Histological and Molecular Characterization of the Musa spp. x Pseudocercospora musae Pathosystem

We thank the reviewers for critically reviewing the manscript. All comments were taken into consideration in order to remakably improve the manuscript.

Reviewer 1:

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General comment

Overall, the manuscript presents interesting findings that describe the histological techniques used to study interaction between banana and yellow sigatoka. However, it is not clear which genes are associated with resistance so that these can be used to develop genetic markers for use by breeders. The authors do not clearly bring out the next steps beyond the findings of this research. Also, there are several typos that need to be corrected. A lot of the content written in the discussion should be transferred to the results section.

Specific comments

1)     Cultivar Yangambi is Km5 and not 45. Also check the spelling of Yangambi and correct it from Yagambi to Yangambi, especially in the abstract.

Corrections made in the text.

 2)     Please rephrase this sentence: As for the WAK2 gene both were initially upregulated but in the Grande Naine, there was a subsequent repression, while in the Yagambi km 45, it remained downregulated.

Correction made in the text.

 3)     Write out BOD in full when mentioning it the first time.

Correction made in the text.

 4)     Line 131: histochemical tets I suppose you mean tests*

Corrections made in the text.

5)     Line 131-134: Why did you choose slides at 120hai? Include this reason in your text.

Corrections made in the text.

 6)     Line 139: What type of leaf samples? Whole leaf? Leaf discs – of what size?

Corrections made in the text.

 7)     Line 177: Rewrite R² as Coefficient of determination (R²).

Corrections made in the text.

 8)     Figure 1: Regardless of the similarity in anatomy of leaves from the two cultivars, it is important to present pictures of Yangambi Km5 at 72hai and 120hai. Infact, you can choose to present pictures of both cultivars at 72hai in one figure and those of 120hai in another figure.

We understand the importance of the request, and unfortunately these figures are not available.  However, Grande Naine fotos were presented for the time-periods stated above since they had infection, as discussed throughtout the manuscript, contrary to Yagambi Km 5, which showed no infection.  Please let us know if you agree with the new detailed discussion presented.

 9)     Table 2: Be consistent in the cultivar names. What is the meaning of I.D. (%) and D.E. est.mm-²

Corrections made in the text accordingly.

I.D (%). = stomatal index; D.E. = stomatal density.  A correção dos nomes também foi realizada.

10)  Please present the pictures for Yangambi and not Grand naine only. You refer to Figure 3E, however, you did not present it.

Please consider it a “typo”. The figure does not exist and this was corrected in the text.

 11)  Figure 4 needs to be improved.

Figure improved

 12)  Some of your discussion should be presented in the result section.

Corrections made in the text accordingly.

 13)  What chemical compound are you referring to in paragraph 312 – 320?

The chemical compounds are phenolic compounds - Corrections made in the text accordingly.

 14)  Question: Can MUSA 3 and 5 be used to develop markers for identifying resistant or susceptible cultivars to yellow sigatoka?

Yes, MUSA 3 and MUSA 5 can be used to develop markers for the identification of cultivars resistant or susceptible to yellow Sigatoka, as significant differences in the expression of these genes can be identified between resistant and susceptible cultivars, with a correlation between specific variants of these genes and resistance/susceptibility to yellow Sigatoka, and the function of these genes in relation to resistance is confirmed by additional experiments.

Reviewer 2

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We thank the reviewer for critically reviewing the manscript. All comments were taken into consideration in order to remakably improve the manuscript.

The manuscript provided by Da Silva et al. aims to better understand the infection process of yellow Sigatoka disease caused by the fungus Pseudocercospora musae in two banana cultivars: the susceptible 'Grande Naine' and the partially resistant 'Yangambi Km 45'. The authors employed histological and microscopy approaches, as well as expression analysis of two banana candidate genes, to elucidate the defense responses of these cultivars during infection.

While the histological experimentation and staining used are interesting to understand the defense response of the Grande Naine variety, it seems not appropriates to monitor the immune response of Yangambi Km 45 since the defense response seems to occur at the leaf surface. The SEM microscopy would have been a better fit to monitored resistance in Yangambi Km 45 if quantified. Several majors' concerns need to be addressed:

1. Stomatal density reduction during infection: The authors claim a reduced number of stomata (stomal index and stomatal density) during infection over time in the 'Grande Naine' variety. However, it is unclear how fungal infection can reduce stomatal density and stomatal index within 24 hours in the susceptible 'Grande Naine" variety. The only explanation for stomata reduction would be a hypersensitive response (HR) that would kill the stomata, but HR occurs in highly resistant cultivars such as Calcutta 4, and even then, stomata would still be visible within 24 hours in such case. Therefore, a reduction in stomatal density by half within 24 hours (between 48- and 72-hours post-infection) in a susceptible 'Grande Naine' variety seems highly unlikely. Can the authors provide an explanation for the developmental reprogramming of stomata in only 24 hours? The authors refer to a previous study (Araujo et al. ) to support their observations in stomata density variation during infection, but this previous paper lacks convincing quantification of stomatal number variation during infection, showing only a series of pictures with different scales. The variation of stomatal density between cultivars is indeed already described, makes sense and can partially explain resistance. But a variation of stomatal density within 24 hours during infection requires additional explanation.

Additional information added to the text accordingly.

2. Defense response in Yangambi Km 45: The authors state they could not observe any defense response in 'Yangambi Km 45' compared to 'Grande Naine', without providing a clear explanation. Indeed, the resistant cultivar is Yangambi Km 45, we would expect a higher immune response compared to Grande Naine and not the opposite as observed by the authors. It is possible that 'Yangambi Km 45' blocks infection at the stomatal level, which would explain the absence of secondary metabolite production inside the leaf. In contrast, the accumulation of phenolic compounds in 'Grande Naine' indicates an active defense response inside the leaf during infection of a susceptible cultivar. Therefore, the authors should have focused more on the stomatal response itself (accumulation of secondary metabolites inside the stomata or epidermal surface or monitoring stomatal closure and not monitoring number of stomata during infection) instead of monitoring the defense response inside the leaf of the two cultivars. Regarding the gene expression analysis, if the aim was to select defense marker genes, the two key genes selected (PRRs) that are monitored by RT-qPCR should be upregulated during infection, not repressed in the resistant cultivar. The authors do not provide a clear explanation regarding the reduction of expression of these two genes in the resistant cultivar. Are these two genes upregulated in the susceptible cultivar in response to infection inside the leaf, but the resistant variety block the infection so early at the stomata level that these two genes are therefore not upregulated? If that's the case, these two genes are not the most representative for comparing the two cultivars immune response to infection.

Additional information added to the text accordingly.

Major Edits:

* Figure 1: Why does the cross-section show only non-infected 'Yangambi' (E)? It should compare the infection processes in both cultivars at the same time-point as for "Grande Naine". Is that because no immune response is observed during infection inside the leaf of Yangambi and therefore indicates the need to monitor the defense response at the epidermal/stomatal level and not inside the leaf.

That is correct. We showed what we observed in the analysis. When there was no infection, it was deemed not so important to show.

* Figure 2: Have the authors quantified the number of penetration events in stomata?

Providing only representative pictures by SEM is insufficient. A figure showing the number of penetration events at different time points in both cultivars would be greatly appreciated and would help to decipher where the immune response of Yangambi is located (blocked at stomata? Or epiphytic growth reduction?).

They were not quantified, only demonstrated by scanning microscopy the hyphae over the stomata as a way to demonstrate their growth. Proof of infection is available in the figures that show the production of phenolic compounds in the times and cultivars analyzed.

* Table 2: The table legend lists the stomatal density (S.D.) and stomatal index (S.I.) of the abaxial surface in paradermal sections of two banana cultivars, but the table also shows

I.D (%) and D.E. est. mm' without definitions. Please clarity these terms, are they the same as S.D. and S.I. ? The authors provide a statistical analysis in the table, but do not include standard deviation or standard error. It would be better for the readers to have a whisker plot or a violin plot, than a table with numbers to better appreciate the observed variation in stomatal density.

Terms and meanings were adjusted in the text as requested.

* Table 3: How were the two genes in Table 3 selected for analysis? The authors refer to an unpublished transcriptomic dataset. Likely, hundreds of genes or more are differentially expressed in their "unshown" transcriptome; why focus on these two genes? Especially since these two genes monitored during infection in Figure 4 are repressed in 'Yangambi' compared to 'Grande Naine', contrary to expectations for defense marker genes in a partially-resistant plant.

The two genes were selected after being validated in the pathosystem under study from all the primers "tested", which showed expression to compare the two genotypes used in the study. Gene expression is dynamic and can vary over time after infection. The specific times when the samples were collected may not coincide with the peaks in the expression of the defense genes in 'Yangambi Km 5. We can consider that 'Yangambi km 5 can use different resistance mechanisms that do not directly involve the high expression of these specific genes. Resistance may be mediated by other genes or signaling pathways that were not addressed in this analysis.

* RT-qPCR Analysis Normalization: Does the normalization of the RT-qPCR analysis to 1 refer to uninfected leaves? Missing statistical analysis.

For the normalization of the expression values for each of the samples, the quantitative variation of expression of the genes of interest was calculated in relation to the reference genes - Tubulin and 25S, according to Pfaffl et al., (2002).

Minors Edits:

- Lines 19, 22, 24, 25, 74, 189, 191, 281, 283, 284, 287, 295, 411: "Yangambi" and not "Yagambi"

Corrections made in the text accordingly.

- Line 69: Pathogenesis-Related proteins are indeed known to be upregulated in response to infection in most of the plant-pathogen interactions. But Cell-Wall Associated Kinase (WAK) or genes related to Leucine-Rich Repeat receptors are classified as Receptor like-proteins (RLPs) or Receptor like-kinases (RLKs) and are related to Pattern Recognition Receptors (PRRs) and not to Pathogenesis-Related (PR) genes. The references 13, 20 and 21 point-out two papers describing transcriptomic analysis where PR genes are described to be overexpressed in resistant banana cultivars, but do not refer to reviews about either PRs or PRRs families as expected.

Corrections made in the text accordingly.

- Line 248: Figure 3E does not exists.

Please consider it a “typo”. The figure does not exist and this was corrected in the text.

- Line 261: The authors refer to Table 2 instead of Table 3

Corrections made in the text accordingly.

Line 304: Authors state "Stomatal index is an indicator of stomatal aperture, which are structures responsible for gas exchange and transpiration in plants." Stomatal index reflects the number of stomata among epidermal cells and not the aperture of stomata.

The measure of stomatal aperture would have been more appropriate in this study to compared immune response in both cultivars.

Corrections made in the text accordingly.

- Line 411: Do the authors mean the repression of the WAK2 gene in Yangambi?

Yes, we are referring to the repression of the WAK2 gene in Yangambi Km 5.