Piatkowski, Bryan

ABSTRACT Microbial colonization of plant roots involves strong selective pressures that shape the structure and function of root-associated communities. In particular, the endosphere represents a highly selective environment requiring host entry and in planta persistence. However, strain-specific microbial traits that enable endosphere colonization remain poorly understood. Here, we use a defined, genome-resolved community of 28 Variovorax strains isolated from the roots of Populus deltoides and Populus trichocarpa (poplar trees) to determine which strains partition between rhizosphere and endosphere compartments and to identify the genomic traits associated with endosphere specialization. By combining strain-resolved metagenomic profiling, comparative genomics, and functional assays, we demonstrate that dominant endosphere colonizers are enriched in genes related to nutrient metabolism, redox balance, transcriptional regulation, and a conserved L-fucose utilization pathway experimentally shown to enhance root colonization. Not all strains succeed through the same strategy. Community-wide functional profiling revealed a distinct and reduced set of traits in the endosphere, including orthogroups associated with low-abundance strains that were overlooked in strain-level analyses. These findings reveal that multiple ecological strategies, such as metabolic competition, regulatory adaptation, and niche specialization, can support endosphere colonization. Our results advance the understanding of how bacterial colonization traits are distributed and deployed within a plant microbiome and suggest that host filtering selects for distinct, and sometimes complementary, microbial strategies. This work supports a shift toward mechanistic, genome-resolved models of microbiome assembly and offers a framework for linking microbial function to host colonization success. IMPORTANCE Plants often depend on diverse microbial partners to support their growth, resilience, and adaptation to changing environments. Among these microbes, some bacteria inhabit the rhizosphere (the narrow zone around roots where microbes interact with the plant) while others are able to enter and persist within root tissues. The traits that distinguish these two lifestyles remain poorly understood. In this study, we examined a group of related Variovorax strains from poplar tree root microbiomes to ask why some rhizosphere-associated strains also become successful endosphere colonizers. We found that strains appear to succeed through different strategies: some may benefit from rapid growth on plant-derived carbon sources, while others may rely on stress tolerance or fine-tuned regulation. These results suggest that there is no single path from the rhizosphere into the root interior, but rather multiple strategies shaped by the host environment. Understanding this diversity can inform efforts to design resilient plant-microbe communities.

ABSTRACT The senescent cell (SC) fate is linked to aging, multiple disorders and diseases, and physical dysfunction. Senolytics, agents that selectively eliminate 30%–70% of SCs, act by transiently disabling the senescent cell antiapoptotic pathways (SCAPs), which defend those SCs that are proapoptotic and pro‐inflammatory from their own senescence‐associated secretory phenotype (SASP). Consistent with this, a JAK/STAT inhibitor, Ruxolitinib, which attenuates the pro‐inflammatory SASP of senescent human preadipocytes, caused them to become “senolytic‐resistant”. Administering senolytics to obese mice selectively decreased the abundance of the subset of SCs that is pro‐inflammatory. In cell cultures, the 30%–70% of human senescent preadipocytes or human umbilical vein endothelial cells (HUVECs) that are senolytic‐resistant (to Dasatinib or Quercetin, respectively) had increased p16 INK4a , p21 CIP1 , senescence‐associated β‐galactosidase (SAβgal), γH2AX, and proliferative arrest similarly to the total SC population (comprising senolytic‐sensitive plus‐resistant SCs). However, the SASP of senolytic‐resistant SCs entailed less pro‐inflammatory/apoptotic factor production, induced less inflammation in non‐senescent cells, and was equivalent or richer in growth/fibrotic factors. Senolytic‐resistant SCs released less mitochondrial DNA (mtDNA) and more highly expressed the anti‐inflammatory immune evasion signal, glycoprotein non‐melanoma‐B (GPNMB). Transplanting senolytic‐resistant SCs intraperitoneally into younger mice caused less physical dysfunction than transplanting the total SC population. Because Ruxolitinib attenuates SC release of proapoptotic SASP factors, while pathogen‐associated molecular pattern factors (PAMPs) can amplify the release of these factors rapidly (acting as “senosensitizers”), senolytic‐resistant and senolytic‐sensitive SCs appear to be interconvertible.

Abstract Extracellular vesicles (EVs) are key mediators of cell–cell communication and are involved in transferring specific biomolecular cargo to recipient cells to regulate their physiological functions. A major challenge in the understanding of EV function in vivo is the difficulty ascertaining the origin of the EV particles. The recent development of the “Snorkel-tag,” which includes EV-membrane-targeted CD81 fused to a series of extra-vesicular protein tags, can be used to mark EVs originating from a specific source for subsequent isolation and characterization. We developed an in vivo mouse model, termed “CAGS-Snorkel,” which expresses the Snorkel-tag under the control of the Cre-lox system, and crossed this mouse with either Prx1-Cre (mesenchymal progenitors) or Ocn-Cre (osteoblasts/osteocytes) and isolated Snorkel-tagged EVs from the mouse bone marrow plasma using a magnetic bead affinity column. miRNA-sequencing was performed on the isolated EVs, and although similar profiles were observed, a few key miRNAs involved in bone metabolism (miR-106b-5p, miRs-19b-3p, and miRs-219a-5p) were enriched in the Ocn-derived relative to the Prx1-derived EV subpopulations. To characterize the effects of these small EVs on a bone cell target, cultured mouse bone marrow stromal cells were treated with Prx1 or Ocn EVs, and mRNA-sequencing was performed. Pathways involved in ossification, bone development, and extracellular matrix interactions were regulated by both EV subpopulations, whereas a few pathways including advanced glycation end-products signaling were uniquely regulated in the Ocn EV subpopulation, underlying important biological effects of specific EV subpopulations within the bone marrow microenvironment. These data demonstrate that EV isolation in vivo using the CAGS-Snorkel mouse model is a useful tool in characterizing the cargo and understanding the biology of tissue-specific EVs. Moreover, while bone mesenchymal cell populations share a common EV secretory profile, we uncover key differences based on the stage of osteoblastic differentiation that may have important biological consequences.