RNA-Seq of Tomato Fruit-Alternaria Chitin Oligomer Interaction Reveals Genes Encoding Chitin Membrane Receptors and the Activation of the Defense Response
, María Elena Báez-Flores 2, Miguel Ángel Hernández-Oñate 3 and Martín Ernesto Tiznado-Hernández 1
Round 1
Reviewer 1 Report
According to the introduction of the manuscript, the author reported L58, 59, 62, many studies earlier reported the chitin and even in tomato, how this study is different from others?
The introduction section is unclear, somehow looks like discussion, specially the last paras, please modify it.
as per the author, according to the reference 33, how this study is novel? or different?
Ref 34, 35, are you sure there are no studies reported, specifically, "Fungal pathogen recognition????
Even similar studies have been reported earlier in tomato, as per the author, explain why this study is necessary??
The data is only extracted and dependent on the RNA-seq and there is no physiological data.. also in the conclusion that this is an effective way of conttrolling disease, did the author or any other study reported the physiological, functional studies that control? if so, then how this study is different from those?
ALso, if reported earlier, then I think change the title and the presentation of the study to... THe molecular mechanism by which plants react to chitin....
The introduction lack clarity and It's confusing.
minor spelling checking required.
Author Response
We thank the reviewers and editor for the constructive analysis of our manuscript. Below are the answers to all the expressed comments and concerns. The revised version of this manuscript has been improved, thanks to you.
Reviewer 1:
Comments and Suggestions for Authors
According to the introduction of the manuscript, the author reported L58, 59, 62, many studies earlier reported the chitin and even in tomato, how this study is different from others?
Response to reviewer: That section of the introduction was modified for clarity in the revised manuscript.
New text reads:
However, in those studies, no information is included about how chitin is perceived, or about the molecular mechanism through which the defense response is induced in fruits.
The introduction section is unclear, somehow looks like discussion, specially the last paras, please modify it.
Response to reviewer: The last paragraphs of the introduction was modified for clarity in the revised manuscript.
as per the author, according to the reference 33, how this study is novel? or different?
Response to reviewer: Reference 33 corresponds to the study by Ai et al. (2023), who analyzed the participation of SlLYK4 and SlLYK1 in resistance to disease caused by the tomato fungal pathogen B. cinerea. Although the authors found that SlLYK4 overexpression enhanced disease resistance not only in leaves but also in fruit, in other studies in which the tomato plant-Pseudomonas (31), tomato plant-arbuscular mycorrhizae (32) interaction has been analyzed, the participation of other chitin receptors has been reported. In that sense, it is still not clear if the response of the fruit when exposed to specific fungal molecules such as chitin oligomers of Alternaria is recognized by the same chitin receptors and if the defense mechanism is induced in the same way as has been shown reported in other studies.
A brief explanation was included in the text
Ref 34, 35, are you sure there are no studies reported, specifically, "Fungal pathogen recognition????
Response to reviewer: That section of the introduction was modified for clarity in the revised manuscript.
Even similar studies have been reported earlier in tomato, as per the author, explain why this study is necessary??
Response to reviewer: Thank you very much for the comment. There are similar studies in tomato, in which genes encoding chitin receptors and genes involved in the defense response were over expressed. These gene expressions were observed in tomato plant as a response to different microorganism, such as Pseudomonas syringae (31), arbuscular mycorrhizae (32), tomato cotyledons infected with Clavibacter michiganensis subsp. michiganensis (35), or in transformed tomato plants, in which was analyzed the chitin receptors SlLYK4 and SlLYK1 in the defense mechanism in tomato plants and fruit (33). In these studies, some genes encoding chitin receptors such as SlLYK1, SlLYK4, SlLYK9, SlLYK11, SlLYK12, SlLYK13, CERK1, Bti9, were over expressed depending of the microorganism that interacts with the different tomato tissues. Furthermore, it is important to comment that treatments with live fungus induce a strong response in plants or fruits because the plant is faced with a very complex response/defense mechanism of the fungus, while when the plant is exposed to molecule isolated from the fungus, its response may be different or to a lesser degree. In this sense, it is unclear if the specific fungal molecules, such as chitin oligomers obtained from Alternaria, are recognized in tomato fruit by the same chitin receptors reported previously and if the defense mechanism is induced in the same way as has been reported in other studies.
A brief explanation was included in the text.
The data is only extracted and dependent on the RNA-seq and there is no physiological data.. also in the conclusion that this is an effective way of conttrolling disease, did the author or any other study reported the physiological, functional studies that control? if so, then how this study is different from those?
Response to reviewer: Thank your for your kind comment. In the present study, tomato responses were evaluated in very short times (30 min post-treatment) to observe the early response to chitin oligomers isolated from the fungus Alternaria, so it was impossible to perform physiological studies. In a previous study carried out in our laboratory, it was observed that these Alternaria chitin oligomers applied to tomato fruits significantly induced the enzymatic activity of the chitinase and glucanase enzymes and also inhibited (78%) the development of the infection caused by A. alternata (Valle-Sotelo et al., 2022).
We would like to carry out the study, including more exposure times and concentrations of chitin oligomers and more analysis of tomato fruits, such as physiological analysis. However, we need to find resources to carry out this study.
ALso, if reported earlier, then I think change the title and the presentation of the study to... THe molecular mechanism by which plants react to chitin....
Response to reviewer: Thank you for your kind comment. Based on our previous responses, the authors consider no change the article title.
The introduction lack clarity and It's confusing.
Response to reviewer: That introduction was modified for clarity in the revised manuscript.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
General comments
The experiments presented in the manuscript aim to elucidate genetic pathways involved in the response of tomato fruits to fungal pathogens in order to improve and design strategies to reduce postharvest loss in tomato. The identification of genes differentially expressed in tomato fruits treated with chitin oligomers from the necrotrophic fungus A. alternata using RNA seq and RT-qPCR is presented. Authors identify upregulation of tomato genes encoding a plasma membrane receptor as well as genes involved in signaling pathways regulated by ethylene and jasmonic acid and pathogenesis-related proteins.
The introduction is well written and informative. Results and discussion may be improved by reorganizing the presentation of the information, which is partly confusing. The quality of the figures is partly deficient. The English writing must be improved.
Detailed comments:
Abstract:
I suggest to shortly comment the method of chitin oligomer preparation and stage of fruit development upon treatment.
Introduction
L55: please correct - Some studies have demonstrated
L63: please correct - showed a significant increase in the enzymatic activity of the chitinase and glucanase
L68: please correct - Due to its characteristics it exhibits
L145: please correct - in which live fungus was used
Materials and Methods:
L160: please correct - They were maintained overnight; L162: Fruits were selected based; L169: Fruits were rinsed with sterile distilled water to remove traces of chlorine and were divided; L167: …groups were exposed…L173: Fruits from the other group were immersed L174:… which was considered; L175:.. the fruit was challenged; L183 The RNA purity was measured; L291:… group were identified…L220:… were considered
L167: These chitin oligomers showed a low molecular weight (≤ 1 kDa), an estimated polymerization degree of < 5, and an acetylation degree of 76.7% - how were these parameters evaluated?
Paragraph 2.3. Postharvest application of chitin oligomers - please indicate: how many fruits were treated/group
Paragraph 2.4. RNA isolation from tomato fruits: Three biological replicates were analyzed for each treatment – this refers to three fruits?
L185: Nuclease-free water was added to the RNA for a final concentration of…? Please add this information.
L213: How many groups were considered as outliers?
L237-238: were the reference genes previously evaluated? (Please add reference)
Results and discussion:
L255: please correct - :…without treatment was performed; L26:.. were mapped to
Letter size in Fig 1B should be increased.
Fig. legend 1 and 2: (F1.1-3) and control (C1.1 and C1.3) – please change as C1.2 is not included
L363: please correct…. which had been shown to have antimicrobial activity
L389: Further, it was also found the Solyc11g010730.1 gene – was this gene found as up-regulated?
Fig. 3: Heatmap includes sample C2 - please clarify if it was considered as outlier or not (see fig. 1 and 2)
L420, L426, L481, L514: on the other side
Table 3: authors should include level of up/down regulation in the present experiment.
L452 Paragraph 2.3 should be 3.3 Please correct title: ….were differentially expressed
L454: …..which play….
L461:… which could be involved in the regulation of….
L489:… these three plant hormones
L498: ….were upregulated
L500: … in the regulation of
Paragraph 3.2. The structure of this paragraph is slightly confusing. I propose to mention together the clone identified, the homologue, and immediately followed by the discussion concerning the gene (function described in other experiments).
Paragraph 2.5 should be 3.5
L484: A model created based on the bibliographic data
The English writing must be improved.
Author Response
We thank the reviewers and editor for the constructive analysis of our manuscript. Below are the answers to all the expressed comments and concerns. The revised version of this manuscript has been improved, thanks to you.
Reviewer 2:
The introduction is well written and informative. Results and discussion may be improved by reorganizing the presentation of the information, which is partly confusing. The quality of the figures is partly deficient. The English writing must be improved.
Response to reviewer: We thank the reviewer and editor for the constructive analysis of our manuscript. Below are the answers to all the expressed comments and concerns. The revised version of this manuscript has been improved thanks to you. The revised manuscript has been checked by editing service to improve the English writing.
Detailed comments:
Abstract:
I suggest to shortly comment the method of chitin oligomer preparation and stage of fruit development upon treatment.
Response to reviewer: Your suggestion was included in the abstract.
Introduction
L55: please correct - Some studies have demonstrated
Response to reviewer: Your suggestion was included in the text.
L63: please correct - showed a significant increase in the enzymatic activity of the chitinase and glucanase
Response to reviewer: Your suggestion was included in the text.
L68: please correct - Due to its characteristics it exhibits
Response to reviewer: Your suggestion was included in the text.
L145: please correct - in which live fungus was used
Response to reviewer: Your suggestion was included in the text.
Materials and Methods:
L160: please correct - They were maintained overnight; L162: Fruits were selected based; L169: Fruits were rinsed with sterile distilled water to remove traces of chlorine and were divided; L167: …groups were exposed…L173: Fruits from the other group were immersed L174:… which was considered; L175:.. the fruit was challenged; L183 The RNA purity was measured; L291:… group were identified…L220:… were considered
Response to reviewer: Your suggestions were included in the text.
L167: These chitin oligomers showed a low molecular weight (≤ 1 kDa), an estimated polymerization degree of < 5, and an acetylation degree of 76.7% - how were these parameters evaluated?
Response to reviewer: The 2.2 section in materials and methods was modified to indicate how the chitin oligomers were characterized.
Paragraph 2.3. Postharvest application of chitin oligomers - please indicate: how many fruits were treated/group
Response to reviewer: The 2.3 section of materials and methods was modified to indicate how many fruits were used per treatment/group.
Paragraph 2.4. RNA isolation from tomato fruits: Three biological replicates were analyzed for each treatment – this refers to three fruits?
Response to reviewer: The 2.4 RNA isolation from tomato fruit was modified to indicate how many fruits were considered as one biological replicate.
L185: Nuclease-free water was added to the RNA for a final concentration of…? Please add this information.
Response to reviewer: Your suggestion was included in the text.
L213: How many groups were considered as outliers?
Response to reviewer: Your suggestion was included in the text.
L237-238: were the reference genes previously evaluated? (Please add reference)
Response to reviewer: The reference was included in the text.
Results and discussion:
L255: please correct - :…without treatment was performed; L26:.. were mapped to
Response to reviewer: Your suggestions were included in the text.
Letter size in Fig 1B should be increased.
Response to reviewer: Your suggestion was included in the Figure 1B.
Fig. legend 1 and 2: (F1.1-3) and control (C1.1 and C1.3) – please change as C1.2 is not included
Response to reviewer: Your suggestions were included in the Fig. legend 1 and 2.
L363: please correct…. which had been shown to have antimicrobial activity
Response to reviewer: Your suggestion was included in the text.
L389: Further, it was also found the Solyc11g010730.1 gene – was this gene found as up-regulated?
Response to reviewer: Your suggestion was included for clarity in the revised manuscript.
Fig. 3: Heatmap includes sample C2 - please clarify if it was considered as outlier or not (see fig. 1 and 2)
Response to reviewer: Figure 3 was modified for clarity in the revised manuscript.
L420, L426, L481, L514: on the other side
Response to reviewer: Your suggestions were included in the revised manuscript.
Table 3: authors should include level of up/down regulation in the present experiment.
Response to reviewer: Your suggestions were included in the Table 3.
L452 Paragraph 2.3 should be 3.3 Please correct title: ….were differentially expressed
Response to reviewer: Your suggestions were included in the revised manuscript.
L454: …..which play….
Response to reviewer: Your suggestion was included in the revised manuscript.
L461:… which could be involved in the regulation of….
Response to reviewer: Your suggestion was included in the revised manuscript.
L489:… these three plant hormones
Response to reviewer: Your suggestion was included in the revised manuscript.
L498: ….were upregulated
Response to reviewer: Your suggestion was included in the revised manuscript.
L500: … in the regulation of
Response to reviewer: Your suggestion was included in the revised manuscript.
Paragraph 3.2. The structure of this paragraph is slightly confusing. I propose to mention together the clone identified, the homologue, and immediately followed by the discussion concerning the gene (function described in other experiments).
Response to reviewer: Your suggestion was included in the text. The structure of the section 3.2 was modified for clarity in the revised manuscript.
Paragraph 2.5 should be 3.5
Response to reviewer: Your suggestion was included in the revised manuscript.
L584: A model created based on the bibliographic data
Response to reviewer: Your suggestion was included in the revised manuscript.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
the author addressed all the concerns I raised before..
