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Below, please find the reviewers' comments for your perusal. Please prepare a response to reviewers detailing how you addressed each of their concerns. I suggest that you perform the experiment requested by the reviewer or revise and discuss your conclusion.
no comment
no comment
no comment
no comment
The authors have improved all the points I have previously suggested, and the additional experiments were sufficiently conducted. I think the manuscript is suitable for publication.
no comment
no comment
no comment
no comment - The experimental design is appropriate.
no comment
I reviewed the first version of this manuscript and would like to commend the authors for addressing the reviews. I think this paper helps to expand understanding of AlgU’s role in virulence. I still have an issue with using the reduction in biofilms to conclude that EPS production was reduced (see lines 253, 273-274). This is a reasonable hypothesis, but based on the data the most that can be said is that biofilm formation was reduced in the algU mutant, possibly because of reduced EPS production. AlgU is a global regulator, and as such regulates many genes. I don’t think that it’s possible to say which are involved in biofilm formation without testing them. There are tests for EPS production if the authors are interested in demonstrating that EPS was reduced. I should also add that demonstrating reduced biofilms is an important result on its own.
I agree with the reviewers that your article has some novelty, but could have been more detailed in the functional verification and evaluation.
I strongly suggest the authors to add experiments to improve the article according to the reviewers' comments, and if they cannot add experiments, please provide evidence to explain the reviewers' questions.
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1. A little confuse of this description. “For Psg gene expression profiles, bacteria were grown in HS medium optimized for COR production (HSC; Palmer and Bender, 1993) for 3 and 48 h, adjusted to an OD600 of 0.1, and grown again in fresh HSC medium for 3 h.”
2. No figure legends for Supplementary figures.
3. The efficiency of the complementation of algU seems not good, as shown in Supplementary Figure 1.
4. The importance and function of AlgU in Pseudomonas has been studied widely. Authors should discussion more with their results.
That is ok.
That is ok.
That is ok.
In this paper, the authors found that the algU mutant showed significantly reduced virulence together with reduced bacterial populations in planta. They showed that AlgU regulated algD, fliC, and hrpL expression and also demonstrated that expression of coronatine-related genes and coronatine production were down-regulated in the algU mutant. The molecular mechanism of the pathogenicity of Pseudomonas species are a recent interesting topic. In particular, this work suggests that stomatal-based defense and flagellin-based defense may contribute to the reduced pathogenisity of the algU-mutant, which will provide important insights into the interactions between plants and pseudomonas in molecular level.
I think the manuscript is suitable for publication in PeerJ, but suggest the following several comments for a higher quality. Additional experiments are not always necessary. If the authors already have the data, adding them will make the paper even better.
In Line 192, the authors demonstrated that the algU mutant showed dramatically reduced bacterial populations in planta. But it may be necessary to investigate whether the bacterial growth ability of the algU mutant on the medium is the same as that of WT.
In Fig 3 and Fig 4, the authors investigated gene expression profiles involved in the virulence and COR quantification of WT and the algU mutant. But did those of algU-complemented strain recover to the same level as the wild strains?
The authors investigated disease symptoms on soybean leaves inoculated with WT and mutant strains in Line 181 and Fig 1. But is the area of chlorotic region inoculated with the mutant strain the same as that of the wild strain? The authors should put a scale bar in the figure.
Line 267
It is interesting to note that AlgU may suppress stomatal-based defense. The authors observed the bacterial populations of Psg by spray-inoculation in Fig. 2, but how about the bacterial populations when inoculated by syringe infiltration in Fig. 1? If there is no difference in the bacterial populations of WT and the algU-mutant when inoculated by syringe infiltration, it is considered that stomatal-based defense may greatly contribute to the reduction of pathogenicity of the algU-mutant.
No comment - my comments are in my review.
No comment - my comments are in my review.
No comment - my comments are in my review.
PeerJ 62630v1
Multiple virulence factors regulated by AlgU contribute to the pathogenicity of Pseudomonas savastanoi pv. glycinea in soybean
This manuscript reports the results of a transposon insertion screen to identify mutants with altered virulence phenotypes of Pseudomonas savastanoi on host plants. From screening 1000 transposon insertion mutants the authors identified 28 mutants with reduced virulence. The majority of these insertions were in genes affecting coronatine phytotoxin production. A mutant with an insertion in algU, a global transcription regulator, was also identified. The authors took advantage of this to test the function of AlgU in regulating virulence in this strain.
This is a concise study that adds to the emerging understanding of AlgU as having a prominent role in helping P. syringae transition to pathogenic growth – including regulating coronatine, hrpL and fliC expression. The paper presents a clear central message and is noteworthy for people interested in P. savastanoi biology specifically, and AlgU function in general. The paper is fairly well written but could use some careful editing to improve readability.
In terms of criticism – the conclusions about alginate production are weakened by the biofilm experiment. For example, the authors use their observation that biofilm formation is reduced in the algU mutant to conclude that alginate biosynthesis is reduced. As presented, this is a circuitous, uninformative (and unnecessary) way to obtain information about alginate production. First, alginate is not a major or necessary component of Pseudomonas biofilms, which suggests that this assay does not inform about alginate production. Second, AlgU regulates on the order of ~1000 genes, additional work is needed to make claims on which of those is responsible for altered biofilm formation. Third, the paper reports results of qRT-PCR analyses that show AlgU regulating the algD gene (which encodes the enzyme responsible for the first committed step in alginate production). With that result and because there is a deep literature on AlgU’s role in regulating alginate production, I would think that would be sufficient for this study to claim that the biosynthetic genes are AlgU dependent. Showing that AlgU regulates algD is basically a positive control, showing that AlgU has the same role in P. savastanoi as it does in almost all Pseudomonads. However, the focus of this paper is on P. savastanoi virulence, considering that, the result is not important because alginate is not considered to be a virulence factor for this strain. With that said, demonstrating that alginate affects either biofilm formation (by comparing wt vs alginate-minus strains) or virulence (WT vs alginate-minus) would be a noteworthy contribution.
Specific comments:
Lines 80-81 – I think it is fair to say that the relative importance of specific virulence functions may vary for each pathovar or for each specific pathovar-host interaction. The observed relative importance of individual virulence factors may also be a function of the type of read out used to assess virulence. For example, in this ms, the authors “screened for mutants with less…chlorosis”. This might explain the disproportionate numbers of mutants that were identified with defects in coronatine production.
Table 1 – Please list the locus tag for the affected gene and the genomic coordinates for the transposon insertions.
Lines 86-88: Which EPS is being referred to here? If the authors are refering to alginate, please consider https://doi.org/10.1099/mic.0.2007/012864-0, which shows that AlgU, but not alginate are important for virulence. Additionally, there are not any results in this MS demonstrating a role of EPS in plant virulence.
Line 31-32, Line 231 -232 – Finding reduced biofilms with the algU mutant is not evidence that
AlgU regulates alginate biosynthesis.
Figure 1. Scale bars would help in comparing virulence of mutants.
Line 252-253 – This reference does not discuss alginate in biofilms. There is evidence that
alginate is not a significant (or necessary) component of biofilms, see: https://doi.org/10.1073/pnas.1231792100
Line 274 – AlgU also regulates hrpL expression in DC3000.
Line 275 – This phrasing is confusing – the genes are in the hrp cluster, but they are not regulated by the hrp cluster.
Line 282-283 – These references (Bao et al. 2020; Lam et al. 2014) are not the original or recent papers dealing directly with how flagellin or the flg22 epitope trigger PTI.
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