Novel Poly-(Lactic-Co-Glycolic Acid) Targeted Nanoparticles Conjunct with Antibody for the Enhancement of Antibacterial Activity against Ralstonia solanacearum

Round 1

Reviewer 1 Report

The research paper entitled ‘Novel poly-(lactic-co-glycolic acid) targeted nanoparticles conjunct with antibody for the enhancement of antibacterial activity against Ralstonia solanacearum’ by Yang et al. presents novel conjugates of antimicrobial properties that are aimed at eradicating Ralstonia solanacearum cells. Ralstonia solanacearum is a phytopathogen of high economic importance and currently the available methods to combat this disease-causing agent are scarce. Therefore, the whole topic and the collected data are of high significance and definitely worth releasing them to the public in the Agronomy Journal. However, I assume that the manuscript was written probably by students with lacks in broad microbiological and molecular biology knowledge and little experience with scientific manuscripts. There are a lot of wrong or superficial parts. In general, the whole manuscript is written in not scientific manner and requires a lot of attention given by more experienced scientists. I recommend to reject it, though after laborious work, it should be reconsidered.  

- For the first time I see “simple summary” section in a manuscript submitted to Agronomy. I think this section is not present in the Word template. It should be deleted as the Abstract has a role of summarizing main findings. Besides, this section is written in really bad English and nearly impossible to understand “pathogenicity of the drug” (line 16), “medicial molecule (line 12)” etc.

- Editing process is needed for this manuscript e.g. lack of italics (line 28), errors “Centrifugation was performede to collect” (line 432) etc

- Although I am not a native English speaker myself I think it is necessary to subject the manuscript to professional English correction. Unprofessional or just incorrect statements appear frequently e.g. “powerful disease” (line 40), “Due to this feature, and severity its notoriously known as “green wilt disease (Qing Ku Bing)” in China by farmers and scientists [1].” (lines 43-44), “Hereby, based on PLGA nanoparticles loaded with CAPE and MC, a pharmaceutical conjugated with an antibody was presented” (lines 85-86), “to produce PLGA target nanoparticles (PLGA-TNP) on R. solanacearum” (lines 85-86), “and the results are reflected in the error bars of the figures” (line 516), “Figure 1 shows that the samples of four PCR tubes connected with R. solanacearum-specific antibodies had obvious bands” (line 94), “best activation time” (line 133), “ordinary PCR” (line 281), “normal conditions” (line 295), “normal electrophoretic band”, “The polygalacturonase gene pehC, electrophoresis related gene pilT” (line 330)

- Line 33 – R. solanacearum causes brown rot and bacterial wilt, not “plant blight disease”

- Introduction: there is not enough literature background and scarce scientific details have been presented in the introduction section. In my opinion it is superficial and many facts are written wrongly or overlooked. For instance: “The process of R. solanacearum from infecting plants to plant disease mainly proceeds through two stages, the pre-infection stage and the later stage of infection which is regulated by Type III and Type II secretion systems, and phenotype conversion systems respectively [4]” (lines 49-54) – not only English language is really bad, but also the infection is not regulated by the secretion systems, but transcriptional regulators. Intensification of disease symptoms is associated with virulence factors secreted via the secretion systems. It makes a huge difference. Similar comments apply to the next sentence “These regulatory genes, such as exopolygalacturonases (PehC), the transcription regulatory factors PhcA and PhcBSR that constitute the sensing system [6] and the motility-related gene PilT all play important roles in the virulence of R. solanacearum” (lines 54-58) – Exopolygalacturonases are proteins associated with virulence, in other words enzymes responsible for degradation of plant tissue, not regulatory genes as stated in the manuscript. Line 59 “abuse of antibiotics” – discuss in more literature details. Line 65 “this disease” – what disease? Line 71 – PLGA is not a transformation. Line 79 – “jeopardize other beneficial microorganisms or plant cells in the soil” – Plant cells in soil? In conclusion, I have an impression that this paper was written by unexperienced students with lack of scientific background and insufficient support from senior researchers from this group. Please, consult phytobacteriologists prior to resubmission of this publication.

- Line 83 – inhibition of growth of R. solanacearum

- Materials & methods: Please provide details on the methods by which the isolate from mulberry tree was identified as R. solanacearum. Is this strain deposited in international bacterial collection? If so, please provide the number and name of the collection. I wouldn’t write from which company was purchased with chemical component – this data should be included in parenthesis e.g. phenethyl caffeate (Macleans, China), DEPC water (Takara, China?) and isopropanol (Takara, China?) of [add here information of which purity grade the ingredient was] were used. Provide more details on the RNA extraction and PCR kits used. Still major language mistakes occur e.g. “The immuno-PCR method was employed to verify the specific capture ability of the antibody as described in according to previous research”, “SEM was used to observe the apparent morphology of the targeted nanomedicine after encountering R. solanacearum, and the inhibitory effect of the bacteriostatic agent was observed through the cell membrane and morphology” (lines 470-473). Line 417 – “R. solanacearum and E. coli were diluted 1000 times” – you mean bacterial cultures or bacterial suspensions? In what medium? Cultured for how long, at what temperature? The culture/suspension was of what OD? Please provide details on the primers used (not the targeted genomic region) and amplification parameters of the PCRs intended for detecting R. solanacearum and E. coli (lines 422-423). Line 476 – “R. solanacearum and E. coli containing EGFP genes were…”. What did you mean by EGFP, this abbreviation was not explained in the manuscript. The whole 4.7 section is written in a manner not clear enough to repeat the experiments. Bacteria were cultured on solid or liquid media? Which ones? They were cultured for how long, at what temperature? The cultures were said to be mixed, but it is unclear which ones (solid/liquid cultures? Overnight ones?). Please provide more details and write this section clearly. By ,,The amplification system was 20 μL,..” you mean the PCR reaction volume? Just volumes are presented without concentrations of the reactants. Not a system was denaturated but DNA during PCR reaction. Provide details on the electrophoretic separation. Line 493- “Forty microlitres of overnight cultured R. solanacearum suspension (OD600=1)” should be “40 µl of OD₆₀₀ = 1 R. solanacearum bacterial suspension”, please add information in what medium/solution were the cells suspended (0.85% NaCl, TSB, LB?). Please add more information on qPCR procedures and selection of genes for evaluation of genes expression. Statistical analysis section is really poorly written – “and the results are reflected in the error bars of the figures” (line 515) - this has no meaning.

- Results & Discussion: The results are described in a wrong, superficial or unprofessional manner e.g. “Figure 1 shows that the samples of four PCR tubes connected with R. solanacearum-specific antibodies had obvious bands”, “lower strain content in the PCR tubes” and many more. The data itself are interesting, however, they need proper scientific evaluation, description and discussion. Besides, regarding gene expression assays I don’t understand, why among genes involved in motility and regulation of transcription, the gene encoding polygalacturonase has been selected for evaluation. Please comment on that.  

Description of the Figures was performed in an unprofessional, wrong manner. These statements should describe what is in the figure, not what was done. Fig 8 – what is shown? Means +-SE/ Means +-SD? Please provide appropriate details.

Author Response

We very much appreciate your comments, and in your opinion necessary revisions that needs to be made in upgrading the standards of the manuscript. The manuscript has been duly revised accordingly. In what follows please find the responses to your comments.

Comment 1: For the first time I see “simple summary” section in a manuscript submitted to Agronomy. I think this section is not present in the Word template. It should be deleted as the Abstract has a role of summarizing main findings. Besides, this section is written in really bad English and nearly impossible to understand “pathogenicity of the drug” (line 16), “medicial molecule (line 12)” etc.

Response: We accept this suggestion and have removed the “simple summary” section and given a detailed introduction of the pathogenicity of the drug in the introduction section (lines 50-71).

Comment 2: Editing process is needed for this manuscript e.g. lack of italics (line 28), errors “Centrifugation was performede to collect” (line 432) etc.

Response: The “Centrifugation was performede to collect” has been modified to “The nanoparticles were collected by centrifugation at 8000xg for 5 min.” (line 506).

Comment 3: Although I am not a native English speaker myself I think it is necessary to subject the manuscript to professional English correction. Unprofessional or just incorrect statements appear frequently e.g. “powerful disease” (line 40), “Due to this feature, and severity its notoriously known as “green wilt disease (Qing Ku Bing)” in China by farmers and scientists [1].” (lines 43-44).

Response: The“powerful disease” has been modified to “severe diseases”, the (lines 43-44) are redundant, and has been duly deleted.

Comment 4: “Hereby, based on PLGA nanoparticles loaded with CAPE and MC, a pharmaceutical conjugated with an antibody was presented” (lines 85-86), “to produce PLGA target nanoparticles (PLGA-TNP) on R. solanacearum” (lines 85-86), “and the results are reflected in the error bars of the figures” (line 516).

Response: We have revised the incorrect statement that appeared above, the (lines 85-86) has been modified to “Hereby, a pharmaceutical conjugated with antibodies is proposed based on PLGA nanoparticles loaded with CAPE and MC”, the results of the error bars were corrected.

Comment 5: “Figure 1 shows that the samples of four PCR tubes connected with R. solanacearum-specific antibodies had obvious bands” (line 94), “best activation time” (line 133), “ordinary PCR” (line 281), “normal conditions” (line 295), “normal electrophoretic bands”, “The polygalacturonase gene pehC, electrophoresis related gene pilT” (line 330).

Response: We have revised the incorrect statement that appeared above. “best activation time” has been modified to “optimal activation time” (line 175), “ordinary PCR” has been modified to “Polymerase Chain Reaction” (line 325), “normal conditions” has been modified to “regularity conditions” (line 339), “normal electrophoretic bands” has been modified to “normal electrophoretic band” (line 342), The polygalacturonase gene pehC and electrophoresis related gene pilT” genes are also redescribed in more detail in the manuscript.

Comment 6: There is not enough literature background and scarce scientific details have been presented in the introduction section.  

Response: We accordingly added the references and and introduced in detail in the background (lines 85-93).

Comment 7: For instance: “The process of R. solanacearum from infecting plants to plant disease mainly proceeds through two stages, the preinfection stage and the later stage of infection which is regulated by Type III and Type II secretion systems, and phenotype conversion systems respectively ” (lines 49-54) – not only English language is really bad, but also the infection is not regulated by the secretion systems, but transcriptional regulators. Intensification of disease symptoms is associated with virulence factors secreted via the secretion systems.

Response: We re-narrated and redescribed these two stages from R. solanacearum infection progress.

Comment 8: It makes a huge difference. Similar comments apply to the next sentence “These regulatory genes, such as exopolygalacturonases (PehC), the transcription regulatory factors PhcA and PhcBSR that constitute the sensing system [6] and the motility-related gene PilT all play important roles in the virulence of R. solanacearum” (lines 54-58) – Exopolygalacturonases are proteins associated with virulence, in other words enzymes responsible for degradation of plant tissue, not regulatory genes as stated in the manuscript.

Response: The respective references has been added to both the  background and scientific details of the literature. In the preinfection period of mulberry trees, when R. solanacearum contacted plant cells, R. solanacearum outer membrane receptor proteins are activated to regulate type III and type II secretion systems via two regulatory pathways. The activation of type II secretion system requires the regulation of polygalacturonase (pehC) environmental tolerance gene expression and the activation of pilT gene expression, a flagellar formation and motility-related gene, to accelerate the colonization process of R. solanacearum in plant cells.

Comment 9: Line 59 “abuse of antibiotics” – discuss in more literature details. Line 65 “this disease” – what disease? Line 71 – PLGA is not a transformation. Line 79 – “jeopardize other beneficial microorganisms or plant cells in the soil” – Plant cells in soil?

Response: We accept this suggestion and have revised the incorrect statement that appeared above, The “abuse of antibiotics” has been discussed in detail, “this disease” means bacterial wilt(line 100).

Comment 10: Line 83 – inhibition of growth of R. solanacearum

Response: We have modified “inhibition of R. solanacearum” to“inhibition of growth of R. solanacearum”.

Comment 11: Materials & methods: Please provide details on the methods by which the isolate from mulberry tree was identified as R. solanacearum. Is this strain deposited in international bacterial collection? If so, please provide the number and name of the collection.

Response: The Ralstonia strain used in this project is R. solanacearum physiological race 5, which was isolated from mulberry bacterial wilt after the pathogenicity test showed that this strain can infect mulberry.

Comment 12: Materials & methods: I wouldn’t write from which company was purchased with chemical component – this data should be included in parenthesis e.g. phenethyl caffeate (Macleans, China), DEPC water (Takara, China?) and isopropanol (Takara, China?) of [add here information of which purity grade the ingredient was were used.

Response: We have indicated the details of the chemical materials used in parentheses.

Comment 13: Provide more details on the RNA extraction and PCR kits used.

Response: The manufacturer of the kit used is indicated in the article, and the method of used was carried out with reference to the instructions(line 472).

Comment 14: Lines 470-473 Still major language mistakes occur e.g. “The immuno-PCR method was employed to verify the specific capture ability of the antibody as described in according to previous research”, “SEM was used to observe the apparent morphology of the targeted nanomedicine after encountering R. solanacearum, and the inhibitory effect of the bacteriostatic agent was observed through the cell membrane and morphology”.

Response: We corrected the syntax errors of the mentioned statements.

Comment 15: Line 417 – “R. solanacearum and E. coli were diluted 1000 times” – you mean bacterial cultures or bacterial suspensions? In what medium? Cultured for how long, at what temperature? The culture/suspension was of what OD?

Response: The culture conditions of R. solanacearum and E. coli have been described in detail, where the medium for R. solanacearum is CPG liquid medium at 30°C and for E. coli is LB liquid medium at 37°C. The OD values of both are 0.8~1.0(lines 487-488).

Comment 16: Lines 422-423 Please provide details on the primers used (not the targeted genomic region) and amplification parameters of the PCRs intended for detecting R. solanacearum and E. coli.

Response: The primers used to detect R. solanacearum and E. coli were 16S rDNA and T7, respectively(lines 494-496).

The 16S rDNA primer sequences were 27F: 5'-AGAGTTTGATCCTGGCTCAG -3'

1492R: 5' -GGTTACCTTGTTACGACTT-3'.

T7 primer sequence is T7: 5'-TAATACGACTCACTATAGGG-3'

T7 ter: 5'-TGCTAGTTATTGCTCAGCGG-3'

Comment 17: Line 476 – “R. solanacearum and E. coli containing EGFP genes were…”. What did you mean by EGFP, this abbreviation was not explained in the manuscript. The whole 4.7 section is written in a manner not clear enough to repeat the experiments. Bacteria were cultured on solid or liquid media? Which ones? They were cultured for how long, at what temperature? The cultures were said to be mixed, but it is unclear which ones (solid/liquid cultures? Overnight ones?). Please provide more details and write this section clearly. By ,The amplification system was 20 μL,..” you mean the PCR reaction volume? Just volumes are presented without concentrations of the reactants. Not a system was denaturated but DNA during PCR reaction. Provide details on the electrophoretic separation.

Response: We have defined EGFP and rewritten this section and explained the culture conditions for R. solanacearum and E. coli.

Comment 18:  Line 493- “Forty microlitres of overnight cultured R. solanacearum suspension (OD₆₀₀=1)” should be “40 µl of OD₆₀₀=1 R. solanacearum bacterial suspension”, please add information in what medium/solution were the cells suspended (0.85% NaCl, TSB, LB?).

Response: In this study, CPG liquid medium was used to culture R. solanacearum (line 574).

Comment 19:  Please add more information on qPCR procedures and selection of genes for evaluation of genes expression.

Response: The detailed procedure of qPCR is mentioned in detail in Ref. 13. Specifically, each reaction system (20μL) contains 10μL of TB Green, 0.8μL of upstream primers, 0.8μL of downstream primers, 6.4μL of RNase-free double-distilled water, and 2μL of cDNA. Reaction conditions are 94°C for 5 min; 94°C, 15s, 60°C, 30s, 72°C, 30s, for 45 cycles; 95°C for 10s, 65°C for 60s, 97°C for 1s. Selection of pathogenicity-related genes is described in Ref. 13(line 564-566).

Comment 20: Results & Discussion: The results are described in a wrong, superficial or unprofessional manner e.g. “Figure 1 shows that the samples of four PCR tubes connected with R. solanacearum-specific antibodies had obvious bands”, “lower strain content in the PCR tubes” and many more. The data itself are interesting, however, they need proper scientific evaluation, description and discussion.

Response: The results and the discussed data are corrected and explained by consulting other claims in the literature.

Comment 21: Besides, regarding gene expression assays I don’t understand, why among genes involved in motility and regulation of transcription, the gene encoding polygalacturonase has been selected for evaluation. Please comment on that. 

Response: We introduced this gene because polygalacturonase is related to the degradation of pectin, and in the process of R. solanacearum infesting plants, the degradation of the plant cell wall is the first thing to be done. For the selection of the gene, we performed experiments and adjusted it with reference to the literature13. Polygalacturonase is capable of degrading pectin and is the first step in the infestation of plants by R. solanacearum for colonization, so we investigated this gene.

Comment 22: Description of the Figures was performed in an unprofessional, wrong manner. These statements should describe what is in the figure, not what was done. Fig 8 – what is shown? Means +-SE/ Means +-SD? Please provide appropriate details.

Response: We have corrected Figure 8 and the figure caption and further analyzed the data.

Reviewer 2 Report

The authors of the study developed the poly-(lactic-co-glycolic acid) nanoparticles loaded with methyl caffeate and caffeic acid phenethyl ester to treat R. solanacearum that is responsible to cause bacterial infection in plants.

The manuscript is interesting. However, the following points should be considered to improve the quality of the manuscript before publication.  

1. The English language of the manuscript should be improved before publication.
2. Legends of figure 4 are incomplete.
3. Figure 6 and 7, it is recommended to remove the extra information from the electron microscopic images and use the ImageJ software to draw scale bars.
4. Typesetting of the manuscript needs improvement.
5. The conclusion is long and has redundant information. It should be improved. Please just provide your conclusion and not the detailed results.
6. The authors should explain the mechanistic antibacterial actions of the PLGA-TNPs.
7. The authors of the study can cite the following reference to understand the antibacterial mechanisms;

Javed, B., Ikram, M., Farooq, F., Sultana, T., Mashwani, Z.-R., & Raja, N. I. (2021). Biogenesis of silver nanoparticles to treat cancer, diabetes, and microbial infections: a mechanistic overview. Applied Microbiology and Biotechnology, 1–15. https://doi.org/10.1007/s00253-021-11171-8

Ikram, M., Javed, B., Raja, N. I., & Mashwani, Z.-R. (2021). Biomedical Potential of Plant-Based Selenium Nanoparticles: A Comprehensive Review on Therapeutic and Mechanistic Aspects. International Journal of Nanomedicine, 16, 249–268. https://doi.org/10.2147/IJN.S295053

Author Response

Thank you for your positive comments and the time spent carefully to coordinate the review process of this manuscript. Please find as follows the necessary revisions and responses to the reviewer comments.

Comment 1： The English language of the manuscript should be improved before publication.

Response: Thank you for your suggestions. The format has been thoroughly checked and the grammar has been improved throughout the manuscript.

Comment 2： Legends of figure 4 are incomplete.

Response: The legend of Figure 4 has been modified, we added the figure title to make the legend complete.

Comment 3： Figure 6 and 7, it is recommended to remove the extra information from the electron microscopic images and use the ImageJ software to draw scale bars.

Response: The electron microscope image has been redrawn, removing the superfluous contents. By using the ImageJ revised Figure 6 and 7.

Comment 4： Typesetting of the manuscript needs improvement.

Response: The typesetting of the manuscript has been checked and revised line by line.

Comment 5： The conclusion is long and has redundant information. It should be improved. Please just provide your conclusion and not the detailed results.

Response: The conclusion has been condensed, by removing some specific data, to make it more concise and refined.

Comment 6： The authors should explain the mechanistic antibacterial actions of the PLGA-TNPs in the introductory section.

Response: The mechanism of bacteriostasis has been explained in detail.

Comment 7： The authors of the study can cite the following reference to understand the antibacterial mechanisms.

Response: We have carefully learned these two references and extracted quotable information to make the antibacterial mechanism much more detailed.

Reviewer 3 Report

The manuscript aims to describe "Novel poly-(lactic-co-glycolic acid) targeted nanoparticles conjunct with antibody for the enhancement of antibacterial activity against Ralstonia solanacearum". The article, generally speaking, is well designed, discussed and presented. It is also an extensive article, showing interesting methods and new results that merit publication.   However, some minor corrections are needed before the final acceptance of the manuscript.

1. Some figures are not well explained. Kindl explains it more as figures are the backbone of the article.
2. There are some typographical and grammatical mistakes. Kindly check the paper line by line from start till end.

Author Response

Thank you for your positive comments and the time spent carefully to coordinate the review process of this manuscript. Please find as follows the necessary revisions and responses to the reviewer comments.

Comment 1： Some figures are not well explained. Kindly explains it more as figures are the backbone of the article.

Response: We accept this suggestions. The analysis of the figures in the manuscript has been revised, especially the part of molecular biology, so that the figures can better support and explain the result of the experiment.

Comment 2： There are some typographical and grammatical mistakes. Kindly check the paper line by line from start till end.

Response: We have carefully checked the manuscript, and the typesetting and grammatical errors in the manuscript have been found and corrected.

Round 2

Reviewer 1 Report

The research paper entitled ‘Novel poly-(lactic-co-glycolic acid) targeted nanoparticles conjunct with antibody for the enhancement of antibacterial activity against Ralstonia solanacearum’ by Yang et al. presents novel conjugates of antimicrobial properties that are aimed at eradicating Ralstonia solanacearum cells. Ralstonia solanacearum is a phytopathogen of high economic importance and currently the available methods to combat this disease-causing agent are scarce. Therefore, the whole topic and the collected data are of high significance and definitely worth releasing them to the public in the Agronomy Journal. In general, the parts referring to physicochemical characterization of the PLGA-TNP are well-prepared, while the fragments related to microbiology or phytopathology require further attention. 

- I don’t understand the enclosed Data Availability Statement: ,,No new data were created or analyzed in this study. Data sharing.” (line 621). Could the authors comment on that?

- Still editing process is needed for this manuscript e.g. lack of/additional spaces, dots, commas, italics (lines 58, 70, 192, 189, 192, 207, 212, 562-562), lack of superscripts (-1 in 2.4 section), upper-cases in the middle of the sentence (lines 493-494), typing errors (line 54) etc occur

- Although I am not a native English speaker myself, still English in this manuscript needs to be improved. Examples: ,,Therefore, PLGA-targeted nanoparticles conjugated with antibodies for enhancing activity against R. solanacearum and successfully provided potential as a new antibacterial agent for controlling bacterial wilt.”; ,,The main pathogenic factors of R. solanacearum include extracellular polysaccharide (EPS), cell wall degrading enzymes (mainly cellulase and pectinase) and type III Hrp secretion system effector protein factors, with a very complex pathogenesis [3]” (proteins cannot be pathogenic. The bacterium is pathogenic, proteins can be only pathogenesis-related); ,,The pathogenesis of R. solanacearum-infected plants” (the plants also cannot be pathogenic. Development of bacterial wilt can be divided into two stages…); ,, Thus, an antibacterial agent with effective inhibitory potential against pathogenicity will aid in the curbing of R. solanacearum.” (the pathogenicity is not to be combated, but the pathogen); ,,Therefore, since the reaction time of OD450 is the highest when the reaction time is 40 min, it indicates effective antibody coupling at a reaction time of 40 min.” -> change second ‘is’ to reaches; ,, Therefore, the optimal conditions for the preparation of targeted nanoparticles were prepared by using magnetic stirring…” -> change ‘prepared’ to ‘achieved’; ,, which greatly improves the utilization rate of API and in effect enhances the research abilities of pesticide reduction.”; the inhibitory effect of compound nanoagents containing MC and CAPE on R. solanacearum not only meets the challenge of producing drug resistance”; ,,Therefore, compared with ordinary nanoparticles, targeted nanoparticles may more easily specifically bind with R. solanacearum and destroy the surface structure of the bacterial membrane, resulting in the overflow of R. solanacearum cell contents and its abilities as an antibacterial agent”; ,,The related genes of the core system-phenotypic transformation system regulating the virulence and pathogenicity of R. solanacearum are phcA and phcB, and the related genes egl, are treated with Triton X-100 as the blank control group”; ,,R. solanacearum regulates the expression of environmental tolerance genes of the pehC gene, destroys the plant defencse system, and promotes root invasion and colonization of R. solanacearum”; ,, The decrease in the expression of phcA because CAPE has a certain inhibitory effect on various transcription factors and transcriptional activation factors in cells”. Please correct the above-listed phrases. Also prepositions and articles in the manuscript are often used wrongly (e.g. line 289, 309, 523).

- Some statements need more details e.g. line 63 ,,which regulates the polygalacturonase” (specify – regulates gene expression, activates the protein? What is the molecular mechanism of this regulation?); line 101 ,,enhanced effect on reactive oxygen species (ROS).” (be more specific – in ROS generation?); line 129 ,, are both used as indicators for investigation” -> were studied; ,, Various high-concentration treatment groups showed high antibacterial rates.” Be more specific;  line 229 ,, which greatly improves the utilization rate of API and in effect enhances the research abilities of pesticide reduction.” -> What did you mean by a pesticide here? The developed PLGA-TNP? Develop the topic. Improve English, add some discussion with the literature.; ,,the inhibitory effect of compound nanoagents containing MC and CAPE on R. solanacearum not only meets the challenge of producing drug resistance” -> What did you mean by drug resistance? Resistance of R. solanacearum to the developed PLGA-TNP? To other pesticides? Please clarify and correct English

- Fig 6 – add names of universal primers and literature reference for amplification conditions.

- There are no information in the manuscript on electrophoretic separation conditions – agarose content, separation buffer, time of separation, voltage, visualization methods. E.g. lines 578-581

- Please correct: line 85 ,,Antibiotics are widely used in agriculture due to their better effects of promoting growth, saving materials, and resisting diseases.” (antibiotics do not resist disease, might prevent disease development, though they are rarely used in plants cultivation (at least in Europe). Beside ,,better effects” seems not too professional); ,,Figure 1 shows that the samples of from four PCR tubes covered connected with R. solanacearum-specific antibodies had yielded characteristic obvious band, while samples containing water and R. solanacearum were used as negative and positive controls, respectively”; Line 188 -> ,,best reaction” -> most suitable reaction conditions; Line 198 ,,lengthy” -> ,,long”; ,,electrophoretic results” line 341 – please be more specific; line 348 ,,results showed that the targeted developed nanoparticles targeted R. solanacearum”. -> correct English

- API should be explained while first mentioned (line 223)

- Lines 256-261 – Is it a continuation of description to Fig 4? Should be presented closer to the picture

- Line 571 – add literature reference for T7 and T7T-based PCR reaction.

- Line 583 - ,,inhibitory ability of gradient agents against” -> ,,inhibitory effects of antibacterial agents applied in different gradients against”

- Add information which SEM and TEM devices were used. Model (Company).

- ,,Analysis of the results showed that the targeted NPs mainly acted on the later stage of R. solanacearum infection, but also had a certain inhibitory effect on the invasion and colonization of R. solanacearum in the early stage of infection.” If I understood correctly, qPCR was done just in vitro on R. solanacearum cells. If gene expression would be performed during plant invasion by R. solanacearum, such assumptions could have been drown. On the data that authors collected just variation in the gene expression might be pointed. Without linkage to plant infection. One phrase on importance of these genes in disease symptoms development could be added later on, but for sure no inhibitory effect on the invasion and colonization of the host was shown.   

- Regarding microscopic visualizations. Statement ,,R. solanacearum (or other bacterium) under normal conditions” (or regularity conditions) is not detailed and professional enough. How was the sample prepared – were R. solanacearum cells suspended in 0.85% NaCl, other buffer or culture media? You can use the term non-treated R. solanacearum cells instead of under normal conditions.

- add reference or contents of the TMB color developing solution and stop solution (section 4.4)

- add information on normalization during qPCR assays (section 4.9). How many repetitions were preformed? Line 603 ,,pathogenicity” lower case

- line 133 Identification of the specific binding ability of the R. solanacearum-targeting antibody

- line 324 – Verification of PLGA-TNP targeting abilities

- Line 392 – please, correct it is wrong ,- , expression of environmental tolerance genes of the pehC gene”

- appropriate literature reference should be provided for statements on acidic character of soil on mulberry plantations (lines 293-294)

- Table 2 – I don’t see data on the quality of MC and CAPE required.

- Figs 6-7. There is no information for how long bacterial cells have been exposed to the treatment prior to visualization.  

- bacterial strains are often mistaken with bacterial cells or bacterial suspension. E.g. line 415 the cells have been treated not strains.; line 500

- ,,The morphological changes of E. coli and R. solanacearum cells after the action” line 326. ,, when the cell morphology surface of R. solanacearum was treated” line 335 Morphology cannot be treated

- line 365. ,,The perforation of the polymer nanoparticles causes many regular circular holes in the R. solanacearum cell membrane.” What did you mean? Release of nanoparticles from the polymer structure leads to formation of regular circular holes in the R. solanacearum cell membrane?

- There is something wrong line 380 ,,electrophoresis related gene pilT” -> rather motility-related

- Line 447 - potentially valuable in the prevention and treatment of R. solanacearum infection.

- ,,the virulence and pathogenicity of R. solanacearum” line 383, 404 -> I would write just about pathogenicity. ,, the virulence and pathogenicity of R. solanacearum infection” I would write about pathogenicity of R. solanacearum and epidemiology of bacterial wilt. Infection cannot be pathogenic. It is just caused by a pathogen.

- line 405 ,, plant cell wall degradase” the proper term is plant cell wall degrading enzyme

- Line 497 – I would write about adsorbance of the cells not membranes

- Line 570 – add the concentration of what?

- Line 572 is messy - ,,T7-peimer, 0.5 μL of primer T7T-primer”. Still no final concentrations of the reactants. Line 575 ,,save” is not correct in this context

There are still unaddressed issues from the former review:

- Materials & methods: Please provide details on the methods by which the isolate from mulberry tree was identified as R. solanacearum. Is this strain deposited in international bacterial collection? If so, please provide the number and name of the collection. If it is an isolate identified to the species level by the laboratory, the identification method should be described in the manuscript. ,,Lab isolating strains” is not in proper English and not detailed enough.

- M&M I wouldn’t write from which company was purchased with chemical component – this data should be included in parenthesis e.g. phenethyl caffeate (Macleans, China), DEPC water (Takara, China?) and isopropanol (Takara, China?) of [add here information of which purity grade the ingredient was] were used. There are no phrases like were used/were utilized etc

- M&M PCR lines 570-574. Just volumes are presented without concentrations of the reactants. ­­This issue was not addressed

- M&M Statistical analysis section is really poorly written – “and the results are reflected in the error bars of the figures” (line 515) - this has no meaning. This issue was not addressed. For instance there are no information on the statistical test used for comparison between data shown in fig 8. No information on normality or equality of variances for checking whether to use parametric or nonparametric analysis is depicted. Under Fig. 8 still there is no information what was shown. Means +/- standard errors? Please add this information. Still the statement ,,results are reflected in the error bars of the figures” is there. I assume that the data were showed as means +/- standard errors. Error bars may only refer to variability in the dataset.

Author Response

Response: We very much appreciate your comments and the manuscript has been revised according to the necessary revisions. In what follows please find the responses to your comments.

Comment 1: - I don’t understand the enclosed Data Availability Statement: ,,No new data were created or analyzed in this study. Data sharing.” (line 621). Could the authors comment on that?

Response: Deleted.

Comment 2: - Still editing process is needed for this manuscript e.g. lack of/additional spaces, dots, commas, italics (lines 58, 70, 192, 189, 192, 207, 212, 562-562), lack of superscripts (-1 in 2.4 section), upper-cases in the middle of the sentence (lines 493-494), typing errors (line 54) etc occur

Response: Thank you for your positive comments and the time spent carefully coordinating the review process of this manuscript. The formatting errors mentioned above have been corrected.

Comment 3: Therefore, PLGA-targeted nanoparticles conjugated with antibodies for enhancing activity against R. solanacearum and successfully provided potential as a new antibacterial agent for controlling bacterial wilt.”; ,,The main pathogenic factors of R. solanacearum include extracellular polysaccharide (EPS), cell wall degrading enzymes (mainly cellulase and pectinase) and type III Hrp secretion system effector protein factors, with a very complex pathogenesis [3]”

Response: We accept this suggestion and have been modified to “Therefore, PLGA-targeted nanoparticles not only enhance the activity against R. solanacearum but also provide a new idea for controlling bacterial wilt”. “type III Hrp secretion system effector protein factors, with a very complex pathogenesis” to“type III Hrp secretory system effector protein factors associated with pathogenesis”. (Line 43-44)

Comment 4: Thus, an antibacterial agent with effective inhibitory potential against pathogenicity will aid in the curbing of R. solanacearum.” (the pathogenicity is not to be combated, but the pathogen); ,,Therefore, since the reaction time of OD450 is the highest when the reaction time is 40 min, it indicates effective antibody coupling at a reaction time of 40 min.” -> change second ‘is’ to reaches;

Response: We have revised the incorrect statement that appeared above. “Thus, this antibacterial agent has potential to effectively inhibit the pathogenicity of R. solanacearum”. “Consequently, the OD₄₅₀ was the highest when the reaction time is 40 min, it indicating that antibody coupling was the best when the reaction time was 40 min” and change second ‘is’ to reaches. (Line 188-190)

Comment 5: Therefore, the optimal conditions for the preparation of targeted nanoparticles were prepared by using magnetic stirring…” -> change ‘prepared’ to ‘achieved’; which greatly improves the utilization rate of API and in effect enhances the research abilities of pesticide reduction.

Response: We modified the issues mentioned above. (Line 193)

Comment 6: the inhibitory effect of compound nanoagents containing MC and CAPE on R. solanacearum not only meets the challenge of producing drug resistance”; ,,Therefore, compared with ordinary nanoparticles, targeted nanoparticles may more easily specifically bind with R. solanacearum and destroy the surface structure of the bacterial membrane, resulting in the overflow of R. solanacearum cell contents and its abilities as an antibacterial agent”;

Response: We have been modified “the inhibitory effect of compound nanoagents containing MC and CAPE on R. solanacearum not only meets the challenge of producing drug resistance” to “the inhibitory effect of compound nanoagents containing MC and CAPE on R. solanacearum not only solved the problem of developing drug resistance”, (line 313) and explained the targeted nanoparticles antibacterial ability has been proven. (Line 338)

Comment 7: The related genes of the core system-phenotypic transformation system regulating the virulence and pathogenicity of R. solanacearum are phcA and phcB, and the related genes egl, are treated with Triton X-100 as the blank control group”;

Response: The error has been corrected. (Line 383)

Comment 8: R. solanacearum regulates the expression of environmental tolerance genes of the pehC gene, destroys the plant defencse system, and promotes root invasion and colonization of R. solanacearum”;

Response:  This sentence has been revised. “R. solanacearum could well complete the process of colonization in plants by regulating the environmental tolerance gene pehC, which could destroy the plant defense system, and promotes root invasion and colonization of R. solanacearum.”. (Line 390-396)

Comment 9: The decrease in the expression of phcA because CAPE has a certain inhibitory effect on various transcription factors and transcriptional activation factors in cells”. Please correct the above-listed phrases. Also prepositions and articles in the manuscript are often used wrongly (e.g. line 289, 309, 523).

Response: We have corrected the issues mentioned above.

Comment 10: Some statements need more details e.g. line 63 ,,which regulates the polygalacturonase” (specify – regulates gene expression, activates the protein? What is the molecular mechanism of this regulation?);

Response: More information has been added in Line 67-71. The molecular mechanism of this regulation is that plant cell signaling is transmitted to PehR via the outer model receptor protein PehS of Ralstonia solanacearum and regulates the expression of environmental tolerance genes such as exopolygalacturonases ( PehC ).

Comment 11: line 101 ,,enhanced effect on reactive oxygen species (ROS).” (be more specific – in ROS generation?); line 129 ,, are both used as indicators for investigation” -> were studied;

Response:  The details have been described. The reactive oxygen species (ROS) produce and increase oxidative stress in cells. (Line 91-92)

Comment 12: Various high-concentration treatment groups showed high antibacterial rates.” Be more specific;  line 229 ,, which greatly improves the utilization rate of API and in effect enhances the research abilities of pesticide reduction.” -> What did you mean by a pesticide here? The developed PLGA-TNP? Develop the topic.

Response: We have been added a detailed discussion and the pesticide here means PLGA-TNP. (Line 223-227)

Comment 13: - Fig 6 – add names of universal primers and literature reference for amplification conditions.There are no information in the manuscript on electrophoretic separation conditions – agarose content, separation buffer, time of separation, voltage, visualization methods. E.g. lines 578-581

Response: The universal primers name has been added in Line 550-558. The electrophoretic separation conditions have already been added in Line 556-558.

Comment 14：Please correct: line 85 ,,Antibiotics are widely used in agriculture due to their better effects of promoting growth, saving materials, and resisting diseases.” (antibiotics do not resist disease, might prevent disease development, though they are rarely used in plants cultivation (at least in Europe). Beside ,,better effects” seems not too professional);

Response: We have replaced it with a more professional expression. (Line 73-75)

Comment 15: Figure 1 shows that the samples of from four PCR tubes covered connected with R. solanacearum-specific antibodies had yielded characteristic obvious band, while samples containing water and R. solanacearum were used as negative and positive controls, respectively”; Line 188 -> ,,best reaction” -> most suitable reaction conditions; Line 198 ,,lengthy” -> ,,long”; ,,electrophoretic results” line 341 – please be more specific; line 348 ,,results showed that the targeted developed nanoparticles targeted R. solanacearum”. -> correct English

Response: We have replaced it with a more correct expression.

Comment 16:API should be explained while first mentioned and add information which SEM and TEM devices were used. Model (Company).

Response: We have carefully checked the whole manuscript, added the explanations and the information.

Comment 17: Lines 256-261 – Is it a continuation of description to Figure 4? Should be presented closer to the picture. Line 583 - , inhibitory ability of gradient agents against” -> “inhibitory effects of antibacterial agents applied in different gradients against”.

Response: After we check the description，we confirm that is not the continuation of the description to Figure 4. And we have replaced the “inhibitory ability of gradient agents against” with a more correct expression.

Comment 18: Various high-concentration treatment groups showed high antibacterial rates.” Be more specific;  line 229, which greatly improves the utilization rate of API and in effect enhances the research abilities of pesticide reduction.” -> What did you mean by a pesticide here? The developed PLGA-TNP? Develop the topic.

Response: We have been added a detailed discussion and the pesticide here means PLGA-TNP. (Line 223-227)

Comment 19: - Regarding microscopic visualizations. Statement ,,R. solanacearum (or other bacterium) under normal conditions” (or regularity conditions) is not detailed and professional enough. How was the sample prepared – were R. solanacearum cells suspended in 0.85% NaCl, other buffer or culture media? You can use the term non-treated R. solanacearum cells instead of under normal conditions.

Response: Thank you for your positive comments and the time spent carefully coordinating the review process of this manuscript. The R. solanacearum cells suspended culture media and “normal conditions” have changed to “non-treated R. solanacearum cells”. (Line 327)

Comment 20: Add reference or contents of the TMB color developing solution and stop solution (section 4.4)- add information on normalization during qPCR assays (section 4.9). How many repetitions were preformed? Line 603 ,pathogenicity” lower case

Response: TMB chromogenic solution was prepared by mixing the dihydrate tetramethylbenzidine solution with citric acid solution in equal amount. The termination solution was 0.5 mol sulfuric acid, which was added at Line 508-513. The internal reference gene of R. solanacearum was 16S rRNA, and three biological replicates were performed.(Line 588)

Comment 21: Line-133 Identification of the specific binding ability of the R. solanacearum-targeting antibody

Line- 324 - Verification of PLGA-TNP targeting abilities

Line 392 - please, correct it is wrong , expression of environmental tolerance genes of the pehC gene”.

Response: We have modified the issues mentioned above.

Comment 22: appropriate literature reference should be provided for statements on acidic character of soil on mulberry plantations (lines 293-294)

Table 2 – I don’t see data on the quality of MC and CAPE required.

Response: We add the literature reference on the acidic character of soil on mulberry plantations; MC and CAPE are encapsulated as pharmacodynamic molecules of PLGA-TNP, and the related information has been added in Table 2.

Comment 23: Figs 6-7. There is no information for how long bacterial cells have been exposed to the treatment prior to visualization. - bacterial strains are often mistaken with bacterial cells or bacterial suspension. E.g. line 415 the cells have been treated not strains.; line 500.

Response: We modified the issues mentioned above. And “strains” has changed to “bacterial cells”.(Line 420)

Comment 24: The morphological changes of E. coli and R. solanacearum cells after the action” line 326. when the cell morphology surface of R. solanacearum was treated” line 335 Morphology cannot be treated.

Response: The time spent carefully to coordinate the review process of this manuscript. The error has been corrected.

Comment 25: line 365. The perforation of the polymer nanoparticles causes many regular circular holes in the R. solanacearum cell membrane.” What did you mean? Release of nanoparticles from the polymer structure leads to formation of regular circular holes in the R. solanacearum cell membrane?

Response: It means that the release of MC and CAPE in nanoparticles will damage the cell membrane and flagellar structure of R. solanacearum, which will lead to morphological changes and pores on the surface of R. solanacearum. More details have been added in Line 362-365.

Comment 26：There is something wrong line 380 “electrophoresis related gene pilT” -> rather motility-relatedthe virulence and pathogenicity of R. solanacearum” line 383, 404 -> I would write just about pathogenicity. “ the virulence and pathogenicity of R. solanacearum infection” I would write about pathogenicity of R. solanacearum and epidemiology of bacterial wilt. Infection cannot be pathogenic. It is just caused by a pathogen.

Response: “electrophoresis related” was replaced with “motility-related” and “virulence” was deleted. (Line 380, 383)

Comment 27：Line 405 “ plant cell wall degradase” the proper term is plant cell wall degrading enzyme. Line 497 – I would write about adsorbance of the cells not membranes. Line 570 – add the concentration of what?

Response: We have carefully checked the whole manuscript，added the explanations and the information.

Comment 28：Line 572 is messy - “T7-peimer, 0.5 μL of primer T7T-primer”. Still no final concentrations of the reactants. Line 575 “save” is not correct in this context.

Response: We delete the “-peimer” and replace the word “save” with “stored”. (Line 554)

Comment 29：Materials & methods: Please provide details on the methods by which the isolate from mulberry tree was identified as R. solanacearum. Is this strain deposited in international bacterial collection? If so, please provide the number and name of the collection. If it is an isolate identified to the species level by the laboratory, the identification method should be described in the manuscript. “Lab isolating strains” is not in proper English and not detailed enough.

Response: We replace the expression of identification method about strains. (Line 455)

Comment 30: The company was purchased with chemical components – this data should be included in parenthesis e.g. phenethyl caffeate (Macleans, China), DEPC water (Takara, China?) and isopropanol (Takara, China?) of [add here information of which purity grade the ingredient was] were used. There are no phrases like were used/were utilized etc.

Response: We modified the issues mentioned above. (Line 457-466)

Comment 31: PCR lines 570-574. Just volumes are presented without concentrations of the reactants. ¬¬This issue was not addressed.

Response: The corresponding substrate concentration is 998 ng/μL. (Line 585)

Comment 32：M&M Statistical analysis section is really poorly written – “and the results are reflected in the error bars of the figures” (line 515) - this has no meaning. This issue was not addressed. For instance there are no information on the statistical test used for comparison between data shown in fig 8. No information on normality or equality of variances for checking whether to use parametric or nonparametric analysis is depicted. Under Fig. 8 still there is no information what was shown. Means +/- standard errors? Please add this information. Still the statement “results are reflected in the error bars of the figures” is there. I assume that the data were showed as means +/- standard errors. Error bars may only refer to variability in the dataset.

Response: We have added the standard errors in the revised manuscript.