Overexpression of DUF538 from Wild Arachis Enhances Plant Resistance to Meloidogyne spp.

Round 1

Reviewer 1 Report

The presented study “Overexpression of DUF538 from wild Arachis enhances plant resistance to Meloidogyne spp.” from Araujo et al. is a continuation of the previous work of the research group.

The manuscript is clearly written. However, it has some repetitions and few formal corrections need to be addressed.

Therefore, I do have very few comments:

Please spell out Meloidogyne in the headings; or keep it at least consistent and use the abbreviated form throughout the manuscript.

 

The format of the references are in accordance to the journal requirements.

 

Introduction

I recommend describing that Arachis stenosperma is a peanut relative in line 59 and not later (line 80).

 

Materials and methods still need more improvement and details.

E.g. qPCR settings and method of J2 extraction

Out of curiosity, why do you count the females 60 DAI as females count could be performed much earlier?

Results

The results are presented well, nevertheless, some parts belong to the material and methods section, e.g. lines: 292-296, 331-333, 336-337, 397-399, 421-423 and the labeling of the in situ Figure 2 is mislabeled (“H” exists twice).

Discussion

The discussion is somewhat well written, however the expression analysis is only an indication for the hormone signaling activity that should be further studied in future.

 

 

 

Author Response

Response to Reviewer #1 Comments

- Please spell out Meloidogyne in the headings; or keep it at least consistent and use the abbreviated form throughout the manuscript.

Response: Thank you for bringing this to our attention. The spell out Meloidogyne is standardized throughout the manuscript, with the full genus name in the section headings and figure titles, and the abbreviated form in the text.

- Introduction: I recommend describing that Arachis stenosperma is a peanut relative in line 59 and not later (line 80).

Response: Arachis stenosperma is now described as a peanut wild relative in its first citation (Introduction section; line 59).

- Materials and methods still need more improvement and details.

- E.g. qPCR settings and method of J2 extraction

Response: As suggested, we provided more detailed information in the Materials and methods section about the qPCR settings and method of J2 extraction (new subsection 2.2. Nematode inoculum).

- Out of curiosity, why do you count the females 60 DAI as females count could be performed much earlier?

Response: Initially, our goal was to evaluate the number of Meloidogyne incognita egg masses, galls, and acid fuchsin stained females in each plant root system in the transgenic Arabidopsis thaliana OE lines at 60 DAI (days after inoculation). Nonetheless, the sand:substrate mixture (2:1; v:v) in which A. thaliana plants were grown, and the bioassays with M. incognita were conducted, proved to be very hard to be washed off the roots, potentially affecting the egg masses and galls integrity. Therefore, in order to avoid escape, we only used the data from the stained female, which was collected at the same time (60 DAI).

- Results:

The results are presented well, nevertheless, some parts belong to the material and methods section, e.g. lines: 292-296, 331-333, 336-337, 397-399, 421-423

Response: We agree with this and have therefore moved parts of the Results (lines 292-296, 336-337, 421-423) to the material and methods section or removed from the Results (lines 331-333, 397-399) parts that had already been described in the material and methods section.

- and the labeling of the in situ Figure 2 is mislabeled (“H” exists twice).

Response: A new Figure 2, with corrected image labeling and revised legend, is now included in the manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

This manuscript reports on the characterization of the Domain of Unknown Function 538 (DUF538) protein in a wild relative of peanut, namely Arachis stenosperma. The authors explored the expression pattern of DUF538 in this species and others of the same genus. Subsequently, they determined the interest of overexpressing the DUF538 protein motif in transformed roots of peanut and soybean and finally in Arabidopsis to reduce the infection by the root-knot nematodes. Interestingly, expression of DUF538 reduced nematode infection in all the three plant species tested. This paper is a follow-up to earlier studies by the same group.

This paper is well written, the results are clearly documented and for the most part easily interpretable. The used statistics are appropriate, pertinent and most of the time clearly described and justified. Even if this study is mostly descriptive with limited insights into explanatory mechanisms (apart from effects on the expression of stress marker genes), it provides interesting observations and results which could be exploited to design plants resistant to root-knot nematodes.

 

I have just a few minor points which could be addressed by the authors:

1-The definition of the DUF538 consensus sequence is not very clear. Indeed, in my pdf file Fig 1A is blurred and difficult to read. Moreover, there seems to be a mix of cDNAs and of coding sequences (ATG to stop) in the aligned sequences. The legend states that “transcript sequences“ were aligned thus cDNAs (?), but then 5’ and 3’ UTRs are missing for some. It would be clearer if only coding sequences are aligned.

2- In Fig 2, the description of panel H showing the control sense probes should probably be rewritten (“no signal was observed with the control sense probe in root sections…” instead of “as signals in root sections …”)

3- Cell wall, apoplast and nuclei should be labelled in Fig 5.

4- How was the relative expression defined for Q-PCRs experiments shown in Fig 6 and Supp Fig 1? The description provided in the methods section is not documented enough. Does it refer to ACT2 expression (corresponding to a value of 1?). It is usually recommended to use at least two independent reference genes. Then in Fig 6 what are the expression levels of the marker genes in the WT untransformed control? Are the values related to induction levels or ratios of the marker genes? This should be better defined. Concerning the statistical analysis, I am a bit surprised that JAZ1 is differentially expressed in the DUF17 line but not EIN3 that seems to be more repressed? Same for MYC2.

5- On which criteria were these Arabidopsis stress-related genes chosen?

6- Was the number of inserted transgenes determined in Arabidopsis? The lines are described as homozygous, how was this established?

Author Response

Dear Editor,

We would like to thank the reviewers for their analysis and suggestions which we believe will improve the quality of the manuscript “Overexpression of DUF538 from wild Arachis enhances plant resistance to Meloidogyne spp.”. We have addressed all the comments and carefully revised the English language and grammar of the manuscript.

Below we provide a point-by-point response to each of the comments made by the reviewer #2.

Response to Reviewer #2 Comments

- 1. The definition of the DUF538 consensus sequence is not very clear. Indeed, in my pdf file Fig 1A is blurred and difficult to read. Moreover, there seems to be a mix of cDNAs and of coding sequences (ATG to stop) in the aligned sequences. The legend states that “transcript sequences“ were aligned thus cDNAs (?), but then 5’ and 3’ UTRs are missing for some. It would be clearer if only coding sequences are aligned.

Response: We agree that the alignment using cDNAs and coding sequences was confusing, and we therefore update Figure 1 to show only the coding sequences (from ATG to TAA). The quality of the new Figure 1 was improved to allow better visualization.

- 2. In Fig 2, the description of panel H showing the control sense probes should probably be rewritten (“no signal was observed with the control sense probe in root sections…” instead of “as signals in root sections …”).

Response: The description of panel H was modified in Figure 2 legend according to the reviewer’s suggestion.

3- Cell wall, apoplast and nuclei should be labelled in Fig 5.

Response: In a new Figure 5, colored arrows were added to discriminate nuclei, cell wall and cytoplasm (instead of apoplast). The Figure 5 legend was changed accordingly.

4- How was the relative expression defined for Q-PCRs experiments shown in Fig 6 and Supp Fig 1? The description provided in the methods section is not documented enough. Does it refer to ACT2 expression (corresponding to a value of 1?).

Response: We have now detailed the methods used for relative expression quantification in the material and methods section. The AtACT2 reference gene was used as the normalizer (corresponding to a value of 1) to determine AsDUF538 transgene expression (mean normalized expression; MNE) in OE lines (Figure S1). As suggested (see below), the transcript levels of the Arabidopsis marker genes in OE lines, relative to WT plants, were reanalyzed (new Figure 6), using now two reference genes as normalizers.

- It is usually recommended to use at least two independent reference genes.

Response: Thank you for bringing this to our attention. We agree and have therefore carried out new qRT-PCR reactions using the same Arabidopsis samples (WT plants and OE lines) as templates with primer pairs of an additional reference gene (AtEF-1α; new Supplementary Table S2). The new relative expression analysis, using both AtACT2 and AtEF-1α as references, revealed that the expression behavior (upregulation) of the five Arabidopsis marker genes did not change when a second reference gene was included (new Figure 6). Interestingly, all relative expression values were slightly higher than that observed when only one reference gene was used (new Figure 6). The text (material and methods and Results sections) was modified in accordance with this new qRT-PCR analysis.

- Then in Fig 6 what are the expression levels of the marker genes in the WT untransformed control? Are the values related to induction levels or ratios of the marker genes? This should be better defined.

Response: It is now stated in the material and methods section that the quantification of mRNA levels of the five Arabidopsis marker genes in OE lines was relative to WT untransformed control plants (corresponding to a value of 1). Therefore, a relative expression value above 1.0 means that the expression of a target gene is induced in OE lines relative to the WT plants.

- Concerning the statistical analysis, I am a bit surprised that JAZ1 is differentially expressed in the DUF17 line but not EIN3 that seems to be more repressed? Same for MYC2.

Response: Despite EIN3 and MYC 2 show lower relative expression levels compared to JAZ1 in OE17 line, all three marker genes (EIN3, MYC 2, and JAZ1) are upregulated (relative expression above 1.0) in all OE lines tested (OE4; 8; and 17).

5- On which criteria were these Arabidopsis stress-related genes chosen?

Response: The five Arabidopsis stress-related marker genes (AtAPX; AtEIN3; AtJAZ1; AtAOC2; and AtMYC2) were chosen based on their involvement with JA (jasmonic acid) and ET (ethylene) phytohormone pathways and the antioxidant defense system, as stated on page 10 (lines 391-394). We assume that JA and ET signaling and ROS (reactive oxygen species) activation are critical processes on defense responses against RKN infection.

6- Was the number of inserted transgenes determined in Arabidopsis?

Response: We did not determine the number of inserted transgenes in OE lines since we consider that these results do not add relevant information to the manuscript. Its focus is the functional validation of AdDUF538 overexpression for enhancing RKN resistance in three heterologous model systems.

- The lines are described as homozygous, how was this established?

The detailed methodology established in our laboratory to screen homozygous Arabidopsis OE lines at T2 generation was recently published [Vinson et al. Scientific Reports 10(1): 1-19 (2020)]. In summary, we consider that the floral dipping produces hemizygous transgenic Arabidopsis plants (T0 primary transformants) selected on antibiotic-containing medium, with a transformation efficiency of around 0.1% in our laboratory conditions. T0 plants were then grown to maturity and allowed to self-pollinate to produce T1 seeds. Assuming a single locus of insertion, T1 would result in a mixture of homozygous WT: heterozygous WT/transgene: homozygous transgene plants in a 1:2:1 ratio, and an antibiotic resistance:sensitivity ratio of 3:1. T1 plants were allowed to grow to maturity and self-pollinate to produce T2 seeds. Importantly, these T1 and T2 seeds were not pooled – they were maintained separate on a T0 mother-plant basis, representing each an overexpressing (OE) line. When we plant T2 seed, therefore, if it came from a homozygous T1 mother plant, all T2 descendants will be antibiotic (hygromycin) resistant. On the other hand, if the T1 mother plant were heterozygous, the T2 descendants would continue to segregate for antibiotic resistance. We used this method to identify homozygous T1 mother plants, and only homozygous T2 seeds deriving from them are further were used in our experiments. For this specific homozygous test, we used 24 seeds of each T1 plant.

The above methodology established by Vinson et al. (2020) was used in the present study to get homozygous transgenic Arabidopsis plants and is appropriately cited in the material and methods section (page 2, lines 190-192).

Author Response File: Author Response.docx