Biology subjects

Pettinga, D.

Publications and source records attributed to Pettinga, D..

6 recordsLinked to original sources

bioRxiv2026

Rational reduction of a sorghum SynCom that preserves growth promotion reveals flavonoid-mediated plant microbe interactions

O_LIPlant growth is influenced by the composition of its associated microbiome. The inherent complexity and functional redundancy of natural plant microbiomes presents a formidable barrier to understanding the myriad biological interactions therein. Efforts have been made to develop synthetic microbial communities (SynComs) that can provide a rigorous and generalizable framework for the rational design of next-generation microbial products for sustainable agriculture. We test multiple strategies for stable, plant growth promoting SynCom design and evaluate the phenotypic and molecular impacts of a successful plant-SynCom interaction. C_LIO_LIWe designed 4 distinct, reduced-complexity variants of SynCom SRC1 and assessed their capacities for colonization, stability, and plant growth promotion. To understand the impact on plant performance of our highest performing SynCom variant, we characterized the hosts longitudinal transcriptional response to SynCom inoculation and corroborated the results with metabolomics analysis. C_LIO_LIThe top performing SynCom stably colonized sorghum roots and rhizospheres, elicited plant growth promotion, and induced dynamic spatiotemporal gene transcription in sorghum roots and shoots defined by modulation of growth-defense tradeoff machinery and enhanced flavonoid production. C_LIO_LIThe resultant reduced-complexity SynCom is a highly stable, soil-independent, plant growth promoting, and demonstrates the utility of colonization-based selection criteria, integrated with longitudinal transcriptomic and metabolomic characterization. C_LI

plant biology↗

bioRxiv2025

Uncovering the hidden diversity and functional roles of root endophytic Streptomyces under drought stress

The genus Streptomyces has consistently been found to be enriched in drought-stressed plant root microbiomes, yet the ecological basis and functional variation underlying this enrichment at the strain and isolate level remain unclear. Using two 16S rRNA sequencing methods with different levels of taxonomic resolution, we confirmed drought-associated enrichment of Streptomyces in field-grown sorghum roots and identified five closely related but distinct ASVs belonging to the genus with variable drought enrichment patterns. From a culture collection of sorghum root endophytes, we selected 12 Streptomyces isolates representing these ASVs for phenotypic and genomic characterization. Whole-genome sequencing revealed substantial variation in gene content, even among closely related isolates, and exometabolomic profiling showed distinct metabolic responses to media supplemented with drought-versus well-watered root tissue. Traits linked to drought survival, including osmotic stress tolerance, siderophore production, and carbon utilization, varied widely among isolates and were not phylogenetically conserved. Using a broader panel of 48 Streptomyces, we demonstrate that drought enrichment (DE) scores, determined through mono-association experiments in gnotobiotic sorghum systems, showed high variability and lacked correlation with plant growth promotion. Pangenome-wide association identified gene clusters involved in osmolyte transport (e.g., proP) and membrane biosynthesis (e.g., fabG) as positively associated with DE, though most associations lacked phylogenetic signal. Collectively, these results demonstrate that Streptomyces enrichment under drought is not a conserved genus-level trait but is instead strain-specific and functionally heterogeneous. Furthermore, enrichment in the root microbiome does not predict beneficial effects on plant growth. This work underscores the need to resolve functional traits at the strain level and highlights the complexity of microbe-host-environment interactions under abiotic stress. ImportanceUnderstanding how beneficial microbes respond to drought is critical for developing microbiome-based strategies to enhance crop resilience. The Streptomyces genus is consistently enriched in drought-stressed root microbiomes, yet our study reveals that this enrichment is not a uniform isolate-level response. Instead, we show that drought enrichment is highly strain-specific and functionally diverse, with key associated traits like osmotic stress tolerance and siderophore production varying independently of phylogeny. Importantly, enrichment does not predict a microbes ability to promote plant growth under stress. By integrating high-resolution taxonomic, genomic, and metabolomic approaches, we uncover the ecological and functional complexity underlying Streptomyces responses to drought. These findings emphasize the importance of resolving microbial function at the strain level and caution against assuming functional traits based on taxonomic identity itself.

microbiology↗

bioRxiv2024

Improving rice drought tolerance through host-mediated microbiome selection

Plant microbiome engineering remains a significant challenge due to challenges associated with accurately predicting microbiome assembly and function in complex, heterogeneous soil environments. However, host-mediated selection can simplify the process by using plant host phenotype as a reporter of microbiome function; by iteratively selecting microbiomes from hosts with desired phenotypes and using them to inoculate subsequent cohorts of hosts, artificial selection can steer the microbiome towards a composition producing optimized plant phenotypes. In this study, we inoculated rice with wild microbial communities from fallow rice field, desert, and serpentine seep field soils. By challenging these plants with drought and iteratively selecting microbiomes from the least drought stressed plants across multiple generations, we derived simplified microbiomes that enhanced both the growth and drought tolerance of rice. Across selection cycles, microbiomes within and between soil treatments became increasingly similar, implicating both dispersal and selection as drivers of community composition. With amplicon sequencing data we identified specific bacterial taxa associated with improved rice drought phenotypes; while many of these taxa have been previously described as plant growth promoters, we also identified novel taxa exhibiting strong positive correlation with improved drought performance. Lastly, we resolved 272 metagenome-assembled genomes (MAGs) and used these MAGs to identify functions enriched in bacteria driving enhanced drought tolerance. The most significantly enriched functions--particularly glycerol-3-phosphate and iron transport--have been previously implicated as potential mediators of plant-microbe interactions during drought. Altogether, these data demonstrate that host-mediated selection provides an efficient framework for microbiome engineering through the identification of both individual taxa and simplified communities associated with enhanced plant phenotypes.

plant biology↗

bioRxiv2023

Defined synthetic microbial communities colonize and benefit field-grown sorghum

The rhizosphere represents a dynamic and complex interface between plant hosts and the microbial community found in the surrounding soil. While it is recognized that manipulating the rhizosphere has the potential to improve plant fitness and health, engineering the rhizosphere microbiome through inoculation has often proved challenging. This is in large part due to the competitive microbial ecosystem in which the added microbes must survive, and lack of adaptation of these added microbes to the specific metabolic and environmental pressures of the rhizosphere. Here, we constructed an inoculation formula using a defined synthetic community (dSynCom) approach that we hypothesized would improve engraftment efficiency and potentially the relationship with the host plant, Sorghum bicolor. The dSynCom was assembled from bacterial isolates that were either: 1) identified to potentially play a role in community cohesion through network analysis, or 2) identified to benefit from host-specific exudate compounds. Growth of the dSynCom was first evaluated in vitro on solid media, secondly in planta under gnotobiotic laboratory conditions, and finally using sorghum plants grown in the field. We demonstrate that the dSynCom performs best in terms of maintaining diversity when grown in the presence of the plant host in lab conditions, and that many lineages are lost from the community when grown either in vitro or in a native field setting. Finally, we demonstrate that the dSynCom is able to promote growth of above- and below-ground plant phenotypes compared to uninoculated controls, both in the lab and when applied to plants grown in the field. These results demonstrate the potential utility of SynComs for supporting crop performance even in the absence of persistence, and the need for a deeper mechanistic understanding of community control of host fitness in agricultural contexts.

synthetic biology↗

bioRxiv2023

Sorgoleone degradation by sorghum-associated bacteria; an opportunity for enforcing plant growth promotion

Metabolite exchange between plant roots and their associated rhizosphere microbiomes underpins plant growth promotion by microbes. Sorghum bicolor is a cereal crop that feeds animals and humans and is used for bioethanol production. Its root tips exude large amounts of a lipophilic benzoquinone called sorgoleone. Sorgoleone is an allelochemical that suppresses the growth of competing plant seedlings and is mineralized by microbes in soil. As an avenue to understand how sorghum and its root microbiome may be connected through root exudates, we identified the molecular determinants of microbial sorgoleone degradation and the distribution of this trait among microbes. We isolated and studied from sorghum-associated soils, three bacterial strains classified as Acinetobacter, Burkholderia, and Pseudomonas species that grow with sorgoleone as a sole carbon and energy source. The genomes of these strains were sequenced and subjected to transcriptomic and gene fitness analyses to identify candidate sorgoleone degradation genes. Follow up mutational analysis showed that sorgoleone catabolism is dependent on four contiguous genes that are conserved among the species we sequenced. Phylogenetic analysis of the sorgoleone degradation gene cluster showed that sorgoleone catabolism is enriched in sorghum-associated Streptomyces strains. The discovery of bacteria that grow on a compound like sorgoleone that is plant specific and not widely distributed in the environment, provides an opportunity to study how a plant exudate can enforce the development of a rhizosphere specific microbiome for the mutual benefit of plant and microbe. SignificanceThe grain crop sorghum exudes an herbicidal compound called sorgoleone from its root tips, which inhibits the growth of other plants. We isolated bacteria that grow on sorogleone and identified a cluster of bacterial genes required for sorogleone degradation that can be used as a biomarker for this trait. An approach to improve the production of crops in stressful conditions such as drought, is to encourage their association with plant growth promoting bacteria. Our discovery of sorgoleone degradation genes opens the door to engineering bacteria that receive benefit from sorghum in the form of a plant-specific growth substrate, and in return promote the growth of this crop.

microbiology↗

bioRxiv2020

Mapping of DNA damage genome-wide at nucleotide resolution by circle damage sequencing (CD-seq)

To establish relationships between mutations, for example in cancer genomes, and possible mechanisms linked to DNA damage, it is necessary to know at what sequence positions of the genome the damage occurs. However, it has been challenging to specifically map DNA damage at the nucleotide level of resolution and genome-wide with high sensitivity. Here, we describe a new method, which we named circle damage sequencing (circle-damage-seq), to accomplish this goal. The method is based on circularization of DNA molecules and DNA damage-selective cleavage of the circularized DNA followed by adapter ligation and sequencing. Based on the design of this approach, only DNA damage-containing molecules are sequenced. We conducted proof-of-principle studies to show that mapping of ultraviolet B-induced cyclobutane pyrimidine dimers (CPDs) can easily be achieved and show a specific tetranucleotide sequence context for CPDs (5PyPy<>PyT/A) with no further sequence enrichment outside of this context. Our approach shows strongly reduced levels of CPDs near transcription start sites and a spike of this damage near the transcription end sites of genes. We then show that 1,N6-etheno-deoxyadenosine DNA adducts formed after treatment of cells with the lipid peroxidation product 4-hydroxynonenal can be mapped genome-wide at adenine positions within a preferred sequence context of 5TAC/G3. The circle-damage-seq method can be adapted for a variety of DNA lesions for which specific excision enzymes are available.

genomics↗