P-body formation is required for yeast proliferation in the phyllosphere

Reviewer #1 (Public review):

Summary:

Stemming from the previous research on the adaptation of methylotrophic microbes in the phyllosphere environment, this paper tested a novel hypothesis on the molecular and cellular mechanisms by which yeast uses biomolecular condensates as unique niches for the regulation of methanol-induced mRNAs. While a few in vivo experiments were conducted in the phyllosphere, more assays were carried out on plates to mimic various stress conditions, diminishing the reliability of the conclusions in supporting the main hypothesis.

Strengths:

This study addressed an interesting and important biological question. Some of the experiments were conducted methodically and carefully. The visualization of both the biomolecular condensates and the mRNAs was helpful in addressing the questions. The results are expected to be useful in paving the way for the future study to directly test its main hypothesis. The results of this study could also have a general implication for the adaptation of a huge population of microbes in the enormous space of the phyllosphere on Earth.

Weaknesses:

The results were often over- and misinterpreted. Given mthat any hypotheses were tested indirectly on plates, the correlative results could only be used to carefully suggest the likelihood of the hypotheses. For example, a single edc3 mutant was used to represent a P-body-defective strain, although it is well known that EDC3 is a critical component in mRNA decapping; hence, the mutant should display a pleiotropic phenotype, rather than a mere reduced P-body phenotype. Using a similar reductionist approach, the study went on to employ a series of plate assays to argue that the conditions were mimicking the phyllosphere, which could be misleading under these circumstances. Furthermore, the low percentage of the colocalization between P-bodies and mimRNA granules and the similar results from negative control mRNAs do not convincingly support the idea that mimRNAs are sequestered between two biomolecular condensates, and P-bodies could serve as regulatory hubs. Given that the abundance of mimRNA granules was positively correlated with the transcript abundance of mimRNAs, and P-body abundance did not change too much under methanol induction, the results could not support an active mimRNA sequestration mechanism from mimRNA granules to P-bodies with a proportional increase of the overlap between the two condensates. More direct experiments conducted in the phyllosphere using multiple P-body defective yeast strains should strengthen the manuscript, assuming all the results turned out to be supportive.

Reviewer #2 (Public review):

Summary:

This article aims to elucidate the potential roles of P-bodies in yeast adaptation to complex environmental conditions, such as the plant leaf phyllosphere. The authors demonstrated that yeast mutants defective in one of the P-body-localized proteins failed to grow in the Arabidopsis thaliana phyllosphere. They conducted detailed imaging analyses, focusing particularly on the co-localization of P-bodies and mRNAs (DAS1) related to the methanol metabolism pathway under various environmental conditions. The study newly revealed that these mRNAs form dot-like structures that occasionally co-localize with a P-body marker. Furthermore, the authors showed that the number of P-body-labeled dots increases under stress conditions, such as H₂O₂ treatment, and that mRNA dots are more frequently localized to P-body-like structures. Based on these detailed observations, the authors hypothesize that P-bodies function to protect mRNAs from degradation, particularly under stress conditions.

Strengths:

I think the authors' attempt to elucidate the potential roles of P-bodies in yeast under stress conditions is novel, and the imaging data are overall very nice.

Weaknesses:

I believe the authors could make additional efforts to more clearly demonstrate that P-bodies are indeed required for yeast proliferation in the phyllosphere, as described below, since this represents the most novel aspect of the study.

Reviewer #3 (Public review):

Summary:

The authors use fluorescent microscopy and fluorescent markers to investigate the requirement of P-bodies during growth on methanol, a common substrate available on plant leaves, by using a yeast edc3 mutant defective in P-body formation. Growth on methanol upregulates the transcription of methanol metabolic genes, which accumulate in granular structures, as observed by microscopy. Co-localization of P-bodies and granules was quantified and described as dynamically enhanced during oxidative stress. Ultimately, the authors suggest a model where methanol induces the accumulation of methanol-induced mRNAs in cytosolic granules, which dynamically interact with P-bodies, especially during oxidative stress, to protect the mRNAs from degradation. However, this model is not strongly supported by the provided data, as the quantification of the co-localization between different markers (of organelles and between P-body and granules) is not well presented or described in the text.

Considering that there is only a small EDC3-dependent overlap between P-bodies and mimRNA granules, the claim that P-bodies regulate mimRNAs is not fully justified. Rather, EDC3 could also be involved in mimRNA granule formation, independent of P-bodies.

Strengths:

(1) The authors could show convincingly that P-bodies (using a P-body-deficient edc3-KO strain) are important for colonizing the plant phyllosphere and for the regulation of methanol-induced mRNAs (mimRNA).

(2) The visualization of mimRNA granules and P-bodies using fluorescent markers is interesting and was validated by alternative methods, such as FISH staining.

(3) The dynamic formation of mimRNA granules and P-bodies was demonstrated during growth on leaves and in artificial medium during oxidative stress. The mimRNA granules showed a similar dynamic as the abundances of several mimRNAs and their corresponding proteins.

(4) A role of EDC3 in the formation of mimRNA granules was demonstrated. However, the link between P-bodies and mimRNA granules was not clearly shown.

Weaknesses:

(1) The study largely relies on fluorescent microscopy and co-localization measurements. However, the subcellular resolution is not very high; it is unclear how dot-like structures were measured and, importantly, how co-localization was quantified.

(2) The text does not clarify to what degree P-bodies and mimRNA granules are different structures. Based on the images, the size of P-bodies and granules seems to be vastly different, making it unclear whether these structures are fused or separate, even if their markers are reported to overlap.

(3) The evidence that mimRNA granules contain ribosome-free and ribosome-associated RNA is only based on inhibitors and microscopy, without providing further evidence measuring granule content by isolation and sequencing approaches.

(4) Similarly, the co-localization with other organelle markers is not supported by quantitative data.