RMH

The rhizobia-legume nitrogen-fixing symbiosis begins when plants perceive rhizobial Nod factors (NF), activating the Common Symbiosis Signalling Pathway (CSSP) that drives nodule organogenesis. Rhizobia also inject Type III effectors (T3E) into host cells through a Type III Secretion System (T3SS) related to those of pathogenic bacteria. These effectors were long viewed only as suppressors of plant immunity and determinants of host compatibility, but this picture has changed. Recent studies revealed that rhizobial effectors (known as Nodulation Outer Proteins, Nops) such as NopT, NopM, NopL, NopP and NopZ directly target NF receptors or associated kinases, thereby regulating CSSP activation. The effector-triggering nodulation proteins (ET-Nods), a separate class of T3Es found in various Bradyrhizobium species, can activate nodule organogenesis independently of NF perception by rewiring host transcriptional and SUMOylation-based signalling. We trace the evolutionary origins of these activities, which may derive either from redirection of ancestral immune-targeting effectors toward symbiotic functions or from rhizobium-specific innovations. We ask whether the T3SS contributed to nodulation. We then consider what this interference means for the host, ranging from reinforced partner choice to partial loss of host control, and argue that rhizobial effectors have evolved as specialised modulators that converge on the signalling hubs governing symbiosis.

Plant growth-promoting Pseudomonas bacteria are promising biocontrol agents whose regulatory and effector genes shape bacterial traits involved in interactions with host plants and soil microorganisms. However, previous research has predominantly examined individual genes or traits in isolation, often making it difficult to compare their relative contributions within a single strain. This reductionist approach limits our understanding of how beneficial bacteria simultaneously engage in interactions with plants, suppress pathogens, and shape microbial communities. To address this gap, we selected 16 representative genes and gene clusters in P. fluorescens 2P24 based on genome annotation and previous knowledge of three PGPR-relevant functional layers: protein secretion systems, regulatory pathways, and secondary metabolite biosynthesis. This panel included the major annotated protein secretion systems in strain 2P24, selected regulatory systems with known or predicted roles in bacterial physiology and ecological interactions, and secondary-metabolite-related genes/gene clusters such as phl (2,4-DAPG biosynthetic gene cluster). We then examined how deletion of these genes or gene clusters affected bacterial traits, plant immune elicitation, and soil or rhizosphere bacterial community composition. Through comprehensive genetic and phenotypic analyses, we identified six genes and gene clusters—lapBCE, gacS, phoP, rsmX, fliC, and phl—that are essential for fundamental bacterial physiological processes and ecological fitness. More importantly, we discovered two regulatory modules, GacS-Phl and GacS-FliC, that coordinately govern multi-interface ecological interactions in the rhizosphere. Specifically, GacS regulates the production of 2,4-diacetylphloroglucinol, an antimicrobial compound that directly suppresses competing soil-dwelling Streptomyces populations. Simultaneously, GacS modulates flagellin expression, and flagellin acts as a microbe-associated molecular pattern triggering plant immune responses that indirectly reshape the composition and structure of rhizosphere microbiome communities. This study establishes a coordinated regulatory model that integrates global regulatory networks, microbe-associated molecular patterns, and antimicrobial secondary metabolites in mediating complex ecological interactions. Our findings reveal that beneficial Pseudomonas strains employ a GacS-mediated regulatory framework to simultaneously manage multiple ecological interfaces, providing mechanistic insights into how these bacteria balance plant-beneficial activities with competitive strategies. This regulatory framework offers a theoretical foundation for rational design and application of microbial biocontrol agents for sustainable agricultural practices.

Since the discovery and application of green fluorescent protein for use as an optical tag, fluorescent proteins (FPs) have changed the way we observe biological signals and processes. FP and fluorescent biosensor design has continued to advance, facilitated by new technologies and developments in chemistry, physics, engineering and screening technologies. We summarize some of the recent developments in FP engineering, focusing on insights that have led to improved photostability or longer wavelengths, among other improvements, highlighting mScarlet as a case study. We also discuss the engineering of new fluorescent biosensors, focusing on the ways in which screening, quantitative imaging, spatial resolution and multiplexing have been improved in recent years. Finally, we conclude with a perspective on what the future holds for the next generation of FPs and biosensors. This Review summarizes recent developments in fluorescent protein technology, including the engineering of fluorescent biosensors.

Candida species are fungal pathogens whose rapidly increasing antifungal resistance poses a substantial public health challenge. High-throughput CRISPR-based screening could accelerate antifungal target discovery, yet its application in Candida has been limited by low DNA transformation efficiency. Chemical transformation exposes cells to environmental stresses to permit DNA uptake, but the physiological constraints on transformability remain poorly defined. Here, we show that genetic transformability in C. albicans is governed by the cellular capacity to withstand and recover from transformation-induced stress. By systematically manipulating preculture conditions and transformation chemistry to modulate cellular stress tolerance, we developed a fungal advanced chemical transformation (FACT) method. The FACT method improves transformation efficiency over 100-fold across diverse Candida species and enables robust pooled CRISPR screening. Systems-level proteomic and physiological analyses reveal that nutrient limitation and optimized lithium acetate formulations prime cells for tolerance to transformation stress, with transformation efficiency strongly correlating with the expression of oxidative stress response proteins. This work overcomes a critical barrier to high‑throughput functional genomics and provides a mechanistic blueprint for enabling genetic manipulation of recalcitrant fungi.

A decade has passed since the initial description of cytosine base editors in 2016. This new class of gene editors, utilizing DNA damage intermediates beyond double-stranded DNA breaks, fundamentally shifted the paradigm of gene editing from stochastic disruption to precise chemical correction. Since then, the field has rapidly matured, with the development of optimized base editors with improved editing efficiency, targeting scope, precision and specificity, as well as the subsequent generation of adenine base editors and prime editors. Base editors have not only become widely used research tools but have served as foundational platforms for biotechnology companies developing human therapies. Today, base editors are being deployed in trials for a diverse list of indications including sickle cell disease, alpha-1 antitrypsin deficiency, inborn errors of metabolism, leukaemias and cardiovascular indications. This Review critically examines the ‘bench-to-bedside’ trajectory of base editors, detailing their mechanistic evolution and expanding role in treating human disease. Base editing, a gene editing technology capable of installing point mutations with high efficiency and precision, was first reported a decade ago. This Review examines the evolution of base editors and their road to clinical translation, highlighting key advances, ongoing challenges and the current clinical trial landscape.

Nitrogen-fixing cyanobacteria are important sources of bioavailable nitrogen on Earth. Some nitrogen-fixing cyanobacteria are endosymbionts of algae, and recently one of them, UCYN-A2, was shown to be an early-stage organelle (nitroplast) of haptophytes. These biological associations are important models for studying co-evolution and organellogenesis. Endosymbioses between Rhopalodiaceae diatoms and nitrogen-fixing cyanobacteria (spheroid bodies, SBs) are widespread in marine and freshwater ecosystems, but their adaptation and evolution remain unclear. We found that the genomes and metabolic characteristics of marine and freshwater SBs are remarkably similar. Purifying selection pressures dominated SB diversification both within marine and freshwater habitats and across habitat boundaries. The metabolic characteristics and genome reduction of SBs are substantially different from UCYN-A, suggesting different evolutionary journeys for the endosymbionts influenced by different hosts. We estimated that the marine SBs diversified ~185 Mya (95% highest posterior density (HPD): 340-88 Mya), which was within a prolonged oligotrophic period in the ocean, during which UCYN-A also diversified. The freshwater SBs diversified from the marine SBs ~137 Mya (95% HPD: 258-66 Mya) and further diversified ~80 Mya (95% HPD: 157-31 Mya), when sea level was increasing. Our study reveals the genetic and metabolic underpinnings characterizing the nitrogen-fixing cyanobacterial endosymbionts of Rhopalodiaceae diatoms.