Bacteria rely on an arsenal of weapons to challenge their opponents in highly competitive environments. To specifically counter closely related bacteria, specialized weapons with a narrow activity spectrum are deployed, particularly contractile phage tail-like particles or R-tailocins. Their production leads to the lysis of the producing cells, indicating that their expression must be carefully orchestrated so that only a small percentage of cells produce R-tailocins for the benefit of the entire population. In this study, we set out to better understand how the production of these phage tail-like weapons is regulated in environmental pseudomonads using the competitive plant root colonizer and environmental model strain Pseudomonas protegens CHA0. Using an RNA sequencing (RNA-seq) approach, we found that genes involved in DNA repair, particularly the SOS response program, are upregulated following exposure of the pseudomonad to the DNA-damaging agents mitomycin C and hydrogen peroxide, while genes involved in cell division and primary metabolism are downregulated. The R-tailocin and prophage gene clusters were also upregulated in response to these DNA damaging agents. By combining reverse genetics, transcriptional reporters and chromatin immunoprecipitation sequencing (ChIP-seq), we show that the R-tailocin locus-specific LexA-like regulator PrtR1 represses R-tailocin gene expression by binding directly to the promoter region of the cluster, while the histone-like nucleoid structuring (H-NS) proteins MvaT and MvaV act as master regulators that indirectly regulate R-tailocin cluster expression. Our results suggest that at least these three regulators operate in concert to ensure tight control of R-tailocin expression and cell lytic release in environmental Pseudomonas protegens strains.

Protein–protein interactions are one of the pillars of all life processes. Many signalling molecules work by promoting and stabilizing these interactions. These molecular ‘glues’ bind simultaneously to two proteins inducing their interaction, which would be otherwise less favorable or non-favorable. Importantly, they can be harnessed for a clinical purpose, but, despite advances in medicine, the wealth of natural molecular glues in plants have only rarely been commercially utilized. These molecular glues may be plant-endogenous or plant-exogenous small molecules or peptides, and they may be involved in many different processes, such as growth promotion or stress response, opening new opportunities for crop protection, along with other applications. In this Review, we analyse the underlying structural motives and molecular interactions in detail, classifying the modes of actions based on their nature (small ligands versus peptides) and receptor classes. We discuss both natural metabolites and mimetics of such compounds, highlighting similarities and differences between signalling pathways and comparing them with relevant mechanisms in mammals. In this Review we discuss the fascinating molecular details of the small molecules and peptides promoting plant protein–protein interactions, and their relevance for plant development and environmental responses.

Host-protective or disease-suppressive microorganisms are emerging as sustainable solutions for controlling crop diseases, such as bacterial wilt. However, the efficacy of biocontrol strategies is often constrained by limited resilience under varying environmental conditions and interactions with native microbial communities in the field. One major challenge is that introduced biocontrol microbes often face suppression by indigenous microbes due to competitive interactions. Synthetic communities (SynCom) offer a promising alternative strategy. However, conventional approaches to assembling SynComs by combining different microbial isolates often result in antagonism and competition among strains, leading to ineffective and inconsistent outcomes. In this study, we assembled a bacterial wilt-suppressive SynCom for tomato, composed of bacterial isolates derived from co-cultured microbial complexes associated with healthy plants. This SynCom demonstrates significant disease-suppressive effects against Ralstonia pseudosolanacearum in tomato seedlings under both axenic and soil conditions. Additionally, our findings suggest the presence of an optimal SynCom colonization level in plants, which is crucial for effective disease suppression. The SynCom also exhibits direct antibiotic activity and modulates the plant-associated microbiome. Our results provide an effective approach to constructing SynComs with consistent and effective disease-suppressive properties within microbial community contexts.

Agrobacterium pathogenesis, which involves transferring T-DNA into plant cells, is the cornerstone of plant genetic engineering. As the applications that rely on Agrobacterium increase in sophistication, it becomes critical to achieve a quantitative and predictive understanding of T-DNA expression at the level of single plant cells. Here we examine if a classic Poisson model of interactions between pathogens and host cells holds true for Agrobacterium infecting Nicotiana benthamiana. Systematically challenging this model revealed antagonistic and synergistic density-dependent interactions between bacteria that do not require quorum sensing. Using various approaches, we studied the molecular basis of these interactions. To overcome the engineering constraints imposed by antagonism, we created a dual binary vector system termed ‘BiBi’, which can improve the efficiency of a reconstituted complex metabolic pathway in a predictive fashion. Our findings illustrate how combining theoretical models with quantitative experiments can reveal new principles of bacterial pathogenesis, impacting both fundamental and applied plant biology. Alamos et al. show that Agrobacterium engages in density-dependent interactions that dictate transformation efficiency and transgene expression. These findings enable a quantitative model to improve metabolic engineering in a predictive manner.

CRISPR-Cas technologies facilitate routine genome engineering of one or a few genes at a time. However, large-scale CRISPR screens with guide RNA libraries remain challenging in plants. Here, we have developed a comprehensive all-in-one CRISPR toolbox for Cas9-based genome editing, cytosine base editing, adenine base editing (ABE), Cas12a-based genome editing and ABE, and CRISPR-Act3.0-based gene activation in both monocot and dicot plants. We evaluated all-in-one T-DNA expression vectors in rice (Oryza sativa, monocot) and tomato (Solanum lycopersicum, dicot) protoplasts, demonstrating their broad and reliable applicability. To showcase the applications of these vectors in CRISPR screens, we constructed guide RNA (gRNA) pools for testing in rice protoplasts, establishing a high-throughput approach to select high-activity gRNAs. Additionally, we demonstrated the efficacy of sgRNA library screening for targeted mutagenesis of ACETOLACTATE SYNTHASE in rice, recovering novel candidate alleles for herbicide resistance. Furthermore, we carried out a CRISPR activation screen in Arabidopsis thaliana, rapidly identifying potent gRNAs for FLOWERING LOCUS T activation that confer an early-flowering phenotype. This toolbox contains 61 versatile all-in-one vectors encompassing nearly all commonly used CRISPR technologies. It will facilitate large-scale genetic screens for loss-of-function or gain-of-function studies, presenting numerous promising applications in plants.

With only a fraction of plastic waste being recovered and the majority incinerated, landfilled or released into the environment, plastic is increasingly accumulating in aquatic and terrestrial ecosystems, leading to widespread pollution. Biocatalysis offers a route to the sustainable recycling of synthetic polyesters, such as polyethylene terephthalate (PET), which is commonly used in textiles, food and beverage packaging, representing a major source of plastic waste. In particular, polyester hydrolases can deconstruct recalcitrant synthetic polymers at scale. This Review discusses the role of biocatalysis in a circular economy of plastics, outlining enzymatic modification and deconstruction strategies for synthetic polyesters. Moreover, protein engineering and computational approaches are examined to design and optimize polyester hydrolases for large-scale recycling in key industrial applications. Finally, the importance of economic viability is highlighted, including strategies to make biocatalysis a cost-effective and sustainable plastics recycling strategy. Plastic pollution could be partly addressed through the biocatalytic recycling of plastic waste streams. This Review discusses the identification and optimization of polyester-degrading enzymes for the large-scale recycling of plastic waste. industry

Circular RNAs (circRNAs) are a class of non-coding RNAs generated through back splicing. High expression of circRNAs is often associated with numerous abnormal cellular biological processes. However, the regulatory factors of circRNAs are not fully understood. In this study, we identified PTBP1 as a crucial regulator of circRNA biogenesis through a comprehensive analysis of the whole transcriptome profiles across 10 diverse cell lines. Knockdown of PTBP1 led to a significant decrease in circRNA expression, concomitant with a distinct reduction in cell proliferation. To investigate the regulatory mechanism of PTBP1 on circRNA biogenesis, we constructed a minigene reporter based on SPPL3 gene. The results showed that PTBP1 can bind to the flanking introns of circSPPL3, and the mutation of PTBP1 motif impedes the back splicing of circSPPL3. Subsequently, to demonstrate that this observation is not an exception, the comprehensive regulatory effects of PTBP1 on circRNAs were confirmed by miniGFP, reflecting the necessity of the binding site in the flanking introns. Analysis of data from clinical samples showed that both PTBP1 and circRNAs exhibited substantial upregulation in acute myeloid leukemia, further demonstrating a potential role for PTBP1 in promoting circRNA biogenesis under in vivo conditions. Competitive endogenous RNA (ceRNA) network revealed that PTBP1-associated circRNAs participated in biological processes associated with cell proliferation. In summary, our study is the first to identify the regulatory effect of PTBP1 on circRNA biogenesis and indicates a possible link between PTBP1 and circRNA expression in leukemia. 

The characterization of antimicrobial susceptibility and other relevant phenotypes in large collections of microbial isolates is a common need across research and clinical microbiology laboratories. Robotization provides unprecedented throughput but involves costs that are prohibitive for the average laboratory. Here, using affordable materials and open-source software, we developed Q-PHAST (Quantitative PHenotyping and Antimicrobial Susceptibility Testing), a unique solution for cost-effective, large-scale phenotyping in a standard microbiology laboratory. Single colonies are grown in a deep 96-well master plate, from which diluted aliquots are used to generate 96 spots on different experimental plates containing solid medium with the substance and concentration of interest. These plates are incubated on inexpensive flatbed scanners that monitor the growth of each spot by obtaining images every 15 min. A simple, python-based software, which can be used via a graphical interface on various operating systems ( https://github.com/Gabaldonlab/Q-PHAST ), analyzes the images to infer growth, fitness (e.g., doubling rate) and susceptibility (e.g., minimum inhibitory concentration) measures. With <120 min of hands-on time per day for three consecutive days, ready-to-use results are obtained and presented in tables or graphs. This solution enables non-experts with limited resources to perform accurate quantitative phenotyping on hundreds of strains in parallel. This Q-PHAST (Quantitative PHenotyping and Antimicrobial Susceptibility Testing) protocol provides a unique solution for cost-effective, large-scale phenotyping in a standard microbiology laboratory using affordable materials and open-source software.

Microbes inhabiting complex porous microenvironments in sediments and aquifers catalyze reactions that are critical to global biogeochemical cycles and ecosystem health. However, the opacity and complexity of porous sediment and rock matrices have considerably hindered the study of microbial processes occurring within these habitats. Here, we generated microbially compatible, optically transparent mineral scaffolds to visualize and investigate microbial colonization and activities occurring in these environments, in laboratory settings and in situ. Using inexpensive synthetic cryolite mineral, we produced optically transparent scaffolds mimicking the complex 3D structure of sediments and rocks by adapting a suspension-based, freeze-casting technique commonly used in materials science. Fine-tuning of parameters, such as freezing rate and choice of solvent, provided full control of pore size and architecture. The combined effects of scaffold porosity and structure on the movement of microbe-sized particles, tested using velocity tracking of fluorescent beads, showed diverse yet reproducible behaviors. The scaffolds we produced are compatible with epifluorescence microscopy, allowing the fluorescence-based identification of colonizing microbes by DNA-based staining and fluorescence in situ hybridization (FISH) to depths of 100 µm. Additionally, Raman spectroscopy analysis indicates minimal background signal in regions used for measuring deuterium and 13C enrichment in microorganisms, highlighting the potential to directly couple D2O or 13C stable isotope probing and Raman-FISH for quantifying microbial activity at the single-cell level. To demonstrate the relevance of cryolite scaffolds for environmental field studies, we visualized their colonization by diverse microorganisms within rhizosphere sediments of a coastal seagrass plant using epifluorescence microscopy. The tool presented here enables highly resolved, spatially explicit, and multimodal investigations into the distribution, activities, and interactions of underground microbes typically obscured within opaque geological materials until now.

Long-term terrestrial ecosystem development is characterized by declining soil phosphorus (P) and a corresponding increase in biological P limitation. The function of P-limited ecosystems relies on efficient use of P by soil microorganisms, but the physiological strategies used by microorganisms to manage P scarcity during ecosystem development are unknown. Here, by applying recent advances in soil metabolomic techniques to samples collected from a ~700,000-year chronosequence of ecosystem development in eastern Australia, we show that soil microbial physiological strategies for P efficiency include a high proportion of non-phosphorous membrane lipids along with substantial intracellular carbon storage. These strategies—which proliferate during primary succession and are maximized in retrogressive, P-depleted ecosystems—uphold microbial carbon limitation, triple modelled P-mineralization potential and can conserve close to double the P contained in the aboveground biomass of vegetation. These findings transform our understanding of terrestrial ecosystems by revealing a strong yet overlooked interplay between the ecophysiology of soil microorganisms and the long-term trajectory of ecosystem development. Microbial physiology conserves phosphorus by reducing phosphorus contributions in membrane lipids and increasing intracellular carbon storage during long-term ecosystem development, according to measurements of a ~700,000–year chronosequence across an Australian dune system.