Write a comment

Summary

The preprint by Kemppinen et al. uses a diverse set of Arabidopsis mutants with impaired stomatal function to monitor bacterial growth, stomatal behavior, and water-soaking capacity after Pst DC3000 infection, with the aim of evaluating the effect of stomatal immunity (closing of stomata at early stages – preventing pathogen entry) versus water immunity (stomatal reopening at later staged – preventing water soaking). The manuscript would overall benefit from a bit more clarity and structure, as well as a broader discussion. It is a bit confusing to jump between different experiments and timepoints thought-out the manuscript rather than presenting the data based on the experiments performed. We have major comments concerning the stomatal aperture and syringe infection experiments performed, as well as some specific points to address.

Major points:

• The authors state that they “evaluate the relative importance of stomatal versus water immunity”, however, the performed syringe infiltrations and stomata aperture assays after 24h to investigate water immunity directly lack the use of most mutants employed in this study. We believe it would be more valuable to see the additional mutant phenotypes.

• It appears a bit confusing to start the paper with Pst infections and only present the stomatal aperture in the second part. Indeed, the authors write in line 122 of the second paragraph “we first assessed the stomatal responses after Pst treatment”. In addition, it is very confusing to jump between timepoints e.g. aperture after 1hpi is presented in the second paragraph in the text, aperture after 24h is presented in the fifth paragraph in text (for only two mutants). Why are the aperture results (for 1h and 24h) not used to formulate expectations for the following pathogen (spray and infiltration) and water soaking capacity assays instead? We think revising the order of results presented could add to the clarity of the paper.

Specific points:

Figure 1:

• Figure 1A: The overall visual presentation could benefit from simplification. Our suggestion would be to highlight the key points related to stomatal opening and closing for each mutant used separately.

• Figure 1C/D: Time points for syringe inoculation were reduced compared to spray inoculation, and only a subset of the mutants were used. We do see the reasoning presented in the text of why to test only those mutants based off the extreme phenotypes, as representatives. However, the authors mention the importance of experimental design when performing pathogen growth assays, and we believe it would be more valuable to see the additional mutant phenotypes.

Figure 2:

• Similarly, only two of the mutants were used to investigate stomatal closure 24 hpi. It would be nice to see if most mutants presented indeed behave the same (as presented in Figure 4) for this timepoint/water immunity.

Figure 3:

• Maintaining consistency in the color schemes for mutant data across figures would be nice here.

Figure 4:

• Presence/Absence of functional closure or reopening of stomata for water immunity is indicated for all the mutants, however, this was only tested for two of the mutants (see comment regarding Figure 2).

Discussion:

• It would be nice to address the observation that e.g. ost1-3 and ost2-2D appear more resistant in Pst assays performed, while they have been reported to show enhanced resistance or wildtype-like pathogen phenotypes (Melotto et al., 2006; Jalakas et al., 2017; Ou et al., 2022; Zhou et al., 2015).

• Could a broader interpretation of the findings within the context of plant immunity and pathogen resistance be included. Based on the results presented here, stomatal immunity appears to be non-existent.

• Discuss the elevated expression of LOX4, which is the only gene upregulated in max2-4, as this observation may hold significance for JA signaling but is not elaborated upon in the discussion.

Competing interests

The authors declare that they have no competing interests.