Improving rice drought tolerance through host-mediated microbiome selection

Reviewer #1 (Public Review):

Summary:
The study claims to explore plant microbiome engineering using host-mediated selection as a strategy to enhance rice growth and drought tolerance.

Strengths:

The authors have derived and identified simplified microbiomes from wild microbial communities of rice fields, deserts, and serpentine seep soils by selecting microbiomes from plants with desired phenotypes across generations. Metagenome-assembled genomes revealed enriched functions, such as glycerol-3-phosphate and iron transport, known to mediate plant-microbe interactions during drought.

Weaknesses:

The findings demonstrate the efficacy of host-mediated microbiome selection, but the engineering part for enhancing rice performance under drought-stress conditions has not been provided. The proposed mechanisms rely on correlations but not direct experimental proofs.

Reviewer #2 (Public Review):

Summary:

In this study, Styer et al. impose artificial selection on root-associated microbiomes to increase drought tolerance in rice plants using different soils as starting microbiomes. Using NDVI and biomass as a proxy for plant health, they find that iterative passaging of the microbiomes of the best-performing plants increased plant resilience to drought stress in a soil-dependent manner. The study makes use of numerous controls. The authors survey the microbiota of the plants across generations, using an array of interesting analyses to characterize their observations. Firstly, the authors find that the acquired microbiomes are divergent towards the beginning of the selection experiment, but nearly converge later suggesting that the selected communities become more similar over time. One reason is that the diversity of the microbiomes severely decreases after only one or two generations of selection AND that microbes from each inoculation source appear to easily disperse across the experiment, leading to microbiome homogeneity. The authors then present an analysis to correlate ASVs with the NDVI and Biomass over the course of the experiment (using the rice soil selection lines) to develop hypotheses about which ASVs may impact plant traits.

Strengths:

The authors set out to refine the understanding of microbiome artificial selection, a topic of recent interest to the plant microbiome field. The authors use an established approach (Mueller et al), expanding upon it by including multiple starting soil inocula to ask whether the strength of selection varies by input microbiome. This is an important and novel question. Using drought resilience as measured by NDVI and plant biomass to select upon was a wise choice for this type of study, given their relative ease and quickness to assess. The inclusion of several types of controls, multiple selection lines, and several starting soil inocula showed a thoughtful experimental design. The analyses were diverse, non-standard, and attempted to address microbiome dynamics on multiple fronts. I am not necessarily convinced by some of the conclusions (see below), however, I think this study examines an important and exciting topic in the area of plant microbiomes. I predict the findings of the experiments will inform a wide audience of researchers attempting similar studies and be helpful in their designs.

Weaknesses:

Although the controls were well designed, the dispersal of the microbiomes erased the utility of the sterile inoculated (SI) controls, at least from my reading of the manuscript. Perhaps the original intent of the SI plants was to contrast the selected microbiomes vs axenic plants to show that plant resilience to drought increased generation after generation. If the controls had worked properly under my presumed scenario, this would allow the authors to account for batch variation across the generations (due to slight differences in MS media prep, water quality, etc.). Instead, the SI lines acquired microbes from the experiment and never appeared to significantly deviate from the SL plants. The dispersal of the microbes amongst soils and selection lines also minimizes any conclusions that can be made about the different starting inocula and how prone to selection they may be.

Reviewer #3 (Public Review):

Summary:

In this work, Styer et al. explore host selection as a means for recruiting microbes that may aid their host under stressful conditions, in this case under drought stress, as an alternative to target-SynCom design. They do so by subjecting rice plants to several generations of soil transplantation, and by using the most successful rice plants as donors for the next generation. By using several NGS approaches and very thorough bioinformatics analysis, the authors identify potential microbial taxa and the associated functions enriched in the conditions of interest.

Strengths:

In general, I think this approach was very much needed in the field as an alternative to SynComs, which are still not readily usable in croplands. This work sets the grounds for future similar approaches, using different stresses and different host plants.

In this work, the experimental setup is well thought-through and well-replicated. In addition, an exhaustive set of preliminary experiments was performed before deciding on the final panel of soils to use and scoring methodology. The figures are clear and well-explained.

Weaknesses:

One of the more unexpected results is that sterile/non-inoculated calcined clay also tends to enrich similar microbes, and the authors did extensive work exploring possible sources and microbial dispersal within the growth chamber. In a future experiment, the work would benefit from including a truly sterile control (same growth chamber but completely isolated from possible contaminations). In this regard, the reader may get to wonder whether these efforts are necessary at all (selection experiments), since plants seem to get from their environment what they need to survive. This is discussed across the paper but not directly addressed and I think the manuscript would benefit from a clear argument for or against this idea.