Coexpression Network Analysis Based Characterisation of the R2R3-MYB Family Genes in Tolerant Poplar Infected with Melampsora larici-populina

Round 1

Reviewer 1 Report

Work by Chen et al. conducted co-expression analysis to discover the MYB transcription factors involvement in E4-infected poplar. 

Revisions:

Figure legends for supplemental figures need to provide detailed information. In FigureS13-S18, there is no information provided such as number of biological samples. There is no relevant statistical analysis performed either. 

In table S1, regarding primer information, are the primers 5' to 3'?

Resolution of the figures needs to be improved. Size of many words in the figure are difficult to see (e.g. Figure 1, Figure 2, Figure 4, etc.). 

Author Response

1. Figure legends for supplemental figures need to provide detailed information. In FigureS13-S18, there is no information provided such as number of biological samples. There is no relevant statistical analysis performed either.

 

Thank you for your comments. We’ve added detailed information for FigureS13-S18. Data are given as mean with s.d. (n=3; *, p value of ‘Intolerant’ and ‘Tolerant 2’ or ‘Tolerant 1’ were both less than 0.05).

 

2. In table S1, regarding primer information, are the primers 5' to 3'?

 

Thank you for your comments. We’ve added primer information. The primers are 5' to 3'.

 

3. Resolution of the figures needs to be improved. Size of many words in the figure are difficult to see (e.g. Figure 1, Figure 2, Figure 4, etc.).

 

Thank you for your comments. We’ve improved the resolution of the figures and enlarged the font in Figure 1, Figure 2, Figure 4 and Figure 6.

Reviewer 2 Report

See attached file.

Comments for author File: Comments.pdf

Author Response

• Title seems a bit clumsy. Please consider rephrasing it. Maybe “Coexpression Network Analysis based Characterization of the R2R3-MYB Family Genes in Tolerant Poplar Infected with Melampsora larici-populina”?

 

Thank you for your comments. We’ve changed the title.

 

• Consider deleting the word “molecularly” in row 13.

 

Thank you for your comments. We’ve deleted it.

 

• Please consider mentioning that R2R3-MYB family genes represent transcription factors in the abstract. It might not be obvious to a reader not familiar with this topic.

 

Thank you for your comments. We’ve added this information in the abstract.

 

• Consider replacing the keyword “E4”. I suggest “transcription factors”. If you wish to keep “E4” in the keywords, you could write it as “Melampsora larici-populina E4”.

 

Thank you for your comments. We’ve replaced E4 with transcription factor.

 

• In row 113, please delete “sequencing reads which containing”.

 

Thank you for your comments. We’ve deleted these words.

 

• Do you mean “transcriptome” in row 122?

 

Yes, we’ve replaced transcript with transcriptome.

 

• Replace “was” with “were” in row 128.

 

We’ve replaced “was” with “were”.

 

• About the sentence “MEs were correlate with external traits (SA and IAA levels) to look for the most significant associations.” – maybe it’s better to write “Correlation analysis between MEs and external traits (SA and IAA levels) were performed to look for the….)

 

Thank you for your comments. We’ve modified this sentence accordingly.

 

• Avoid using “Real-Time Quantitative PCR” in rows 184 and 185. You do in fact mean Reverse transcription Quantitative PCR!

 

Thank you for your comments. We’ve modified these words.

 

• I suggest changing the title of section 3.1 to represent the step of the workflow (identification of R2R3-MYBs), not the achieved result.

 

Thank you for your comments. We’ve modified the title of section 3.1.

 

• In row 212 the term “grey module” appears. It is not explained before or later what it is. Besides, in table S4 there is no “grey module”. There is the Dark gray module and Grey 60 module. Clarify this situation.

 

We should not ignore the discussion of grey modules here. Grey module was reserved for unassigned genes. We’ve added the information in the materials and methods parts (line 144) and grey module was included in table S4.

 

• In row 220 you refer to Figure 1C but there is no C section in Figure1! Correct this situation!

 

Here we made a mistake, the correct way to write should be Figure 1A.

 

• In row 222, before “mainly” add “observable” or “present” or “detectable”, check the punctuation of this sentence.

 

Thank you for your comments. We’ve added “observable” before “mainly”.

 

• Are the sentences “The ME took on low values as many of module genes were underexpressed. The ME took on high values as many module genes were overexpressed.” necessary?

 

Thank you for your comments. We’ve deleted these sentences.

 

• What values are the ME values mentioned in row 230? ME expression values? Maybe this needs a bit of clarification.

 

ME is considered representative of the gene expression profiles in a module. “ME value” refers to the calculated result. To avoid ambiguity, we delete “value” and write them as ME. At the same time, we also delete the "value" in “MM value”, “IC value” and “GS value”, and write only MM, IC and GS.

 

• I suggest to change the title of section 3.3 to “Differential Expression of R2R3-MYB Genes”.

 

Thank you for your comments. We’ve changed the title of section 3.3.

 

• Description of figure 6 could be improved a bit – you don’t explain what’s depicted with red and black lines in figure 6C.

 

Thank you for your comments. We’ve changed figure 6.

 

• The first sentence after Figure 6 (rows 439-442) could be shortened. I think that part of the information provided in this sentence is not necessary.

 

Thank you for your comments. We’ve deleted the unnecessary information in this sentence.

 

• Rephrase the title of section 3.5 to reflect on the step of the workflow, not the results

 

Thank you for your comments. We’ve changed the title of section 3.5 into “Analysis of Interactions between R2R3-MYB Genes, IAA and Free SA”.

 

I enjoyed reading your work. I recommend it for publication with minor changes. Still, one thing that doesn’t leave my mind is that both tolerant individuals are a result of crossing P. deltoides with P. trichocarpa while the intolerant individual is a hybrid of P. nigra and P. deltoides. Couldn’t this fact introduce significant differences in the regulatory 5’ flanking regions of the genes involved and, thereby, influence the results of your study? I am truly not a specialist when it comes to the Populus genus so I don’t know whether it’s possible to find a P. deltoides x P. trichocarpa which is intolerant but, if that is possible, it could provide even more accurate data.

 

Thank you very much for your extremely good and useful advices on “identifying of a P. deltoides × trichocarpa that is intolerant” and “5′-flanking region differences”. Unfortunately, the rust intolerant P. deltoides × trichocarpa is hard to be found at present. So far, we have identified many poplar families of P. deltoides × trichocarpa and P. trichocarpa × deltoides, all of them are tolerent to E4 rust. The tolerance or intolerance of the poplar depends on the interaction of the poplar and the rust. Perhaps after the emergence of a new rust strain, P. deltoides × trichocarpa will be intolerant to it. Such as P. nigra × deltoides is tolerant to rust (E1~E3) until the appearance of the E4.

 

On the other hand, the promoter and other cis-acting transcription regulatory sequence elements in the 5′-flanking regions of those genes, is an excellent research direction to reveal the mechanism of poplar tolerance to the rust. Next, we plan to perform the genome sequencing of these poplars which should be able to comprehensively reveal the tolerance mechanism in the field of 5′-flanking region.