Plants perceive danger signals, pathogen- or damage-associated molecular patterns (PAMPs/DAMPs), to activate immune responses such as transient apoplastic alkalinization and reactive oxygen species (ROS) production. However, how these pH and redox changes occur across organs and tissues during pattern-triggered immunity (PTI) remains poorly understood. Using genetically-encoded biosensors (GEBs), we monitored cytosolic pH and redox dynamics across whole Arabidopsis seedlings with spatiotemporal resolution. Global treatments with diverse danger signals first induced cytosolic acidification and oxidation in roots, followed by petioles and, later, the hypocotyl, revealing organ-specific responsiveness and a bidirectional response gradient. By contrast, Pseudomonas syringae pv. tomato (Pto) DC3000 induced sustained cytosolic alkalinization and suppressed redox responses in inoculated shoots, even when co-treated with PAMPs. Mutants either lacking a functional flagellum (ΔfliC) or Type-3 secretion system (ΔhrcC) induced opposite or no long-term responses in the cytosol, respectively. Together, these findings highlight that the plant's immune response is not uniform but instead follow organ- and tissue-specific patterns which are trigger-dependent, and reflect distinct capacities of seedling cells to activate PTI.

Abstract Extracellular vesicles (EVs) can deliver RNA and proteins into host cells to manipulate immunity; but how EVs transverse the cell wall is unknown. Using the fungal pathogen Botrytis cinerea that induces cross-kingdom RNA interference in plants, we uncovered that EV-mediated RNA delivery was dependent on cell wall degrading enzymes. Through fluorescence and transmission electron microscopy, and molecular genetic techniques, we demonstrate that EV-associated proteins compromise the plant cell wall, thereby facilitating RNA delivery. Notably, cell wall degrading enzymes are commonly associated with EVs across plant-colonizing bacterial, fungal and oomycete species, indicating a conserved role in EV transport across cell walls. These findings uncover a formerly unknown mechanism by which cell wall degrading enzymes facilitate EVs to transverse the cell wall for cargo delivery in cross-kingdom communication.

Plants constantly encounter adverse environmental interactions. One organelle is particularly specialized in stress signaling and phytohormone synthesis: the plastid. Calcium (Ca2+), a key second messenger, is known to intersect with cellular phytohormone signaling networks. While cytosolic Ca2+ dynamics have been studied extensively, the physiological relevance of stromal Ca2+ transients and the identity of channels mediating rapid Ca2+ flux into plastids remain largely unexplored. In this study, we provide evidence for PLASTID ENVELOPE ION CHANNELS (PECs) as long-sought mediators of fast-activating cation channel-like currents. We show that PEC expression is jasmonic acid (JA)-induced to augment stromal Ca2+ transients under stress. Loss of PECs results in decreased JA priming and failure to elicit full defense responses after wounding. In turn, PEC1 overexpression improves Botrytis cinerea tolerance. Our findings link stromal Ca2+ signaling with JA-dependent stress responses and position PECs as modulators of plant defense.

The outcome of infection can be seen as a race between the pathogen and the plant immune system. In this review, we use the case study of Xylella fastidiosa (Xf) and explain how the bacterium exploits its slow and fastidious growth to remain below detection thresholds, thereby delaying immune activation. Its self-limiting behavior in the sessile state provides a temporal window for transitioning into a more exploratory lifestyle, enabling systemic colonization of the host. As bacterial populations expand, vessel occlusion and immune overactivation at late stages occur, often propagating beyond directly infected tissues, leading to hydraulic collapse and drought-like symptoms. We highlight how Xf's adaptations to a nutrient-poor and rapid-flow environment contribute to its persistence. Furthermore, we discuss how resistant plant genotypes, possessing broader or more sensitive repertoires of immune receptors, can detect the pathogen earlier and restrict its systemic spread in the xylem. Understanding the dynamics of these "catch me if you can" strategies may guide novel approaches to reduce Xf survival and mitigate disease progression in susceptible crops while also providing broader insights into plant responses and the infection strategies of other xylem-inhabiting microbes.

Olive trees are severely threatened by the Olive Quick Decline Syndrome (OQDS), caused by the xylem-dwelling bacterium Xylella fastidiosa. While many cultivars are highly susceptible, ‘Leccino’ exhibit partial resistance. To study the underlying immune mechanisms, we established stable callus cultures from vegetative tissues of susceptible and resistant cultivars. These cultures enable functional profiling of immune responses to known pathogen-associated molecular patterns (PAMPs) and Xf-derived triggers. Combined with genomic analysis, this system provides insights into cultivar-specific immunity and supports future strategies to enhance resistance in olive and related crops.

Xylella fastidiosa (Xf) is a Gram-negative bacterial plant pathogen responsible for severe diseases in a variety of economically important crops. A critical aspect of its virulence is the production of extracellular vesicles (EVs). In this study, we discovered that DNA-binding proteins and nonribosomal RNA-binding proteins are abundant in the corona of Xf-EVs. DNA-seq revealed enrichment of three genomic islands (GIs) in EVs, which carry molecular signatures indicative of horizontal gene transfer (HGT). The most abundant GI encodes five homologous small RNAs designated sXFs. RNA sequencing revealed a distinct pattern of noncoding RNAs enriched in EVs, including four island-encoded sXFs. One of the sXF's stem-loops contains motifs for binding the RNA chaperone Hfq, which is also abundant in EVs. Predicted target analysis suggests that sXFs play a role in regulation of natural competence in bacteria. Additionally, sXF plant target prediction identifies a coiled-coil nucleotide-binding domain leucine-rich repeat receptor (CNL) immune gene that is downregulated following Xf infection and Xf-EV treatment. We propose a model where Xf releases nucleic acid carrying EVs with two functions: one to deliver RNA-related cargo that regulates gene expression in both bacterial and plant cells, and another to deliver DNA-related cargo for the genetic transfer of genomic islands. We highlight island-encoded sXFs as potential virulence factors and vesiduction as a mechanism of HGT of sXFs in Xf. Taken together, our data on Xf-EV cargoes provide a molecular framework for understanding the virulence of Xf.

Plants detect pathogens using cell-surface pattern recognition receptors (PRRs) such as ELONGATION Factor-TU (EF-TU) RECEPTOR (EFR) and FLAGELLIN SENSING 2 (FLS2), which recognize bacterial EF-Tu and flagellin, respectively. These PRRs belong to the leucine-rich repeat receptor kinase (LRR-RK) family and activate the production of reactive oxygen species via the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD). The PRR-RBOHD complex is tightly regulated to prevent unwarranted or exaggerated immune responses. However, certain pathogen effectors can subvert these regulatory mechanisms, thereby suppressing plant immunity. To elucidate the intricate dynamics of the PRR-RBOHD complex, we conducted a comparative coimmunoprecipitation analysis using EFR, FLS2, and RBOHD in Arabidopsis thaliana. We identified QIAN SHOU KINASE 1 (QSK1), an LRR-RK, as a PRR-RBOHD complex-associated protein. QSK1 downregulated FLS2 and EFR abundance, functioning as a negative regulator of PRR-triggered immunity (PTI). QSK1 was targeted by the bacterial effector HopF2Pto, a mono-ADP ribosyltransferase, reducing FLS2 and EFR levels through both transcriptional and transcription-independent pathways, thereby inhibiting PTI. Furthermore, HopF2Pto transcriptionally downregulated PROSCOOP genes encoding important stress-regulated phytocytokines and their receptor MALE DISCOVERER 1-INTERACTING RECEPTOR-LIKE KINASE 2. Importantly, HopF2Pto requires QSK1 for its accumulation and virulence functions within plants. In summary, our results provide insights into the mechanism by which HopF2Pto employs QSK1 to desensitize plants to pathogen attack.

Infectious fungi send small RNAs into plant cells to enhance their virulence by silencing defense-related genes. In this issue of Cell Host & Microbe, Wang and colleagues show that full-length messenger RNA is transported in vesicles from plants to fungi, becoming translated by fungal ribosomes and reducing fungal pathogenicity.

In recent years, extracellular vesicles (EVs) have emerged as novel key players in plant-microbe interactions. While it is immensely useful to draw on the established "minimal information for studies of extracellular vesicles" (MISEV) guidelines and precedents in mammalian systems, working with plants and their associated microbes poses specific challenges. To navigate researchers through these obstacles, we offer detailed step-by-step suggestions for those embarking on EV research in the context of plant-microbe interactions. The advice is based on recent publications and our collective experience from the diverse plant and microbe systems studied in a dedicated research consortium. We provide considerations for experimental design, optimization, quality control, and recommendations on how to increase yield, purity, and reproducibility of EV isolation. With this perspective article, we aim not only to assist researchers in our field but also to promote discussions on plant and microbe EVs in the broader EV community.

Das Mikrobiom spielt eine große Rolle für das Wachstum oder auch die Widerstandsfähigkeit von Pflanzen. Bei der Besiedlung pflanzlichen Lebensraums bildet eine Vielzahl von Mikroorganismen komplexe Gemeinschaften mit bestimmten Zusammensetzungen und räumlichen Anordnungen. Welche Gene von welchem Mikroorganismus im Zusammenspiel mit dem Wirt reguliert werden, blieb bisher wegen experimenteller Herausforderungen unbekannt.

RNA interference (RNAi) is a crucial mechanism in immunity against infectious microbes through the action of DICER-LIKE (DCL) and ARGONAUTE (AGO) proteins. In the case of the taxonomically diverse fungal pathogen Botrytis cinerea and the oomycete Hyaloperonospora arabidopsidis, plant DCL and AGO proteins have proven roles as negative regulators of immunity, suggesting functional specialization of these proteins. To address this aspect in a broader taxonomic context, we characterized the colonization pattern of an informative set of DCL and AGO loss-of-function mutants in Arabidopsis thaliana upon infection with a panel of pathogenic microbes with different lifestyles, and a fungal mutualist. Our results revealed that, depending on the interacting pathogen, AGO1 acts as a positive or negative regulator of immunity, while AGO4 functions as a positive regulator. Additionally, AGO2 and AGO10 positively modulated the colonization by a fungal mutualist. Therefore, analyzing the role of RNAi across a broader range of plant-microbe interactions has identified previously unknown functions for AGO proteins. For some pathogen interactions, however, all tested mutants exhibited wild-type-like infection phenotypes, suggesting that the roles of AGO and DCL proteins in these interactions may be more complex to elucidate.

Members of the NETWORKED (NET) family are involved in actin-membrane interactions. Here we show that two members of the NET family, NET4A and NET4B, are essential for normal guard cell actin reorganization, which is a process critical for stomatal closure in plant immunity. NET4 proteins interact with F-actin and with members of the Rab7 GTPase RABG3 family through two distinct domains, allowing for simultaneous localization to actin filaments and the tonoplast. NET4 proteins interact with GTP-bound, active RABG3 members, suggesting their function being downstream effectors. We also show that RABG3b is critical for stomatal closure induced by microbial patterns. Taken together, we conclude that the actin cytoskeletal remodelling during stomatal closure involves a molecular link between actin filaments and the tonoplast, which is mediated by the NET4-RABG3b interaction. We propose that stomatal closure to microbial patterns involves the coordinated action of immune-triggered osmotic changes and actin cytoskeletal remodelling likely driving compact vacuolar morphologies.

Plant receptor kinases are key transducers of extracellular stimuli, such as the presence of beneficial or pathogenic microbes or secreted signaling molecules. Receptor kinases are regulated by numerous post-translational modifications.1,2,3 Here, using the immune receptor kinases FLS24 and EFR,5 we show that S-acylation at a cysteine conserved in all plant receptor kinases is crucial for function. S-acylation involves the addition of long-chain fatty acids to cysteine residues within proteins, altering their biochemical properties and behavior within the membrane environment.6 We observe S-acylation of FLS2 at C-terminal kinase domain cysteine residues within minutes following the perception of its ligand, flg22, in a BAK1 co-receptor and PUB12/13 ubiquitin ligase-dependent manner. We demonstrate that S-acylation is essential for FLS2-mediated immune signaling and resistance to bacterial infection. Similarly, mutating the corresponding conserved cysteine residue in EFR suppressed elf18-triggered signaling. Analysis of unstimulated and activated FLS2-containing complexes using microscopy, detergents, and native membrane DIBMA nanodiscs indicates that S-acylation stabilizes, and promotes retention of, activated receptor kinase complexes at the plasma membrane to increase signaling efficiency.

Vesiculation is a process employed by Gram-negative bacteria to release extracellular vesicles (EVs) into the environment. EVs from pathogenic bacteria play functions in host immune modulation, elimination of host defenses, and acquisition of nutrients from the host. Here, we observed EV production of the bacterial speck disease causal agent, Pseudomonas syringae pv. tomato (Pto) DC3000, as outer membrane vesicle release. Mass spectrometry identified 369 proteins enriched in Pto DC3000 EVs. The EV samples contained known immunomodulatory proteins and could induce plant immune responses mediated by bacterial flagellin. Having identified two biomarkers for EV detection, we provide evidence for Pto DC3000 releasing EVs during plant infection. Bioinformatic analysis of the EV-enriched proteins suggests a role for EVs in antibiotic defense and iron acquisition. Thus, our data provide insights into the strategies this pathogen may use to develop in a plant environment. IMPORTANCE The release of extracellular vesicles (EVs) into the environment is ubiquitous among bacteria. Vesiculation has been recognized as an important mechanism of bacterial pathogenesis and human disease but is poorly understood in phytopathogenic bacteria. Our research addresses the role of bacterial EVs in plant infection. In this work, we show that the causal agent of bacterial speck disease, Pseudomonas syringae pv. tomato, produces EVs during plant infection. Our data suggest that EVs may help the bacteria to adapt to environments, e.g., when iron could be limiting such as the plant apoplast, laying the foundation for studying the factors that phytopathogenic bacteria use to thrive in the plant environment.