Morphogene-assisted transformation of Sorghum bicolor allows more efficient genome editing.

Sorghum bicolor (L.) Moench, the fifth most important cereal worldwide, is a multi-use crop for feed, food, forage and fuel. To enhance the sorghum and other important crop plants, establishing gene function is essential for their improvement. For sorghum, identifying genes associated with its notable abiotic stress tolerances requires a detailed molecular understanding of the genes associated with those traits. The limits of this knowledge became evident from our earlier in-depth sorghum transcriptome study showing that over 40% of its transcriptome had not been annotated. Here, we describe a full spectrum of tools to engineer, edit, annotate and characterize sorghum's genes. Efforts to develop those tools began with a morphogene-assisted transformation (MAT) method that led to accelerated transformation times, nearly half the time required with classical callus-based, non-MAT approaches. These efforts also led to expanded numbers of amenable genotypes, including several not previously transformed or historically recalcitrant. Another transformation advance, termed altruistic, involved introducing a gene of interest in a separate Agrobacterium strain from the one with morphogenes, leading to plants with the gene of interest but without morphogenes. The MAT approach was also successfully used to edit a target exemplary gene, phytoene desaturase. To identify single-copy transformed plants, we adapted a high-throughput technique and also developed a novel method to determine transgene independent integration. These efforts led to an efficient method to determine gene function, expediting research in numerous genotypes of this widely grown, multi-use crop.

Transcriptomic analysis of field-droughted sorghum from seedling to maturity reveals biotic and metabolic responses.

Drought is the most important environmental stress limiting crop yields. The C4 cereal sorghum [Sorghum bicolor (L.) Moench] is a critical food, forage, and emerging bioenergy crop that is notably drought-tolerant. We conducted a large-scale field experiment, imposing preflowering and postflowering drought stress on 2 genotypes of sorghum across a tightly resolved time series, from plant emergence to postanthesis, resulting in a dataset of nearly 400 transcriptomes. We observed a fast and global transcriptomic response in leaf and root tissues with clear temporal patterns, including modulation of well-known drought pathways. We also identified genotypic differences in core photosynthesis and reactive oxygen species scavenging pathways, highlighting possible mechanisms of drought tolerance and of the delayed senescence, characteristic of the stay-green phenotype. Finally, we discovered a large-scale depletion in the expression of genes critical to arbuscular mycorrhizal (AM) symbiosis, with a corresponding drop in AM fungal mass in the plants' roots.

Keep your friends close: Host compartmentalisation of microbial communities facilitates decoupling from effects of habitat fragmentation.

Root-associated fungal communities modify the climatic niches and even the competitive ability of their hosts, yet how the different components of the root microbiome are modified by habitat loss remains a key knowledge gap. Using principles of landscape ecology, we tested how free-living versus host-associated microbes differ in their response to landscape heterogeneity. Further, we explore how compartmentalisation of microbes into specialised root structures filters for key fungal symbionts. Our study demonstrates that free-living fungal community structure correlates with landscape heterogeneity, but that host-associated fungal communities depart from these patterns. Specifically, biotic filtering in roots, especially via compartmentalisation within specialised root structures, decouples the biogeographic patterns of host-associated fungal communities from the soil community. In this way, even as habitat loss and fragmentation threaten fungal diversity in the soils, plant hosts exert biotic controls to ensure associations with critical mutualists, helping to preserve the root mycobiome.

Drought delays development of the sorghum root microbiome and enriches for monoderm bacteria.

Drought stress is a major obstacle to crop productivity, and the severity and frequency of drought are expected to increase in the coming century. Certain root-associated bacteria have been shown to mitigate the negative effects of drought stress on plant growth, and manipulation of the crop microbiome is an emerging strategy for overcoming drought stress in agricultural systems, yet the effect of drought on the development of the root microbiome is poorly understood. Through 16S rRNA amplicon and metatranscriptome sequencing, as well as root metabolomics, we demonstrate that drought delays the development of the early sorghum root microbiome and causes increased abundance and activity of monoderm bacteria, which lack an outer cell membrane and contain thick cell walls. Our data suggest that altered plant metabolism and increased activity of bacterial ATP-binding cassette (ABC) transporter genes are correlated with these shifts in community composition. Finally, inoculation experiments with monoderm isolates indicate that increased colonization of the root during drought can positively impact plant growth. Collectively, these results demonstrate the role that drought plays in restructuring the root microbiome and highlight the importance of temporal sampling when studying plant-associated microbiomes.

The generalizability of water-deficit on bacterial community composition; Site-specific water-availability predicts the bacterial community associated with coast redwood roots.

Experimental drought has been shown to delay the development of the root microbiome and increase the relative abundance of Actinobacteria, however, the generalizability of these findings to natural systems or other diverse plant hosts remains unknown. Bacterial cell wall thickness and growth morphology (e.g., filamentous or unicellular) have been proposed as traits that may mediate bacterial responses to environmental drivers. Leveraging a natural gradient of water-availability across the coast redwood (Sequoia sempervirens) range, we tested three hypotheses: (a) that site-specific water-availability is an important predictor of bacterial community composition for redwood roots and rhizosphere soils; (b) that there is relative enrichment of Actinobacteria and other monoderm bacterial groups within the redwood microbiome in response to drier conditions; and (c) that bacterial growth morphology is an important predictor of bacteria response to water-availability, where filamentous taxa will become more dominant at drier sites compared to unicellular bacteria. We find that both α- and ß-diversity of redwood bacterial communities is partially explained by water-availability and that Actinobacterial enrichment is a conserved response of land plants to water-deficit. Further, we highlight how the trend of Actinobacterial enrichment in the redwood system is largely driven by the Actinomycetales. We propose bacterial growth morphology (filamentous vs. unicellular) as an additional mechanism behind the increase in Actinomycetales with increasing aridity. A trait-based approach including cell-wall thickness and growth morphology may explain the distribution of bacterial taxa across environmental gradients and help to predict patterns of bacterial community composition for a wide range of host plants.

Holo-omics for deciphering plant-microbiome interactions.

Host-microbiome interactions are recognized for their importance to host health. An improved understanding of the molecular underpinnings of host-microbiome relationships will advance our capacity to accurately predict host fitness and manipulate interaction outcomes. Within the plant microbiome research field, unlocking the functional relationships between plants and their microbial partners is the next step to effectively using the microbiome to improve plant fitness. We propose that strategies that pair host and microbial datasets-referred to here as holo-omics-provide a powerful approach for hypothesis development and advancement in this area. We discuss several experimental design considerations and present a case study to highlight the potential for holo-omics to generate a more holistic perspective of molecular networks within the plant microbiome system. In addition, we discuss the biggest challenges for conducting holo-omics studies; specifically, the lack of vetted analytical frameworks, publicly available tools, and required technical expertise to process and integrate heterogeneous data. Finally, we conclude with a perspective on appropriate use-cases for holo-omics studies, the need for downstream validation, and new experimental techniques that hold promise for the plant microbiome research field. We argue that utilizing a holo-omics approach to characterize host-microbiome interactions can provide important opportunities for broadening system-level understandings and significantly inform microbial approaches to improving host health and fitness. Video abstract.

Genome-resolved metagenomics reveals role of iron metabolism in drought-induced rhizosphere microbiome dynamics.

Recent studies have demonstrated that drought leads to dramatic, highly conserved shifts in the root microbiome. At present, the molecular mechanisms underlying these responses remain largely uncharacterized. Here we employ genome-resolved metagenomics and comparative genomics to demonstrate that carbohydrate and secondary metabolite transport functionalities are overrepresented within drought-enriched taxa. These data also reveal that bacterial iron transport and metabolism functionality is highly correlated with drought enrichment. Using time-series root RNA-Seq data, we demonstrate that iron homeostasis within the root is impacted by drought stress, and that loss of a plant phytosiderophore iron transporter impacts microbial community composition, leading to significant increases in the drought-enriched lineage, Actinobacteria. Finally, we show that exogenous application of iron disrupts the drought-induced enrichment of Actinobacteria, as well as their improvement in host phenotype during drought stress. Collectively, our findings implicate iron metabolism in the root microbiome's response to drought and may inform efforts to improve plant drought tolerance to increase food security.

Drought Drives Spatial Variation in the Millet Root Microbiome.

Efforts to boost crop yield and meet global food demands while striving to reach sustainability goals are hindered by the increasingly severe impacts of abiotic stress, such as drought. One strategy for alleviating drought stress in crops is to utilize root-associated bacteria, yet knowledge concerning the relationship between plant hosts and their microbiomes during drought remain under-studied. One broad pattern that has recently been reported in a variety of monocot and dicot species from both native and agricultural environments, is the enrichment of Actinobacteria within the drought-stressed root microbiome. In order to better understand the causes of this phenomenon, we performed a series of experiments in millet plants to explore the roles of drought severity, drought localization, and root development in provoking Actinobacteria enrichment within the root endosphere. Through 16S rRNA amplicon-based sequencing, we demonstrate that the degree of drought is correlated with levels of Actinobacterial enrichment in four species of millet. Additionally, we demonstrate that the observed drought-induced enrichment of Actinobacteria occurs along the length of the root, but the response is localized to portions of the root experiencing drought. Finally, we demonstrate that Actinobacteria are depleted in the dead root tissue of Japanese millet, suggesting saprophytic activity is not the main cause of observed shifts in drought-treated root microbiome structure. Collectively, these results help narrow the list of potential causes of drought-induced Actinobacterial enrichment in plant roots by showing that enrichment is dependent upon localized drought responses but not root developmental stage or root death.