Adaptation of Bacillus thuringiensis to Plant Colonization Affects Differentiation and Toxicity

ABSTRACT The Bacillus cereus group (Bacillus cereus sensu lato) has a diverse ecology, including various species that are vertebrate or invertebrate pathogens. Few isolates from the B. cereus group have however been demonstrated to benefit plant growth. Therefore, it is crucial to explore how bacterial development and pathogenesis evolve during plant colonization. Herein, we investigated Bacillus thuringiensis (Cry−) adaptation to the colonization of Arabidopsis thaliana roots and monitored changes in cellular differentiation in experimentally evolved isolates. Isolates from two populations displayed improved iterative ecesis on roots and increased virulence against insect larvae. Molecular dissection and recreation of a causative mutation revealed the importance of a nonsense mutation in the rho transcription terminator gene. Transcriptome analysis revealed how Rho impacts various B. thuringiensis genes involved in carbohydrate metabolism and virulence. Our work suggests that evolved multicellular aggregates have a fitness advantage over single cells when colonizing plants, creating a trade-off between swimming and multicellularity in evolved lineages, in addition to unrelated alterations in pathogenicity. IMPORTANCE Biologicals-based plant protection relies on the use of safe microbial strains. During application of biologicals to the rhizosphere, microbes adapt to the niche, including genetic mutations shaping the physiology of the cells. Here, the experimental evolution of Bacillus thuringiensis lacking the insecticide crystal toxins was examined on the plant root to reveal how adaptation shapes the differentiation of this bacterium. Interestingly, evolution of certain lineages led to increased hemolysis and insect larva pathogenesis in B. thuringiensis driven by transcriptional rewiring. Further, our detailed study reveals how inactivation of the transcription termination protein Rho promotes aggregation on the plant root in addition to altered differentiation and pathogenesis in B. thuringiensis.

With great interest I've read this manuscript by Lin et al. It is very well written and structured and therefore easy to follow. The figures and data presentation are excellent. I would like to congratulate the authors with this nice study and have only a few small comments/suggestions to further enhance reading: -In the abstract, toxicity is called 'unrelated' to pathogenesis as these changes in pathogenicity are supposed to be unrelated to the enhanced virulence towards insects in view of the EE on the plant (I suppose). This makes sense to me as well, but the sentence in the abstract could benefit from some more detail.
-It would be nice to explain why the Cry-strain was used. What was the reason? -L335: It would be nice to include some more details on the representative images of the hemolytic activity. Given this is a paper of general interest, especially also to plant scientists, it would be beneficial to explain a little bit more for non-experts. What are we looking at? NB: In general this would be beneficial for some other detailed phenotypes such as the pellicle formation, too.
-L359: Maybe it would be better to split the first (A-C) and second halves (D-F) of figure 4? Easier interpretation and integration with the separate sections in the manuscript.
-L409: Lineage E isn't shown in Fig S7 (it would be beneficial though; for interpretation as well as in line with the rest of the manuscript)? -L682: It would be nice to include methodological and material (like plasmids) details on how the fluorescent reporters were created.
-L760: The indicated bioproject only contains data of the 16 genomes; please also add the transcriptome data (or the relevant bioproject number).

Adaptation of Bacillus thuringiensis to plant colonization affects differentiation and toxicity.
Much research has been performed in recent years to characterize the composition and diversity of the plant microbiome, especially in the model plant Arabidopsis thaliana. However, the molecular mechanism of bacterial colonization and adaptation to the plant environment is still limited. In this paper, the authors utilized experimental evolution to explore B. thuringiensis to the root environment. The authors characterized two evolved lineages and demonstrated that they harbor mutations in the transcription termination factor Rho leading to a transcriptional rearrangement. The authors further suggested that this mutation makes these isolates better root colonizers by promoting multicellular behaviors, like aggregation and swarming, and better utilization of plant sugars.

Major issues:
The paper gives an excellent demonstration of the utility of experimental evolution for understanding mechanisms of root colonization.
1. However, the experimental setting was devised for evolving increased biofilm formation and dispersal on polystyrene beads. It is not clear that the plant plays any role in this adaptation as "a plant," not just as a substrate for biofilm attachment. In addition, the condition of the experiment including shaking, and switching from plant to plant in each cycle, seem very artificial regarding root colonization in more natural settings. The authors need to strengthen the notion that the bacteria adapted to plant colonization, as this is the paper's main point. This may be done by monitoring root colonization of the evolved population in other conditions like agar plates and soil.
2. The only proof that bacteria adapted to plant colonization is that the evolved bacteria respond differently to plant material compared with the ancestor. Two controls are lacking to prove this point 1) biofilm formation in LB media without Xylan 2) Biofilm formation on MSN medium with non-plant sugars (Beauregard P, PNAS 2013).
3. The differences in biofilm formation shown in figures 3A-3B and 5C need to be quantified.
These are major results for the paper, and the pictures alone are not compelling enough Minor issues: 1. The legend of figure 1A says: "plates were agitated at 50 rpm," while in the methods section, it is written that "plates were put on a laboratory shaker at 90 rpm". that Lineage E and rhoGlu54stop share the expression pattern with the ancestor. The truth is that they both share the expression pattern differences compared to the ancestor.

Reviewer #1 (Comments for the Author):
With great interest I've read this manuscript by Lin et al. It is very well written and structured and therefore easy to follow. The figures and data presentation are excellent. I would like to congratulate the authors with this nice study and have only a few small comments/suggestions to further enhance reading: -In the abstract, toxicity is called 'unrelated' to pathogenesis as these changes in pathogenicity are supposed to be unrelated to the enhanced virulence towards insects in view of the EE on the plant (I suppose). This makes sense to me as well, but the sentence in the abstract could benefit from some more detail.
We agree with the Reviewer that toxicity and pathogenesis might be confusing in the abstract, therefore we decided to only state pathogenesis, as this has been tested in our experiments.
-It would be nice to explain why the Cry-strain was used. What was the reason?
A: Thank you for pointing this out. Lereclus et al. (1) firstly obtained Bt407 Cry-strain by shifting the Cry+ bacterial culture to 42 °C. The benefits of Cry-strain were significant such as stable heterologous expression and easy molecular modification (1, 2). Since then, this acrystalliferous derivative has been widely used as a model strain in many studies (3)(4)(5)(6)(7)(8). The informative background is the first reason we used Cry-strain.
Another important reason is that considering the ecological importance of B. cereus group species, acrystalliferous bt407 will be an ideal agent that resembles B. cereus, thus providing broader impact of this study.
We have now referred to this information in the article. -L303: Not entirely clear why panel D is included with S4. Wouldn't it be better placed as inset with main Figure 3?
A: Thanks for the suggestion. We have moved the Fig. S4D to Fig. 3 as panel E. It did help we improve the flow of the manuscript and make the paper easier to follow.
-L335: It would be nice to include some more details on the representative images of the hemolytic activity. Given this is a paper of general interest, especially also to plant scientists, it would be beneficial to explain a little bit more for non-experts. What are we looking at? NB: In general this would be beneficial for some other detailed phenotypes such as the pellicle formation, too.
A: Thanks for the suggestions. We have added a description beside the representative images of the hemolytic activity results, which should enhance the understanding of how hemolytic index was calculated. However, only qualitative analysis exists for pellicle formation and used in the Bacillus field in the last 2 decades, which describes the structural complexity of pellicles.  Table 1 and description of the methodology was added in Materials and methods. The authors characterized two evolved lineages and demonstrated that they harbor mutations in the transcription termination factor Rho leading to a transcriptional rearrangement. The authors further suggested that this mutation makes these isolates better root colonizers by promoting multicellular behaviors, like aggregation and swarming, and better utilization of plant sugars.

Major issues:
The paper gives an excellent demonstration of the utility of experimental evolution for understanding mechanisms of root colonization.
1. However, the experimental setting was devised for evolving increased biofilm formation and dispersal on polystyrene beads. It is not clear that the plant plays any role in this adaptation as "a plant," not just as a substrate for biofilm attachment. In addition, the condition of the experiment including shaking, and switching from plant to plant in each cycle, seem very artificial regarding root colonization in more natural settings. The authors need to strengthen the notion that the bacteria adapted to plant colonization, as this is the paper's main point. This may be done by monitoring root colonization of the evolved population in other conditions like agar plates and soil.
A: Thank you for pointing this issue. The EE setup in this study was indeed a modified version of the well know bead model that was previously used to test adaptation on abiotic surfaces. In the meantime, we have also experimentally evolved the bacteria in the bead system to test adaptation to abiotic biofilm formation. The work has been now deposited to the bioRxiv (9).
We reasoned that the microbe has distinct evolutionary patterns in these two EE setups. For example, in the bead system, evolved variants with the highest fitness mutated in a guanylyltransferase gene related to the cell surface properties. The mutation has very specific effects on the adhesiveness of the cells to the abiotic surfaces. On the contrary, the mutation in plant-associated evolved bacteria was more related to the carbohydrate utilization, the effects of which were more pervasive.
Furthermore, the mutational profiles of the microbes in these two setups were distinct as well. In plant-associated evolved variants, 70 SNPs were identified. For bead-associated variants, the number was 27. Importantly, there were few overlapping mutations among them in both setups.
Taking together, we believed this evolutionary pattern was specifically shaped by plant-associated biofilms. Thus, comparison of the two setups, biotic (plant) and abiotic (bead) and the derived conclusions in the two manuscripts clearly demonstrate distinct adaptation patterns.
2. The only proof that bacteria adapted to plant colonization is that the evolved bacteria respond differently to plant material compared with the ancestor. Two controls are lacking to prove this point 1) biofilm formation in LB media without Xylan 2) Biofilm formation on MSN medium with nonplant sugars (Beauregard P, PNAS 2013).
A: Thank you for the very constructive comment. We agree that these two controls would be necessary in the results. Not surprisingly, in LB and MSN plus non-plant sugar (glycerol in this case), Bt407 could not form robust biofilms. The representative images were included in the supplementary Fig. S4. 3. The differences in biofilm formation shown in figures 3A-3B and 5C need to be quantified. These are major results for the paper, and the pictures alone are not compelling enough.
A: Thank you for the comment. Generally, pellicle development provides only a qualitative measure for biofilm formation. It is well accepted approach in the Bacillus field for the last two decades. No true quantitative measure has been developed for the pellicles of Bacilli, thus we cannot provide such measure. Importantly, the complexity and presence of B. subtilis pellicle formed at the air-medium interface directly correlate with plant colonization ability and biocontrol, as described in (11).
Minor issues: 1. The legend of figure 1A says: "plates were agitated at 50 rpm," while in the methods section, it is written that "plates were put on a laboratory shaker at 90 rpm".
A: Thanks for pointing out this mistake. The shaking condition of the EE setup was at 90 rpm. It is now corrected in the legend of Fig. 1A.  Thank you for submitting your revised manuscript to mSystems. We have completed our review and I am pleased to inform you that, in principle, we expect to accept it for publication in mSystems. The reviewers were overall very satisfied with the revisions in your resubmitted manuscript. The only final request is that you explicitly refer to the BioRxiv manuscript you refer to in your rebuttal (9. Lin Y et al. 2021. Adaptation and phenotypic diversification of Bacillus thuringiensis biofilm... bioRxiv 2021.09.03.458824) and its relevance to your experiments within the main text of your revised manuscript. Addressing this comment is essential as this pre-print directly addresses the remaining major concern of this reviewer.
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