Pseudomonas aeruginosa (PA) is an opportunistic pathogen frequently isolated from cutaneous chronic wounds. How PA, in the presence of oxidative stress (OS), colonizes chronic wounds and forms a biofilm is still unknown. The purpose of this study is to investigate the changes in gene expression seen when PA is challenged with the high levels of OS present in chronic wounds. We used a biofilm-forming PA strain isolated from the chronic wounds of our murine model (RPA) and performed a qPCR to obtain gene expression patterns as RPA developed a biofilm in vitro in the presence of high levels of OS, and then compared the findings in vivo, in our mouse model of chronic wounds. We found that the planktonic bacteria under OS conditions overexpressed quorum sensing genes that are important for the bacteria to communicate with each other, antioxidant stress genes important to reduce OS in the microenvironment for survival, biofilm formation genes and virulence genes. Additionally, we performed RNAseq in vivo and identified the activation of novel genes/pathways of the Type VI Secretion System (T6SS) involved in RPA pathogenicity. In conclusion, RPA appears to survive the high OS microenvironment in chronic wounds and colonizes these wounds by turning on virulence, biofilm-forming and survival genes. These findings reveal pathways that may be promising targets for new therapies aimed at disrupting PA-containing biofilms immediately after debridement to facilitate the treatment of chronic human wounds.

Seeds are complex structures composed of three regions, embryo, endosperm, and seed coat, with each further divided into subregions that consist of tissues, cell layers, and cell types. Although the seed is well characterized anatomically, much less is known about the genetic circuitry that dictates its spatial complexity. To address this issue, we profiled mRNAs from anatomically distinct seed subregions at several developmental stages. Analyses of these profiles showed that all subregions express similar diverse gene numbers and that the small gene numbers expressed subregion specifically provide information about the biological processes that occur in these seed compartments. In parallel, we profiled RNAs in individual nuclei and identified nuclei clusters representing distinct cell identities. Integrating single-nucleus RNA and subregion mRNA transcriptomes allowed most cell identities to be assigned to specific subregions and cell types and/or cell states. The number of cell identities exceeds the number of anatomically distinguishable cell types, emphasizing the spatial complexity of seeds. We defined gene coexpression networks that underlie distinct biological processes during seed development. We showed that network distribution among subregions and cell identities is highly variable. Some networks operate in single subregions and/or cell identities, and many coexpression networks operate in multiple subregions and/or cell identities. We also showed that single subregions and cell identities possess several networks. Together, our studies provide unique insights into the biological processes and genetic circuitry that underlie the spatial landscape of the seed.

Significance: How plant embryos are differentiated into embryo proper and suspensor regions following fertilization is a major unanswered question. The suspensor is unique because it can vary in morphology in different plant species. We hypothesized that regulatory genes controlling the specification of embryo proper and suspensor regions should be shared by all plants irrespective of embryo morphology. We compared embryo proper and suspensor transcriptomes of plants with distinct suspensor morphologies. Scarlet runner bean and common bean have highly specialized giant suspensor regions; soybean and Arabidopsis suspensors are smaller and less specialized. We uncovered a small set of embryo proper– and suspensor-specific transcription factors shared by all embryos irrespective of morphology, suggesting that they play an important role in early embryo differentiation.

In plants, RNA-directed DNA methylation (RdDM) uses small interfering RNAs (siRNAs) to target transposable elements (TEs) but usually avoids genes. RNA polymerase IV (Pol IV) shapes the landscape of DNA methylation through its pivotal role in siRNA biogenesis. However, how Pol IV is recruited to specific loci, particularly how it avoids genes, is poorly understood. Here, we identified a Pol IV–interacting protein, ZMP (zinc finger, mouse double-minute/switching complex B, Plus-3 protein), which exerts a dual role in regulating siRNA biogenesis and DNA methylation at specific genomic regions. ZMP is required for siRNA biogenesis at some pericentromeric regions and prevents Pol IV from targeting a subset of TEs and genes at euchromatic loci. As a chromatin-associated protein, ZMP prefers regions with depleted histone H3 lysine 4 (H3K4) methylation abutted by regions with H3K4 methylation, probably monitoring changes in local H3K4 methylation status to regulate Pol IV’s chromatin occupancy. Our findings uncover a mechanism governing the specificity of RdDM.

Chronic wounds (CWs) in humans affect millions of people in the US alone, cost billions of dollars, cause much suffering, and still there are no effective treatments. Patients seek medical care when wound chronicity is already established, making it impossible to investigate factors that initiate chronicity. In this study, we used a diabetic mouse model of CWs that mimics many aspects of chronicity in humans. We performed scRNAseq to compare the cell composition and function during the first 72 h post-injury and profiled 102,737 cells into clusters of all major cell types involved in healing. We found two types of fibroblasts. Fib 1 (pro-healing) was enriched in non-CWs (NCWs) whereas Fib 2 (non-healing) was in CWs. Both showed disrupted proliferation and migration, and extracellular matrix (ECM) deposition in CWs. We identified several subtypes of keratinocytes, all of which were more abundant in NCWs, except for Channel-related keratinocytes, and showed altered migration, apoptosis, and response to oxidative stress (OS) in CWs. Vascular and lymphatic endothelial cells were both less abundant in CWs and both had impaired migration affecting the development of endothelial and lymphatic microvessels. Study of immune cells showed that neutrophils and mast cells are less abundant in CWs and that NCWs contained more proinflammatory macrophages (M1) whereas CWs were enriched in anti-inflammatory macrophages (M2). Also, several genes involved in mitochondrial function were abnormally expressed in CWs, suggesting impaired mitochondrial function and/or higher OS. Heat shock proteins needed for response to OS were downregulated in CWs, potentially leading to higher cellular damage. In conclusion, the initiation of chronicity is multifactorial and involves various cell types and cellular functions, indicating that one type of treatment will not fix all problems, unless the root cause is fundamental to the cell and molecular mechanisms of healing. We propose that such a fundamental process is high OS and its association with wound infection/biofilm.

Transposon silencing via RNA-directed DNA methylation (RdDM) is mediated primarily through 24-nucleotide (nt) small-interfering RNAs (siRNAs) and 2 plant-specific RNA polymerases, Pol IV and Pol V. We generated and characterized RNA-interference (RNAi) lines targeting the largest subunit of Pol IV and Pol V and the second-largest subunit shared by Pol IV and Pol V in soybean. Our analyses showed that the canonical roles of Pol IV and Pol V in RdDM as described in Arabidopsis, whereby Pol IV produces 24-nt siRNAs and Pol V recruits siRNAs to homologous loci to trigger DNA methylation, are conserved in soybean. Our analyses also uncovered previously unknown functions of Pol IV and Pol V in that they repress defense response genes en masse. These genes normally undergo RdDM and are silenced, but they are de-repressed when Pol IV and Pol V are knocked down. Furthermore, the de-repression of a set of defense-related genes channeled their RNAs into the RNAi pathway to produce 21- to 22-nt siRNAs. Knocking down Pol IV and Pol V either singly or together led to increased resistance against the oomycete pathogen Phytophthora sojae, suggesting that Pol IV- and Pol V-mediated gene silencing regulates plant immunity.