have provided the detailed experimental procedures in Materials and Methods (2.3. TRV-Mediated Silencing Assays), including specific details such as the mixing ratios, inoculation conditions, and cultivation temperatures.  

Comments 2: The description of statistical analysis and data visualization requires improvement. The text currently notes the use of Student’s t-test and GraphPad Prism software but omits the significance threshold (e.g., p < 0.05) and the format for data presentation (e.g., mean ± SE).

Response 2: We agree with this comment. Therefore, we have supplemented the significant threshold for statistical analysis and the format of data presentation in Materials and Methods (2.4. RNA Isolation and RT-qPCR; 2.5. Hormone Quantification; 2.6. Selection Preference of WFTs for Host Plants).

Comments 3: The significance test results in Tables 1 and 2 should be indicated using asterisks (*) rather than letters.

Response 3: Thank you for your suggestion. We have made the corresponding modifications in Table 1 and Table 2.

Comments 4: We recommend retain only the essential context in Discussion, thereby allowing greater emphasis on the novelty and applied significance of the present findings.

Response 4: We agree with this comment. Therefore, we removed the fourth paragraph of the Discussion and added content highlighting the practical significance of the current research results.

Reviewer 2 Report

Comments and Suggestions for Authors

This study investigated the role of the inhibitor Spr2 in JA-mediated defense mechanisms against tobacco mosaic virus in pepper. Through a comprehensive approach incorporating virus-induced gene silencing (VIGS), transcriptional analysis, plant hormone quantification, insect behavioral assays, and life history investigations, it was demonstrated that silencing CaSpr2 could significantly reduce JA and JA-Ile accumulation, while WFT exhibits a strong feeding preference for plants with CaSpr2 gene silencing. These findings provide profound insights and practical significance for breeding thrips-resistant varieties in non-model plants. Therefore, I recommend accepting it after minor revisions. In particular, there are a few areas in the article that require modification and supplementation:

1. The first paragraph of the introduction provides additional information on the damage caused by western flower thrips to sweet peppers.
2. L76–79, additional information is provided on the latest research progress regarding the functional characteristics of the Suppressor Protein System Mediating Reaction 2 (Spr2) gene.
3. Once abbreviations for relevant names are first introduced in the text, they need not be reintroduced thereafter, e.g., western flower thrips (WFT), suppressor of prosystemin-mediated responses 2 (Spr2), jasmonic acid (JA), virus-induced gene silencing (VIGS).

The parts that need to be revised in the manuscript have been marked in red for reference only.

Comments for author File: Comments.pdf

Author Response

Comments 1: The first paragraph of the introduction provides additional information on the damage caused by western flower thrips to sweet peppers.

Response 1: We agree with this comment. Therefore, we have added more relevant information about the damage caused by western flower thrips to sweet peppers. Specifically includes: In pepper crops, thrips feeding leads to leaf curling and deformation, flower drop, a reduced fruit set rate, and abnormal fruit development [4]. Beyond direct feeding damage, WFT also acts as a vector for plant viruses such as tomato spotted wilt virus (TSWV) and impatiens necrotic spot virus (INSV), with TSWV being a major contributor to pepper yield losses [5,6].

Comments 2: L76–79, additional information is provided on the latest research progress regarding the functional characteristics of the Suppressor Protein System Mediating Reaction 2 (Spr2) gene.

Response 2: We agree with this comment. Therefore, we have added more relevant information about Spr2 gene. Specifically includes: This study focuses on the Spr2 gene, which encodes a chloroplast fatty acid desaturase (FAD7) involved in JA biosynthesis and systemic wound signaling [25]. FAD7 appears to modulate JA-dependent defenses against chewing insects and SA-dependent defenses against aphids via distinct effects on JA synthesis and SA signaling [26]. Arbuscular mycorrhizal fungi (AMF) colonization is significantly reduced in spr2 mutant tomato plants, aligning with other phenotypic impacts of impaired FAD7 function [27]. In tomato, spr2 mutant plants show reduced volatile organic compound levels and compromised resistance to herbivorous insects [28,29].

Comments 3: Once abbreviations for relevant names are first introduced in the text, they need not be reintroduced thereafter, e.g., western flower thrips (WFT), suppressor of prosystemin-mediated responses 2 (Spr2), jasmonic acid (JA), virus-induced gene silencing (VIGS).

Response 3: Thank you for your suggestion. I have made revisions in the main text and have also added an abbreviation list in the supplementary document.

Reviewer 3 Report

Comments and Suggestions for Authors

In this work, the authors perform functional characterization of the gene encoding suppressor of prosystemin-mediated responses2 (Spr2) in a non-model plant, pepper, in the context of western tomato thrips infestation. They show that the homolog they found indeed functions like its counterparts from other better studied plant species, and also show that its inactivation promotes growth and longevity in thrips feeding in the corresponding plant. This is a very interesting result with potential implications for pest management.

 

The manuscript is definitely well-written, and the study is credible and scientifically sound. However, I have several questions on the applications, as well as suggestions which could make its easier to comprehend.

 

1. To the statement that “Enhancing endogenous JA-mediated resistance through genetic or molecular breeding strategies offers an environmentally friendly alternative that can be integrated into sustainable pest management programs”: I do not argue with this statement, but in my opinion, it very much lack more detailed suggestions on how this could be done. Basically, this work shows that disruption of Spr2 function leads to problems for the plant both in terms of photosynthetic activity and pathogen (particularly WFT) resistance. I would say it does not automatically mean that overproduction of Spr2 would improve pathogen resistance (even though it is likely), and more importantly, how would such overproduction influence the phenotype and fitness of the corresponding plant? Is there any evidence for other plant species? Or is there an alternative idea on how to use the obtained data in pest management? Adding more detailed explanation and ideas to the Discussion section would greatly enhance the impact of the study.

 

2. To “DNA fragments corresponding to partial sequences of CaPDS and CaSpr2”: please explain what PDS (phytoene desaturase?) is and why it is used as positive control. I understand there must have been a previous study successfully showing PDS silencing in pepper using this system, but I could not find it easily. Please add short information and a reference.

 

3. Which cultivar of Capsicum annuum was used in the study? Formal designation is not crucial, but it would be helpful to add some information on its origin and/or characteristics, e.g. if it is sweet or hot, used for food or ornamental?

 

3. “2.3. TRV-Mediated Silencing Assays”: please add appropriate references to this method.

 

4. Please consider dding an abbreviation list to the manuscript, as there are several abbreviations specific to the field.

 

5. Please also consider adding a small commentary on Ja-Ile conjugate for non-specialists to explain what it is, how it differs from Ja and why these two forms of this hormone were chosen.

 

6. In Fig3, it is hard to compare curves in A with the same color curves in B, but in fact what we need to compare is A vs. B, not different life stages. I understand that adding all groups to the same panel and encoding them with different line types can heavily crowd the plot, but I wonder if plotting median survival rates could help.

 

Very small text-related issues:

 

L27 “The western flower thrips (WFT),” excessive comma

 

L141 “The statistical comparisons of hormone contents among different groups” should be “The statistical comparisons of hormone contents between different groups”

 

L158 “Li et al. (2022)” seems to be an incorrect reference format

 

Author Response

Comments 1: To the statement that “Enhancing endogenous JA-mediated resistance through genetic or molecular breeding strategies offers an environmentally friendly alternative that can be integrated into sustainable pest management programs”: I do not argue with this statement, but in my opinion, it very much lack more detailed suggestions on how this could be done. Basically, this work shows that disruption of Spr2 function leads to problems for the plant both in terms of photosynthetic activity and pathogen (particularly WFT) resistance. I would say it does not automatically mean that overproduction of Spr2 would improve pathogen resistance (even though it is likely), and more importantly, how would such overproduction influence the phenotype and fitness of the corresponding plant? Is there any evidence for other plant species? Or is there an alternative idea on how to use the obtained data in pest management? Adding more detailed explanation and ideas to the Discussion section would greatly enhance the impact of the study.

Response 1: We agree with this comment. However, up to now, no studies have yet reported Spr2 overexpression in a wild-type background. Li et al. previously demonstrated that the LeFad7 could compensate for spr2 mutant phenotypes by expressing a LeFad7 fragment under the control of the cauliflower mosaic virus 35S promoter in the spr2 mutant. The potential application of these findings for pest management is discussed in the conclusion. Specifically as follows: From an applied perspective, CaSpr2 should be considered a strategic target for the fine-tuned modulation of JA signaling rather than a single-gene solution. For instance, tissue-specific, inducible, or stress-responsive regulation of CaSpr2 or its downstream components could enhance resistance during herbivory while mitigating potential growth penalties under non-stress conditions. Beyond direct genetic manipulation, these findings suggest alternative routes for translating CaSpr2-associated mechanisms into pest management. CaSpr2 and related JA biosynthetic elements may serve as molecular markers in breeding programs to select pepper genotypes with optimized basal defense. Moreover, targeting specific downstream outputs of CaSpr2-mediated JA signaling, such as herbivore-induced volatiles or defensive metabolites, could provide a more selective means of deterring WFT without broadly activating JA responses. This approach would be particularly valuable within an integrated pest management framework.

Li, C.Y.; Liu, G.H.; Xu, C.C.; Lee, G.I.; Bauer, P.; Ling, H.Q.; Ganal, M.W.; Howe, G.A. The tomato suppressor of prosystemin-mediated responses2 gene encodes a fatty acid desaturase required for the biosynthesis of jasmonic acid and the production of a systemic wound signal for defense gene expression. The Plant Cell 2003, 15, 1646-1661, doi:http://10.1105/tpc.012237.

Comments 2: To “DNA fragments corresponding to partial sequences of CaPDS and CaSpr2”: please explain what PDS (phytoene desaturase?) is and why it is used as positive control. I understand there must have been a previous study successfully showing PDS silencing in pepper using this system, but I could not find it easily. Please add short information and a reference.

Response 2: Thank you for your suggestion. Phytoene desaturase (PDS) is a key enzyme specific to carotenoid biosynthesis in plants. Inactivating PDS disrupts this pathway, leading to leaf chlorosis. Consequently, it is frequently employed as a positive control in VIGS experiments. The following is the relevant reference.

Chung, E.; Seong, E.; Kim, Y.C.; Chung, E.J.; Oh, S.K.; Lee, S.; Park, J.M.; Joung, Y.H.; Choi, D. A method of high frequency virus-induced gene silencing in chili pepper (Capsicum annuum L. cv. Bukang). Molecules and Cells 2004, 17, 377-380, doi:https://doi.org/10.1016/S1016-8478(23)13054-8.

Comments 3: Which cultivar of Capsicum annuum was used in the study? Formal designation is not crucial, but it would be helpful to add some information on its origin and/or characteristics, e.g. if it is sweet or hot, used for food or ornamental?

Response 3: Thank you for your suggestion. The chili varieties used in this study were the hot pepper of the edible variety "Bola" that we obtained from the Hunan Xinsu Seed Company. The detailed information is included in Materials and Methods (2.1. Insects and Plants).

Comments 4: “2.3. TRV-Mediated Silencing Assays”: please add appropriate references to this method.

Response 4: Thank you for your suggestion. I have included the relevant references in Materials and Methods (2.3. TRV-Mediated Silencing Assays) and have also listed them below.

Liu, Y.L.; Schiff, M.; Dinesh-Kumar, S.P. Virus-induced gene silencing in tomato. The Plant Journal 2002, 31, 777-786, doi:http://10.1046/j.1365-313x.2002.01394.x.

Liu, Y.L.; Schiff, M.; Marathe, R.; Dinesh-Kumar, S.P. Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus. The Plant Journal 2002, 30, 415-429, doi:http://10.1046/j.1365-313x.2002.01297.x.

Ratcliff, F.; Martin-Hernandez, A.M.; Baulcombe, D.C. Tobacco rattle virus as a vector for analysis of gene function by silencing. The Plant Journal 2001, 25, 237-245, doi:http://10.1046/j.0960-7412.2000.00942.x.

Comments 5: Please consider adding an abbreviation list to the manuscript, as there are several abbreviations specific to the field.

Response 5: Thank you for your suggestion. We have added an abbreviation list in the supplementary document.

Comments 6: Please also consider adding a small commentary on Ja-Ile conjugate for non-specialists to explain what it is, how it differs from Ja and why these two forms of this hormone were chosen.

Response 6: Thank you for your suggestion. We have provided corresponding explanations in the main text. JA-Ile, a conjugate of JA and isoleucine, represents the most bioactive form of JAs. Its perception in the nucleus by the coreceptor complex SCFCOI1-JAZ, an Skp1-Cullin1-F-box (SCF)-type ubiquitin ligase, relieves the transcriptional repression of target genes (Li et al., 2021). JA and JA-Ile were selected for assay because they are core components of the JA signaling pathway and fulfill complementary, indispensable roles in plant defense, particularly against herbivorous pests and pathogen infections.

Li, M.Y.; Yu, G.H.; Cao, C.L.; Liu, P. Metabolism, signaling, and transport of jasmonates. Plant Communications 2021, 2, 100231, doi:https://doi.org/10.1016/j.xplc.2021.100231.

Comments 7: In Fig3, it is hard to compare curves in A with the same color curves in B, but in fact what we need to compare is A vs. B, not different life stages. I understand that adding all groups to the same panel and encoding them with different line types can heavily crowd the plot, but I wonder if plotting median survival rates could help.

Response 7: Thank you for your suggestion. Figure 3 presents seven parameters: the age-stage-specific survival rate (s_xj), age-specific survival rate (l_x), age-stage-specific fecundity (f_x), age-specific fecundity (m_x), age-specific net maternity (l_x*m_x), life expectancy (e_xj), and age-stage reproductive value (v_xj). This visualization captures the overlapping generations and survival status of the thrips population across its developmental stages. Key demographic values are represented by the areas and positions within the two-dimensional axes. Including the median survival rate in this figure would add complexity without clarifying the primary results. The median survival rate is better suited for comparative plots focusing on a single developmental stage across different treatment groups. Developmental durations and population parameters for each treatment group are further detailed in Tables 1 and 2. Significance analysis of these data provides a clear demonstration of the treatment effects on the thrips.

Comments 8: L27 “The western flower thrips (WFT),” excessive comma

Response 8: Thank you for your suggestion. I have made the necessary revisions in the main text.

Comments 9: L141 “The statistical comparisons of hormone contents among different groups” should be “The statistical comparisons of hormone contents between different groups”

Response 9: Thank you for your suggestion. I have made the necessary revisions in the main text.

Comments 10: L158 “Li et al. (2022)” seems to be an incorrect reference format

Response 10: Thank you for your suggestion. I have made the necessary revisions in the main text.