Phylogenetic Analyses and Transcriptional Survey Reveal the Characteristics, Evolution, and Expression Profile of NBS-Type Resistance Genes in Papaya

Round 1

Reviewer 1 Report

The work represents a contribution to the knowledge of  R-genes (also known as NB-LRRs) in terms of the evolutionary process that allowed this gene family to be contracted while conserving their resistance function in the papaya. The characterization and micro-colinearity analyses of NB-LRR gene families allow them to postulate a mechanism of insertion-derived origin in papaya genome. The characterization of NB-LRRs generates additional information to improve the understanding of this important gene family.

 

Major point:

- In the discussion section, the shortcomings of this study should be also discussed.

- The authors should, at the very least, cite and discuss the recent literature (Andolfo et al., 2022 - https://doi.org/10.1111/tpj.15756).

Author Response

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Reviewer 2 Report

Review of the manuscript: “Phylogenetic Analysis and Transcriptional Survey Reveal the characteristics, evolution and expression profile of NBS0type resistance genes in Papaya”.

Plant represents several types of defense against infection. The most promoted one is gene by gene interaction. The common sequences carrying RGAs motives have the impact in evolutionary changes of the resistance mechanisms. The phylogenetic of inheritance of plant resistance genes is always the target of breeder research.

The presented manuscript summarizes the new value of the analysis based on in silica application. Comparison of the collected data with different species performed in this study gave the huge information about the papaya genome speciation in plants, and explains the trait complexity of papaya resistance to different stressors (such as fungi, drought, viruses et cet.).

In my opinion the manuscript gives big insight in papaya genome characterization. In precisely uncover the mechanisms of plant diversification. It shows directly the power of the plant modeling using collected data and available databases.

The manuscript can be accepted in this form.

Suggestion to change the sentence in line 267: WGCNA was performed based on transcription data of papaya…….

The NBS sequence name is sunset03G0022700 --- perhaps there is a mistake and should be sunset03G0022770 – line 274.

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Reviewer 3 Report

R genes are one of the most difficult gene families to study in plants. Jiang et al., identified a set of high-confidence NBS-type R genes in papaya from the latest high-quality contiguous papaya genome. In addition, they investigated gene structure, gene family evolution, expression profile during stress conditions of this group of genes, providing insights into breeding new resistant cultivars. I have two major comments and several minor ones for this manuscript:

 

Major comments:

Section 3.4 and 3.5:

From the current structure, it’s hard to grasp the significance of section 3.4, i.e. what new information can be learned from this section. If the authors are intended to discussion the evolution of this gene family, I’d suggest to combine section 3.4 and 3.5, and then change the heading to: evolutionary origin of NLR genes in papaya. 

The authors NEED to add more species in the phylogenetic tree. Gene family tree from one single species is not sufficient to infer the evolutionary history of this gene family. I’d suggest the authors to re-run the phylogenetic tree with genes from Table 1. An taxon rich phylogenetic tree plus the synteny analysis from section 3.5 will provide stronger evidence towards the evolutionary origin of papaya NLRs.

 

Supplementary material:

I do not see any supplementary material and the link in the manuscript doesn’t work. Please provide those files in the revision.

 

Minor comments (listed in the same order as the manuscript):

Abstract:

The last sentence is hard to understand.

 

Introduction:

Lines 26-27: what new information do the two new genomes provide? Does it validate some of those statements from version 1 of the genome? Since the new genome will be used in this research, might as well describe the improvement here.

 

 

Materials and Methods:

 

Lines 72-73: “EMBOSS Needle used the Needleman-Wunsch algorithm” can be changed to: “EMBOSS Needle with the Needleman-Wunsch algorithm”

 

Line 77: which basal angiosperms were selected as the outgroup? Can you explain why these specific species were selected?

 

Section 2.3: 1) Why were Rhodophyta proteins used to construct phylogenetic trees? 2) The method does not describe how the phylogenetic tree in Figure 4 was generated. 

 

Lines 112-113: Please include the BioProject IDs.

 

Results:

 

Line 141-142: 0.03% of what? Please be more clear in the text.

 

Line 146: How did the author come up with the conclusion that those 59 NLR genes is “a set of complete and simplified NLRs”? I do not see enough evidence supporting either “complete” or “simplified”. In addition, what does simplified mean here? Fewer genes? Or simpler structure? Please be more specific. 

 

Figure 4: what does the numbers at the nodes mean? Is that bootstrap values? How are those numbers generated? How many bootstrap replicates were performed? 

 

Line 244, “Colletotrichum brevisporum” need to be italicized. 

 

Figure 7. The spelling of “drought”is incorrect. 

 

General language comments:

The writing in some parts of this manuscript is too colloquial (e.g. Line 40: Do not use ‘they’, use ‘AUTHOR et al’ instead.). The authors need to go over the entire manuscript and improve the language. 

Author Response

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Reviewer 4 Report

Dear Authors

I have few major suggestions to improve the manuscript.

1.       To identify candidate genes (NBS- type Resistanace) qRT-PCR is needed to validate the hypothesis as claim by authors, as in-silico data is only the prediction model.

2.       I have seen many diagrams of in silico data of the manuscript, these diagrams need to discuss properly in the whole manuscript with clear information.

3.       Conclusion part is missing in the manuscript. It should be added as separate heading with outcome and future directions of work.

4.       References need to be update.

Author Response

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Reviewer 5 Report

 

In this study, the authors use genome data to dig the NLR gene family in papaya. The aims of this study are clear and the data mining is a good job to figure out the whole view of the NLR gene family in papaya. I have some suggestions for this study:

First, the link between papaya and other species is not clear. It is hard to understand the orthologous or paralogous between papaya and other taxa. There is a lot of bio-information, but it is hard to link in this section. In addition, the authors like to use “in papaya and related species”, but papaya and other species used in this manuscript are deeply divergence. 

Second, the authors identified 59 NLR genes in papaya. It is important information for future study. However, the authors write the “3.3 Chromosome distribution and duplication modes of NLRs in papaya” and illustrate they find “79 duplication gene pairs”. Here, it is really confusing the total number of NLR genes in papaya. 

Third, the authors reconstructed the phylogeny of NLRs in papaya with 28 papaya NLRs (R/T/CNLs). Here, it is hard to catch the point that the authors do not use 59 NLR genes that they find but selected 28 papaya NLRs (R/T/CNLs) to do this analysis. 

Author Response

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Round 2

Reviewer 3 Report

Nice work! Thanks you for making edits to the suggestions. I only have some minor suggestions on the phylogeny: 

What does the color boxes mean in Figure 4A? Are those colors highlighting the same classes as in Figure A1? Please make the colors between these two figures consistent.

The motifs in Figure 4B are very hard to see, please make the colors more clear and bigger.

Since you have basal angiosperms in your tree (Figure A1), I don’t think you need to have red algae to help root the tree. But it’s Ok to keep them in. 

Lines 319-320: Where are those two sequences in the big tree (Figure A1)? 

Author Response

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Reviewer 4 Report

Authors have revised the manuscript very nicely. Therefore, their manuscript can be accepted for publication in agronomy. 

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