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The authors have provided satisfying responses to the comments sent by the reviewers and therefore this paper is considered as acceptable for publication.
I am satisfied with the authors response to my comments
The paper has been appreciated by the reviewers and positive comments have been expressed. However, the reviewers have proposed some modifications you should address to improve the quality of the paper.
The article is well written and easy to follow and adheres to PeerJ policies to the best of my judgement. The introduction gives a good background of the methods used to this end in monitoring immunity-related PCD - HR, and refers to existing literature adequately. Figures are clear and relevant, and overall the study represents a coherent and wide investigation of the suitability of a particular method development.
The research question is clearly defined as to whether vacuum infiltration can substitute syringe infiltration when studying HR elicited by P.s. DC3000 secreting avirulence proteins avrRpm1, Rpt2, Rps4. I appreciate in depth investigation of how the used bacterial density affects HR, and contribution of media and cultivation as we many times see variability in the strength of HR between experiment, evident also from the raw data comparing vacuum with syringe infiltration between 13 independent experiments. Except for the deviation graph that compares vacuum with syringe infiltration, I find that the experiment design did not compare syringe and vacuum infiltration. I suggest that the raw data of figure 4 is used to show variability in the strength of HR between syringe and vacuum infiltration which appears to be quite in the same range, but also use the 13 experiments to illustrate the high variability between experiments despite the effort of standardizing.
Overall I feel confident about the validity of the presented data. Because this is a methods paper, I think reproducibility between experiments need to be emphasized, as I also interpret the authors wish. I assume that the experiments have been repeated, and I propose that the raw data from at least one repetition is given in the raw data sheet as done with figure 6 + supplement 2. Additionally, I would have appreciated that avrRpt2 and Rps4 triggered ion leakage would have been compared vacuum vs. syringe, but this is not very essential as the leakage curves look "syringe-like".
I think you have addressed a critical problem how to reduce variability by standardizing the ion leakage HR assay. An important issue that you should discuss is how variable the amplitude of HR is also in your experiments, assuming that I interpret figure 4 raw data correctly, and figure 6/raw data S2.This reflects on the true variability we face with this assay and should be discussed in the conclusions.
See general comments
The authors propose an improved method to quantify the plant hypersensitive response that will not only increase the throughput but also reduce time limitations and supress the labour intensive step. Cell death following pathogen elicitation is evaluated through electrolyte leakage, measured as an increase in conductivity. As such study of this kind is very much pertinent and of interest to the plant/micro-organisms research community.
However the authors suggest that (lines 23-26, 43-44, 61-62, …) PCD is mandatorily activated during incompatible interaction. This point, namely the importance of cell death to resistance, remains to be resolved even today. Furthermore significant electrolyte leakage also occurs following the production of necrotic lesions during a compatible interaction. It also appears that the dynamics of the signal i.e, the changes in conductivity do not allow to discriminate between PCD induced by different effectors (see fig. 2 & 5 ), and necrotic lesions. So the method appears to measure cell death whatever the underlying mechanism.
I think this point so be further discussed in the MS before publication.
Line 105-108. The use of a Pseudomonas syringae with an empty vector should be mentioned in the MM section too.
Line 170. A bacterial suspension: more appropriate than bacterial solution, see line 176.
Line 267. Pseudomonas syringae DC300 expressing ? , carrying ? empty vector
Line 268. Uninfiltrated instead of unifiltrated
Lines 306-310. Not clear to me
Line 322- 326. Here again I agree that the method is “suitable for detection of subtle differences in electrolyte leakage “but it is by no means that these changes in electrolyte leakage are associated with HR in all cases.
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