Cucurbit Crops Acquired Silencing: Virus-Induced Post-Transcriptional Silencing Is Transmitted Across the Graft Union

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper entitled "Cucurbit crops acquired silencing: virus-induced post-transcriptional silencing is transmitted across the graft union" concerns developing the methods for the infection of RNA virus vectors to the germinating melon seeds and grafting after the emergence of the gene- silencing phenotype in the rootstock, resulting in the gene-silencing of diverse cucurbit crops in the scion.

In this study, two different RNA viral vectors (TRSV - Tobacco ringspot virus and ALSV - Apple latent spherical virus) were used to illustrate that the viral vectors move long-distance across the graft union and induce the gene silencing of the scion. Unfortunately, the authors do not discuss in details why silencing of endogenous genes in scion is dependent only from virus long-distance movement but not from silencing signals formed in rootstocks. 

In general, scientific soundness of this work is rather weak because of absence two important control experiments:

1) The authors should show that the virus progeny moving to scion retains insertion of plant PDS gene. In other words, the experiment in scion with semi-quantitative RT-PCR for virus genome region with foreign insertion, like that for virus CP gene, should be done. 

2) Silencing of eIF4E gene in infected scion was performed with VIGS induced by recombinant TRSV vector. As a result, TRSV replication should be inhibited. Indeed, it was shown that the grafted scion after VIGS does not contain CP gene (see fig. 5E). However, it is not clear how silenced state of scion is supported after blocking virus genome replication. Because of this, the special time course experiment in pre-infected scion with simultaneous detection of plant eIF4E  gene and virus CP gene should be done.

Author Response

Comments1: The paper entitled "Cucurbit crops acquired silencing: virus-induced post-transcriptional silencing is transmitted across the graft union" concerns developing the methods for the infection of RNA virus vectors to the germinating melon seeds and grafting after the emergence of the gene- silencing phenotype in the rootstock, resulting in the gene-silencing of diverse cucurbit crops in the scion. In this study, two different RNA viral vectors (TRSV - Tobacco ringspot virus and ALSV - Apple latent spherical virus) were used to illustrate that the viral vectors move long-distance across the graft union and induce the gene silencing of the scion. Unfortunately, the authors do not discuss in details why silencing of endogenous genes in scion is dependent only from virus long-distance movement but not from silencing signals formed in rootstocks. In general, scientific soundness of this work is rather weak because of absence two important control experiments: The authors should show that the virus progeny moving to scion retains insertion of plant PDS gene. In other words, the experiment in scion with semi-quantitative RT-PCR for virus genome region with foreign insertion, like that for virus CP gene, should be done.

Response1: Thank you for pointing this out. We agree with this comment. Therefore, primers were designed to encompass both the virus genome and the inserted fragment. According to the results of semi-quantitative experiments, TRSV virus with foreign insertion were detected in the scions of melon, cucumber and watermelon but not in the scions of pumpkin in the TRSV grafting experiment. Meanwhile, ALSV virus with foreign insertion were detected in the in the scions of pumpkin in the ALSV grafting experiment. The above results were consistent with the results of CP detection, confirming that virus progeny containing foreign insertion can move from the rootstock to the scion. In Figure 5A, we included the aforementioned results, and we included the relevant description in the revised manuscript.

Comments2: Silencing of eIF4E gene in infected scion was performed with VIGS induced by recombinant TRSV vector. As a result, TRSV replication should be inhibited. Indeed, it was shown that the grafted scion after VIGS does not contain CP gene (see fig. 5E). However, it is not clear how silenced state of scion is supported after blocking virus genome replication. Because of this, the special time course experiment in pre-infected scion with simultaneous detection of plant eIF4E gene and virus CP gene should be done.

Response2: I appreciate your insightful comments. It has been indicated that the melon plant initiated its own antiviral response approximately a week after TRSV-CmPDS infection based on the evidence that the first true leaf showed a photobleaching phenotype. From this result, it can be inferred that the melon scion's CmeIF4E was temporarily silenced during grafting, along with the TRSV virus's diminished capacity to replicate after infection with TRSV-CmeIF4E. This phase maintained the silenced state and inhibited virus genome replication. The infection process was finished before the grafting operation since we employed the seed vacuum infection. As a result, detection of the CmeIF4E gene and virus CP gene could not be provided before to scion infection. Instead, we constantly observed the scions' performance for a long period after grafting (Figure 5B, the revised manuscript). Surprisingly, small photobleached spots or strips occurred on the upper leaves of the melon scions (20 and 28 dag). Simultaneously, quantitative results showed that the silencing effect of CmeIF4E was gradually lost (Figure 5D, the revised manuscript). Consistent with the results, the CP mRNA increased significantly (Figure 5E, the revised manuscript). These results suggested that the expression of CmeIF4E was positively correlated with the CP content, and that the silencing effect of the virus in the scion depended on the expression of CmeIF4E. We have made this addition in the revised manuscript.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

Overall, the work is well-structured, and this contribution should be considered for publication after addressing the following comments.

 

1. This is a well-structured abstract, but several areas can be improved for clarity, grammar, statistical significance to strengthen the findings, minor revisions for clarity, and emphasis on the novelty and potential applications of the research. Please modify the abstract as a whole.

2. In the introduction, The text could have a smoother flow between topics (history, purpose, examples, and relevance). Plant grafting is an ancient horticultural technique with at least 2300 years of documented history in China [1]. This method involves joining two plants ........

3. In section 2,  Standardize Units and Nomenclature, Ensure consistent naming for hybrids (e.g., "Zhemi No. 2" and "Tianbanli No. 2" should match formatting).

4. In section 2.3 The use of black plastic film to maintain high humidity is effective, but it might be interesting to note if any temperature control was involved during this period and how it affected graft success rates.

5. In discussion section, What specific resistance mechanism does pumpkin exhibit against the TRSV virus? Are there any molecular markers or pathways involved in this resistance that could be studied further to improve virus-vector interactions?

Author Response

Comments 1: Overall, the work is well-structured, and this contribution should be considered for publication after addressing the following comments. This is a well-structured abstract, but several areas can be improved for clarity, grammar, statistical significance to strengthen the findings, minor revisions for clarity, and emphasis on the novelty and potential applications of the research. Please modify the abstract as a whole.

Response 1: Thank you very much for your suggestion. We asked a native English-speaking expert to revise our manuscript for grammar and language.

Comments 2: In the introduction, the text could have a smoother flow between topics (history, purpose, examples, and relevance). Plant grafting is an ancient horticultural technique with at least 2300 years of documented history in China [1]. This method involves joining two plants ........

Response 2: Thank you for your suggestion. We have revised the Introduction according to your suggestion, and we invited native English-speaking experts to revise our manuscript, making it smoother and more structured to read.

 

Comments 3: In section 2,  Standardize Units and Nomenclature, Ensure consistent naming for hybrids (e.g., "Zhemi No. 2" and "Tianbanli No. 2" should match formatting).

Response 3: Thank you very much for your reminder. We have revised the format of the hybrid names in the manuscript.

 

Comments 4: In section 2.3 The use of black plastic film to maintain high humidity is effective, but it might be interesting to note if any temperature control was involved during this period and how it affected graft success rates.

Response 4: Our grafting operation was carried out in an artificial climate chamber with a constant temperature setting of 22 °C. According to our experience, the grafting success rates are strongly related to the grafting operation. It takes multiple grafting trainings to acquire the grafting operation. There is undoubtedly a link between temperature and grafting success rates. The higher the temperature after grafting, the faster the transpiration rate, which may reduce grafting success. Because we did not compare the temperatures here, the precise impact requires additional investigation, which will be another topic.

 

Comments 5: In discussion section, What specific resistance mechanism does pumpkin exhibit against the TRSV virus? Are there any molecular markers or pathways involved in this resistance that could be studied further to improve virus-vector interactions?

Response 5: Thank you for pointing this out. We were amazed that only pumpkin showed strong resistance to the TRSV virus among these Cucurbitaceae crops. Our semi-quantitative approach failed to detect the TRSV virus genome in the pumpkin scion. In other words, the TRSV virus was not present in the scion pumpkin after grafting onto melon. According to the main conclusion of this manuscript, the ability of the scion to achieve VIGS depends on the movement of the virus itself from the rootstock to the scion. TRSV can achieve VIGS from melon rootstock to watermelon or cucumber scion, implying that the virus can be transmitted to the scion via phloem. Our experimental evidence also showed the presence of TRSV viruses in watermelon or cucumber scions. Therefore, the absence of TRSV in pumpkin scions indicated that the movement of the TRSV virus through the phloem was completely inhibited in pumpkin. We modified some sentences in the Discussion section to make this process clearer in the revised manuscript. Further research is needed to determine the specific strategy by which pumpkin inhibits TRSV movement. The specific molecular mechanisms or pathways involved in this resistance need further research.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The modified version of the manuscript can be accepted in current form.