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The aboveground parts of terrestrial plants are colonized by a variety of microbes that collectively constitute the phyllosphere microbiota. Decades of pioneering work using individual phyllosphere microbes, including commensals and pathogens, have provided foundational knowledge about how individual microbes adapt to the phyllosphere environment and their role in providing biological control against pathogens. Recent studies have revealed a more complete repertoire of phyllosphere microbiota across plant taxa and how plants respond to and regulate the level and composition of phyllosphere microbiota. Importantly, the development of several gnotobiotic systems is allowing causative and mechanistic studies to determine the contributions of microbiota to phyllosphere health and productivity. New insights into how the phyllosphere carries out key biological processes, including photosynthesis, biomass accumulation, reproduction, and defense against biotic and abiotic insults, in either the presence or absence of a normal microbiota could unleash novel plant- and microbiota-based technologies to improve agriculturally relevant traits of crop plants.

Phytohormone signalling pathways have an important role in defence against pathogens mediated by cell-surface pattern recognition receptors and intracellular nucleotide-binding leucine-rich repeat class immune receptors

The brown planthopper (BPH) is the most destructive insect pest in rice. Through a stylet, BPH secrets a plethora of salivary proteins into rice phloem cells as a crucial step of infestation. However, how various salivary proteins function in rice cells to promote insect infestation is poorly understood. Among them, one of the salivary proteins is predicted to be a carbonic anhydrase (NlCA). The survival rate of theNlCA-RNA interference (RNAi) BPH insects was extremely low on rice, indicating a vital role of this salivary protein in BPH infestation. We generatedNICAtransgenic rice plants and found thatNlCAexpressed in rice plants could restore the ability ofNlCA-RNAi BPH to survive on rice. Next, we produced rice plants expressing the ratiometric pH sensor pHusion and found thatNlCA-RNAi BPH induced rapid intracellular acidification of rice cells during feeding. Further analysis revealed that bothNlCA-RNAi BPH feeding and artificial lowering intracellular pH activated plant defense responses, and that NICA-mediated intracellular pH stabilization is linked to diminished defense responses, including reduced callose deposition at the phloem sieve plates and suppressed defense gene expression. Given the importance of pH homeostasis across the kingdoms of life, discovery of NICA-mediated intracellular pH modulation uncovered a new dimension in the interaction between plants and piecing/sucking insect pests. The crucial role of NICA for BPH infestation of rice suggests that NICA is a promising target for chemical or trans-kingdom RNAi-based inactivation for BPH control strategies in plants.Significance StatementInsect pests pose a serious biotic threat to crop production worldwide. Understanding how insect pests attack plants could inspire innovative pest control measures to enhance global food security. The brown planthopper (BPH) is the most devastative insect pest in rice. In this study, we discovered that BPH secretes a salivary carbonic anhydrase (NICA) to regulate the intracellular pH of the rice cell to facilitate its feeding and survival on rice plants and that NICA-mediated intracellular pH stabilization is linked to diminished defense responses. These findings not only uncovered that intracellular pH homeostasis is a previously uncharacterized battleground in plant-piecing/sucking insect interactions, but also open a door to potentially using NICA as a valuable molecular tool to gain insights into the broad impact of intracellular pH homeostasis on plant cell physiology.

Walking through a garden or a crop field, you may notice that plants damaged by pests (insects or pathogens) look smaller than the same kind of plants nearby that are not damaged. An obvious explanation would be that damaged plants may have lost substantial photosynthetic tissue due to insect and pathogen activities. As such, plants may have a reduced ability to capture light and perform photosynthesis, which fuels the growth of plants. While this is likely part of the reason why damaged plants look smaller, there is also another and perhaps more fascinating explanation that we would like to discuss here in this primer. It turns out that plants attacked by insects, pathogens and other biotic stressors may 'purposely' slow down their growth and that this response is often systemic, meaning that it occurs throughout the plant and beyond the tissue that is damaged by pests. Interestingly, some chemicals or plant genetic mutations that simulate insect or pathogen attacks without causing a loss of photosynthetic tissue can also slow plant growth, suggesting the physical loss of photosynthetic tissue per se is not always a prerequisite for slowing down plant growth. In contrast, there are conditions under which plants need to grow rapidly. For example, plants grow quickly when searching for light during germination or under a shaded canopy due to crowding from neighboring plants. Under these conditions, rapid plant growth is often accompanied by increased susceptibility to pests, presumably because growth is prioritized over defense. This inverse growth-defense relationship is commonly known as the 'growth-defense trade-off' and may be considered one of the most fundamental principles of 'plant economics' that allows plants to adjust growth and defense based on external conditions (Figure 1). As plants must both grow and defend in order to reproduce and survive in the natural world, growth-defense trade-offs have important ecological consequences. In agricultural settings, crops have often been bred to maximize growth-related traits, which could inadvertently result in the loss of useful genetic traits for biotic defenses. Thus, deciphering the molecular mechanisms underlying growth-defense trade-off phenomena could impact future crop breeding strategies aimed at designing superior crop plants with high yields as well as the ability to defend against biotic stressors. Here, we discuss some of the prevailing hypotheses about growth-defense trade-offs, our current understanding of the underlying mechanisms, and the ongoing efforts to optimize growth-defense trade-offs in crop plants.

Over the past three decades, researchers have isolated plant mutants that display constitutively activated defense responses in the absence of pathogen infection. These mutants are called autoimmune mutants and are typically dwarf and/or bearing chlorotic/necrotic lesions. From a genetic screen forArabidopsisgenes involved in maintaining a normal leaf microbiota, we identifiedTIP GROWTH DEFECTIVE 1(TIP1), which encodes a S-acyltransferase, as a key player in guarding leaves against abnormal microbiota level and composition under high humidity conditions. Thetip1mutant has several characteristic phenotypes of classical autoimmune mutants, including a dwarf stature, displaying lesions, and having a high basal level of defense gene expression. Gnotobiotic experiments revealed that the autoimmune phenotypes of thetip1mutant are largely dependent on the presence of microbiota as axenictip1plants have markedly reduced autoimmune phenotypes. We found that the microbiota dependency of autoimmune phenotypes is shared by several “lesion mimic”-type autoimmune mutants inArabidopsis. Interestingly, autoimmune phenotypes caused by mutations inNLRgenes do not require the presence of microbiota and can even be partially alleviated by microbiota. Our results therefore suggest the existence of two classes of autoimmunity (microbiota-dependent vs. microbiota-independent) in plants. The observed interplay between autoimmunity and microbiota in the lesion mimic class of autoimmunity is reminiscent of the interactions between autoimmunity and dysbiosis in the animal kingdom.

Although many studies have shown that microbes can ectopically stimulate or suppress plant immune responses, the fundamental question of whether the entire preexisting microbiota is indeed required for proper development of plant immune response remains unanswered. Using a recently developed peat-based gnotobiotic plant growth system we found thatArabidopsisgrown in the absence of a natural microbiota lacked age-dependent maturation of plant immune response and were defective in several aspects of pattern-triggered immunity. Axenic plants exhibited hypersusceptibility to infection by the foliar bacterial pathogenPseudomonas syringaepv.tomatoDC3000. Microbiota-mediated immunocompetence was suppressed by rich nutrient conditions, indicating a tripartite interaction between the host, microbiota, and abiotic environment. A synthetic microbiota composed of 48 culturable bacterial strains from the leaf endosphere of healthyArabidopsisplants was able to substantially restore immunocompetence similar to plants inoculated with a soil-derived community. In contrast, a 52-member dysbiotic synthetic leaf microbiota overstimulated the immune transcriptome. Together, these results provide evidence for a causal role of a eubiotic microbiota in gating proper immunocompetence and age-dependent immunity in plants.

The complex structure and function of a plant microbiome are driven by many variables, including the environment, microbe–microbe interactions and host factors. Likewise, resident microbiota can influence many host phenotypes. Gnotobiotic growth systems and controlled environments empower researchers to isolate these variables, and standardized methods equip a global research community to harmonize protocols, replicate experiments and collaborate broadly. We developed two easily constructed peat-based gnotobiotic growth platforms: the FlowPot system and the GnotoPot system. Sterile peat is amenable to colonization by microbiota and supports growth of the model plant Arabidopsis thaliana in the presence or absence of microorganisms. The FlowPot system uniquely allows one to flush the substrate with water, nutrients and/or suspensions of microbiota via an irrigation port, and a mesh retainer allows for the inversion of plants for dip or vacuum infiltration protocols. The irrigation port also facilitates passive drainage, preventing root anoxia. In contrast, the GnotoPot system utilizes a compressed peat pellet, widely used in the horticultural industry. GnotoPot construction has fewer steps and requires less user handling, thereby reducing the risk of contamination. Both protocols take up to 4 d to complete with 4–5 h of hands-on time, including substrate and seed sterilization. In this protocol, we provide detailed assembly and inoculation procedures for the two systems. Both systems are modular, do not require a sterile growth chamber, and cost less than US$2 per vessel. This protocol describes two peat-based plant growth systems for microbiome research in Arabidopsis. While both systems support microbe-free plants and input microbiota control, GnotoPots have advantages for throughput and FlowPots for versatility.

The plant immune system is fundamental for plant survival in natural ecosystems and for productivity in crop fields. Substantial evidence supports the prevailing notion that plants possess a two-tiered innate immune system, called pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). PTI is triggered by microbial patterns via cell surface-localized pattern-recognition receptors (PRRs), whereas ETI is activated by pathogen effector proteins via predominantly intracellularly localized receptors called nucleotide-binding, leucine-rich repeat receptors (NLRs)1–4. PTI and ETI are initiated by distinct activation mechanisms and involve different early signalling cascades5,6. Here we show that Arabidopsis PRR and PRR co-receptor mutants—fls2 efr cerk1 and bak1 bkk1 cerk1 triple mutants—are markedly impaired in ETI responses when challenged with incompatible Pseudomonas syrinage bacteria. We further show that the production of reactive oxygen species by the NADPH oxidase RBOHD is a critical early signalling event connecting PRR- and NLR-mediated immunity, and that the receptor-like cytoplasmic kinase BIK1 is necessary for full activation of RBOHD, gene expression and bacterial resistance during ETI. Moreover, NLR signalling rapidly augments the transcript and/or protein levels of key PTI components. Our study supports a revised model in which potentiation of PTI is an indispensable component of ETI during bacterial infection. This revised model conceptually unites two major immune signalling cascades in plants and mechanistically explains some of the long-observed similarities in downstream defence outputs between PTI and ETI. Bacteria elicit two distinct immune responses in Arabidopsis thaliana, mediated by diverse signalling receptors but working in a synergistic manner.

Pattern-triggered immunity and effector-triggered immunity are two primary forms of innate immunity in land plants. The molecular components and connecting nodes of pattern-triggered immunity and effector-triggered immunity are not fully understood. Here, we report that the Arabidopsis calcium-dependent protein kinase CPK3 is a key regulator of both pattern-triggered immunity and effector-triggered immunity. In vitro and in vivo phosphorylation assays, coupled with genetic and cell biology-based analyses, show that actin-depolymerization factor 4 (ADF4) is a physiological substrate of CPK3, and that phosphorylation of ADF4 by CPK3 governs actin cytoskeletal organization associated with pattern-triggered immunity. CPK3 regulates stomatal closure induced by flg22 and is required for resistance to Pst DC3000. Our data further demonstrates that CPK3 is required for resistance to Pst DC3000 carrying the effector AvrPphB. These results suggest that CPK3 is a missing link between cytoskeleton organization, pattern-triggered immunity and effector-triggered immunity., Remodelling of the actin cytoskeleton occurs during plant immune responses to pathogens. Here Lu et al. show that this process requires the calcium-dependent kinase CPK3 which phosphorylates actin depolymerizing factor 4 and is required for both PAMP and effector-triggered immunity in Arabidopsis.

Citrus Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (Las), is one of the most destructive citrus diseases worldwide, yet how Las causes HLB is poorly understood. Here we show that a Las-secreted protein, SDE15 (CLIBASIA_04025), suppresses plant immunity and promotes Las multiplication. Transgenic expression of SDE15 in Duncan grapefruit (Citrus × paradisi) suppresses the hypersensitive response induced by Xanthomonas citri ssp. citri (Xcc) and reduces the expression of immunity-related genes. SDE15 also suppresses the hypersensitive response triggered by the Xanthomonas vesicatoria effector protein AvrBsT in Nicotiana benthamiana, suggesting that it may be a broad-spectrum suppressor of plant immunity. SDE15 interacts with the citrus protein CsACD2, a homolog of Arabidopsis (Arabidopsis thaliana) ACCELERATED CELL DEATH 2 (ACD2). SDE15 suppression of plant immunity is dependent on CsACD2, and overexpression of CsACD2 in citrus suppresses plant immunity and promotes Las multiplication, phenocopying overexpression of SDE15. Identification of CsACD2 as a susceptibility target has implications in genome editing for novel plant resistance against devastating HLB.

The aboveground parts of terrestrial plants, collectively called the phyllosphere, have a key role in the global balance of atmospheric carbon dioxide and oxygen. The phyllosphere represents one of the most abundant habitats for microbiota colonization. Whether and how plants control phyllosphere microbiota to ensure plant health is not well understood. Here we show that the Arabidopsis quadruple mutant (min7 fls2 efr cerk1; hereafter, mfec)1, simultaneously defective in pattern-triggered immunity and the MIN7 vesicle-trafficking pathway, or a constitutively activated cell death1 (cad1) mutant, carrying a S205F mutation in a membrane-attack-complex/perforin (MACPF)-domain protein, harbour altered endophytic phyllosphere microbiota and display leaf-tissue damage associated with dysbiosis. The Shannon diversity index and the relative abundance of Firmicutes were markedly reduced, whereas Proteobacteria were enriched in the mfec and cad1S205F mutants, bearing cross-kingdom resemblance to some aspects of the dysbiosis that occurs in human inflammatory bowel disease. Bacterial community transplantation experiments demonstrated a causal role of a properly assembled leaf bacterial community in phyllosphere health. Pattern-triggered immune signalling, MIN7 and CAD1 are found in major land plant lineages and are probably key components of a genetic network through which terrestrial plants control the level and nurture the diversity of endophytic phyllosphere microbiota for survival and health in a microorganism-rich environment. Mutations in genes involved in immune signalling and vesicle trafficking cause defects in the leaf microbiome of Arabidopsis thaliana that result in damage to leaf tissues, suggesting mechanisms by which terrestrial plants control the level and diversity of endophytic phyllosphere microbiota.

Significance Plants evolved in an environment colonized by a vast number of microbes, which collectively constitute the plant microbiota. The majority of microbiota taxa are nonpathogenic and may be beneficial to plants under certain ecological or environmental conditions. We conducted experiments to understand the features of long-term interactions of nonpathogenic microbiota members with plants. We found that a multiplication–death equilibrium explained the shared long-term static populations of nonpathogenic bacteria and that in planta bacterial transcriptomic signatures were characteristic of the stationary phase, a physiological state in which stress protection responses are induced. These results may have significant implications in understanding the bulk of “nonpathogenic” plant–microbiota interactions that occur in agricultural and natural ecosystems.

Global crop production is greatly reduced by vascular diseases. These diseases include bacterial blight of rice and crucifer black rot caused by Xanthomonas oryzae pv. oryzae ( Xoo ) and Xanthomonas campestris pv. campestris ( Xcc ). The molecular mechanisms that activate vascular defense against such pathogens remains underexplored. Here, we show that an Arabidopsis MAPK phosphatase 1 (MKP1) mutant has increased host susceptibility to the adapted pathogen Xcc and is compromised in nonhost resistance to the rice pathogen Xoo . MKP1 regulates MAPK-mediated phosphorylation of the transcription factor MYB4 that negatively regulates vascular lignification through inhibiting lignin biosynthesis. Induction of lignin biosynthesis is, therefore, an important part of vascular-specific immunity. The role of MKP-MAPK-MYB signaling in lignin biosynthesis and vascular resistance to Xoo is conserved in rice, indicating that these factors form a tissue-specific defense regulatory network. Our study likely reveals a major vascular immune mechanism that underlies tissue-specific disease resistance against bacterial pathogens in plants.

High atmospheric humidity levels profoundly impact host-pathogen interactions in plants by enabling the establishment of an aqueous living space that benefits pathogens. The effectors HopM1 and AvrE1 of the bacterial pathogen Pseudomonas syringae have been shown to induce an aqueous apoplast under such conditions. However, the mechanisms by which this happens remain unknown. Here, we show that HopM1 and AvrE1 work redundantly to establish an aqueous living space by inducing a major reprogramming of the Arabidopsis thaliana transcriptome landscape. These effectors induce a strong abscisic acid (ABA) signature, which promotes stomatal closure, resulting in reduced leaf transpiration, and water-soaking lesions. Furthermore, these effectors preferentially increase ABA accumulation in guard cells, which control stomatal aperture. Notably, a guard-cell specific ABA transporter, ABCG40, is necessary for HopM1 induction of water-soaking lesions. This study provides molecular insights into a chain of events of stomatal manipulation that create an ideal microenvironment to facilitate infection.

Rice false smut caused by Ustilaginoidea virens is becoming one of the most recalcitrant rice diseases worldwide. However, the molecular mechanisms underlying rice immunity against U. virens remain unknown. Using genetic, biochemical and disease resistance assays, we demonstrated that the xb24 knockout lines generated in non-Xa21 rice background exhibit an enhanced susceptibility to the fungal pathogens U. virens and Magnaporthe oryzae. Consistently, flg22- and chitin-induced oxidative burst and expression of pathogenesis-related genes in the xb24 knockout lines were greatly attenuated. As a central mediator of energy signaling, SnRK1A interacts with and phosphorylates XB24 at Thr83 residue to promote ATPase activity. SnRK1A is activated by pathogen-associated molecular patterns and positively regulates plant immune responses and disease resistance. Furthermore, the virulence effector SCRE1 in U. virens targets host ATPase XB24. The interaction inhibits ATPase activity of XB24 by blocking ATP binding to XB24. Meanwhile, SCRE1 outcompetes SnRK1A for XB24 binding, and thereby suppresses SnRK1A-mediated phosphorylation and ATPase activity of XB24. Our results indicate that the conserved SnRK1A-XB24 module in multiple crop plants positively contributes to plant immunity and uncover an unidentified molecular strategy to promote infection in U. virens and a novel host target in fungal pathogenesis.

Extreme weather conditions associated with climate change affect many aspects of plant and animal life, including the response to infectious diseases. Production of salicylic acid (SA), a central plant defence hormone1–3, is particularly vulnerable to suppression by short periods of hot weather above the normal plant growth temperature range via an unknown mechanism4–7. Here we show that suppression of SA production in Arabidopsis thaliana at 28 °C is independent of PHYTOCHROME B8,9 (phyB) and EARLY FLOWERING 310 (ELF3), which regulate thermo-responsive plant growth and development. Instead, we found that formation of GUANYLATE BINDING PROTEIN-LIKE 3 (GBPL3) defence-activated biomolecular condensates11 (GDACs) was reduced at the higher growth temperature. The altered GDAC formation in vivo is linked to impaired recruitment of GBPL3 and SA-associated Mediator subunits to the promoters of CBP60g and SARD1, which encode master immune transcription factors. Unlike many other SA signalling components, including the SA receptor and biosynthetic genes, optimized CBP60g expression was sufficient to broadly restore SA production, basal immunity and effector-triggered immunity at the elevated growth temperature without significant growth trade-offs. CBP60g family transcription factors are widely conserved in plants12. These results have implications for safeguarding the plant immune system as well as understanding the concept of the plant–pathogen–environment disease triangle and the emergence of new disease epidemics in a warming climate.

The Xanthomonas outer protein C2 (XopC2) family of bacterial effectors is widely found in plant pathogens and Legionella species. However, the biochemical activity and host targets of these effectors remain enigmatic. Here we show that ectopic expression of XopC2 promotes jasmonate signaling and stomatal opening in transgenic rice plants, which are more susceptible to Xanthomonas oryzae pv. oryzicola infection. Guided by these phenotypes, we discover that XopC2 represents a family of atypical kinases that specifically phosphorylate OSK1, a universal adaptor protein of the Skp1-Cullin-F-box ubiquitin ligase complexes. Intriguingly, OSK1 phosphorylation at Ser53 by XopC2 exclusively increases the binding affinity of OSK1 to the jasmonate receptor OsCOI1b, and specifically enhances the ubiquitination and degradation of JAZ transcription repressors and plant disease susceptibility through inhibiting stomatal immunity. These results define XopC2 as a prototypic member of a family of pathogenic effector kinases and highlight a smart molecular mechanism to activate jasmonate signaling., XopC2 effectors are present in many plant bacterial pathogens. Here the authors show that XopC2 has kinase activity and enhances disease susceptibility by phosphorylating the OSK1 protein, which increases its interaction with the jasmonate receptor COI1b to promote JA signaling and stomatal opening.

Bacterial phytopathogens deliver effector proteins into host cells as key virulence weapons to cause disease. Extensive studies revealed diverse functions and biochemical properties of different effector proteins from pathogens. In this study, we show that the Pseudomonas syringae effector AvrE, the founding member of a broadly conserved and pathologically important bacterial effector family, binds to phosphatidylinositides (PIPs) in vitro and shares some properties with eukaryotic PROPPINs (β-propellers that bind polyphosphoinositides). In planta pull down experiments with transgenic Arabidopsis plants expressing AvrE revealed that AvrE is associated with several plant proteins including plasma membrane lipid-raft proteins. These results shed new light on the properties of a bacterial effector that is crucial for bacterial virulence in plants.

For the past 4 decades, intensive molecular studies of mostly leaf mesophyll cell-infecting pathogens and chewing insects have led to compelling models of plant-pathogen and plant-insect interactions. Yet, some of the most devastating pathogens and insect pests live in or feed on the phloem, a systemic tissue belonging to the plant vascular system. Phloem tissues are difficult to study, and phloem-inhabiting pathogens are often impossible to culture, thus limiting our understanding of phloem-insect/pathogen interactions at a molecular level. In this Perspective, we highlight recent literature that reports significant advances in the understanding of phloem interactions with insects and prokaryotic pathogens and attempt to identify critical questions that need attention for future research. It is clear that study of phloem-insect/pathogen interactions represents an exciting frontier of plant science, and influx of new scientific expertise and funding is crucial to achieve faster progress in this important area of research that is integral to global food security.

In the past four decades, tremendous progress has been made in understanding how plants respond to microbial colonization and how microbial pathogens and symbionts reprogram plant cellular processes. In contrast, our knowledge of how environmental conditions impact plant-microbe interactions is less understood at the mechanistic level, as most molecular studies are performed under simple and static laboratory conditions. In this review, we highlight research that begins to shed light on the mechanisms by which environmental conditions influence diverse plant-pathogen, plant-symbiont, and plant-microbiota interactions. There is a great need to increase efforts in this important area of research in order to reach a systems-level understanding of plant-microbe interactions that are more reflective of what occurs in nature.

Microbial pathogen contamination is a leading cause of impairment for urban rivers and streams in Michigan. Reports on the ability of green infrastructure best management practices to remove microbial pathogens have been highly variable. This study evaluated the influence of a detention basin (Kreiser Pond) on microbial dynamics in the Plaster Creek watershed in West Michigan. High levels of fecal indicator bacteria and coliphage were documented in influent and effluent water, with significant increases in indicator microbe concentrations during storm events. In dry conditions, Kreiser Pond efficiently reduced the number of indicator microbes flowing through the basin. Rainfall volume had a greater influence on the diversity of bacteria than sampling location. Antibiotic resistance was prevalent in culturable E. coli from Kreiser Pond, demonstrating a potential public health risk and highlighting the need for identifying the ultimate sources of microbial pollution.

Plants are in constant association with a variety of microbes, and although much is known about how symbiotic and pathogenic microbes interact with plants, less is known about the population dynamics, adaptive traits, and transcriptional features of endophytic commensal microbes that live inside leaves. In this study, we evaluated the long-term population and transcriptional dynamics of two bacterial microbiota endophytes and compared them to those of a commensal-simulating non-pathogenic mutant of the bacterial pathogen Pseudomonas syringae. We found that population densities of all three endophytic phyllosphere bacteria remained static over a long period of time, which was caused by a continual equilibrium between bacterial multiplication and death, as evidenced by treatment of plants with antibiotics that only targeted dividing bacteria or by in planta visualization of bacteria carrying a fluorescent division reporter. Population stasis could not be explained by a lack of resources, as Arabidopsis leaves could support population densities up to 100 times higher than the normal microbiota populations, nor was population stasis reversed by significantly quenching PAMP-triggered immunity. Long-term temporal in planta transcriptomic analysis of these three bacterial endophytes revealed up-regulation of protein translation, the generation of energy, and the response to stress, and interestingly, for the microbiota strains, the longer the bacteria remained inside plants, the greater the up-regulation of some of these processes. Further transcriptomic analysis of in planta populations of commensal-simulating Pseudomonas syringae revealed a remarkable resemblance to those of in vitro bacteria in stationary phase, a metabolically active physiological state in which the production of secondary metabolites and stress responses are induced. This study provides novel insight into how endophytic bacteria survive and thrive inside plant leaves, and reshapes our current understanding of what it means to be part of the endophytic microbiota in the phyllosphere.

Stomata exert considerable effects on global carbon and water cycles by mediating gas exchange and water vapour(1,2). Stomatal closure prevents water loss in response to dehydration and limits pathogen entry(3,4). However, prolonged stomatal closure reduces photosynthesis and transpiration and creates aqueous apoplasts that promote colonization by pathogens. How plants dynamically regulate stomatal reopening in a changing climate is unclear. Here we show that the secreted peptides SMALL PHYTOCYTOKINES REGULATING DEFENSE AND WATER LOSS (SCREWs) and the cognate receptor kinase PLANT SCREW UNRESPONSIVE RECEPTOR (NUT) counter-regulate phytohormone abscisic acid (ABA)- and microbe-associated molecular pattern (MAMP)-induced stomatal closure. SCREWS sensed by NUT function as immunomodulatory phytocytokines and recruit SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) co-receptors to relay immune signalling. SCREWS trigger the NUT-dependent phosphorylation of ABA INSENSITIVE 1 (ABI1) and ABI2, which leads to an increase in the activity of ABI phosphatases towards OPEN STOMATA 1 (OST1)–a key kinase that mediates ABA- and MAMP-induced stomatal closure(5,6)–and a reduction in the activity of S-type anion channels. After induction by dehydration and pathogen infection, SCREW–NUT signalling promotes apoplastic water loss and disrupts microorganism-rich aqueous habitats to limit pathogen colonization. The SCREW–NUT system is widely distributed across land plants, which suggests that it has an important role in preventing uncontrolled stomatal closure caused by abiotic and biotic stresses to optimize plant fitness.

Maintaining microbiome structure is critical for the health of both plants and animals. By re-screening a collection of Arabidopsis mutants affecting root immunity and hormone crosstalk, we identified a FERONIA (FER) receptor kinase mutant (fer-8) with a rhizosphere microbiome enriched in Pseudomonas fluorescens without phylum-level dysbiosis. Using microbiome transplant experiments, we found that the fer-8 microbiome was beneficial. The effect of FER on rhizosphere pseudomonads was largely independent of its immune scaffold function, role in development and jasmonic acid autoimmunity. We found that the fer-8 mutant has reduced basal levels of reactive oxygen species (ROS) in roots and that mutants deficient in NADPH oxidase showed elevated rhizosphere pseudomonads. The addition of RALF23 peptides, a FER ligand, was sufficient to enrich P. fluorescens. This work shows that FER-mediated ROS production regulates levels of beneficial pseudomonads in the rhizosphere microbiome.

Interview with Sheng Yang He, who studies the molecular biology of plant-pathogen interactions at Michigan State University.

Maintaining microbiome structure is critical for the health of both plants1 and animals2. In plants, enrichment of beneficial bacteria is associated with advantageous outcomes including protection from biotic and abiotic stress3,4. However, the genetic and molecular mechanisms by which plants enrich for specific beneficial microbes without general dysbiosis have remained elusive. Here we show that through regulation of NADPH oxidase, FERONIA kinase negatively regulates beneficial Pseudomonas fluorescens in the Arabidopsis rhizosphere microbiome. By rescreening a collection of Arabidopsis mutants that affect root immunity under gnotobiotic conditions, followed by microbiome sequencing in natural soil, we identified a FERONIA mutant (fer-8) with a rhizosphere microbiome enriched in P. fluorescens without phylum-level dysbiosis. Using microbiome transplant experiments, we found that the fer-8 microbiome was beneficial and promoted plant growth. The effect of FER on rhizosphere Pseudomonads was independent of its immune coreceptor function, role in development, and jasmonic acid autoimmunity. We found that the fer-8 mutant has reduced basal levels of reactive oxygen species (ROS) in roots and that mutants deficient in NADPH oxidase showed elevated rhizosphere Pseudomonad levels. Overexpression of the ROP2 gene (encoding a client of FER and positive regulator of NADPH oxidase5) in fer-8 plants suppressed Pseudomonad overgrowth. This work shows that FER-mediated ROS production regulates levels of beneficial Pseudomonads in the rhizosphere microbiome.

The complex structure and function of a plant microbiome are driven by many variables, including the environment, microbe-microbe interactions and host factors. Likewise, resident microbiota can influence many host phenotypes. Gnotobiotic growth systems and controlled environments empower researchers to isolate these variables, and standardized methods equip a global research community to harmonize protocols, replicate experiments and collaborate broadly. We developed two easily constructed peat-based gnotobiotic growth platforms: the FlowPot system and the GnotoPot system. Sterile peat is amenable to colonization by microbiota and supports growth of the model plant Arabidopsis thaliana in the presence or absence of microorganisms. The FlowPot system uniquely allows one to flush the substrate with water, nutrients and/or suspensions of microbiota via an irrigation port, and a mesh retainer allows for the inversion of plants for dip or vacuum infiltration protocols. The irrigation port also facilitates passive drainage, preventing root anoxia. In contrast, the GnotoPot system utilizes a compressed peat pellet, widely used in the horticultural industry. GnotoPot construction has fewer steps and requires less user handling, thereby reducing the risk of contamination. Both protocols take up to 4 d to complete with 4-5 h of hands-on time, including substrate and seed sterilization. In this protocol, we provide detailed assembly and inoculation procedures for the two systems. Both systems are modular, do not require a sterile growth chamber, and cost less than US$2 per vessel.

The plant immune system is fundamental to plant survival in natural ecosystems and productivity in crop fields. Substantial evidence supports the prevailing notion that plants possess a two-tiered innate immune system, called pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). PTI is triggered by microbial patterns via cell surface-localized pattern-recognition receptors (PRRs), whereas ETI is activated by pathogen effector proteins via mostly intracellularly-localized receptors called nucleotide-binding, leucine-rich repeat proteins (NLRs)1-4. PTI and ETI are initiated by dist 30 inct activation mechanisms and are considered to act independently and have evolved sequentially5,6. Here we show that, contrary to the perception of PTI and ETI being separate immune signaling pathways, Arabidopsis PRR/co-receptor mutants, fls2/efr/cerk1 and bak1/bkk1/cerk1 triple mutants, are greatly impaired in ETI responses when challenged with incompatible Pseudomonas syrinage bacteria. We further show that the NADPH oxidase (RBOHD)-mediated production of reactive oxygen species (ROS) is a critical early signaling event connecting PRR and NLR cascades and that PRR-mediated phosphorylation of RBOHD is necessary for full activation of RBOHD during ETI. Furthermore, NLR signaling rapidly augments the transcript and protein levels of key PTI components at an early stage and in a salicylic acid-independent manner. Our study supports an alternative model in which PTI is in fact an indispensable component of ETI during bacterial infection, implying that ETI halts pathogen infection, in part, by directly co-opting the anti-pathogen mechanisms proposed for PTI. This alternative model conceptually unites two major immune signaling pathways in the plant kingdom and mechanistically explains the long-observed similarities in downstream defense outputs between PTI and ETI.

Citrus Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (Las), is one of the most destructive citrus diseases worldwide, yet how Las causes HLB is poorly understood. Here we show that a Las-secreted protein, SDE15 (CLIBASIA_04025), suppresses plant immunity and promotes Las multiplication. Transgenic expression of SDE15 in Duncan grapefruit (Citrus × paradisi) suppresses the hypersensitive response induced by Xanthomonas citri ssp. citri (Xcc) and reduces the expression of immunity-related genes. SDE15 also suppresses the hypersensitive response triggered by the Xanthomonas vesicatoria effector protein AvrBsT in Nicotiana benthamiana, suggesting that it may be a broad-spectrum suppressor of plant immunity. SDE15 interacts with the citrus protein CsACD2, a homolog of Arabidopsis (Arabidopsis thaliana) ACCELERATED CELL DEATH 2 (ACD2). SDE15 suppression of plant immunity is dependent on CsACD2, and overexpression of CsACD2 in citrus suppresses plant immunity and promotes Las multiplication, phenocopying overexpression of SDE15. Identification of CsACD2 as a susceptibility target has implications in genome editing for novel plant resistance against devastating HLB.

Application of nanopesticides may substantially increase surface attachment and internalization of engineered nanoparticles (ENPs) in food crops. This study investigated the role of stomata in the internalization of silver nanoparticles (Ag NPs) using abscisic acid (ABA)-responsive ecotypes (Ler and Col-7) and ABA-insensitive mutants (ost1-2 and scord7) of Arabidopsis thaliana in batch sorption experiments, in combination with microscopic visualization. Compared with those of the ABA-free control, stomatal apertures were significantly smaller for the Ler and Col-7 ecotypes (p ˂ 0.05) but remained unchanged for the ost1-2 and scord7 mutants, after exposure to 10 μM ABA for 1 h. Generally Ag NP sorption to the leaves of the Ler and Col-7 ecotypes treated with 10 μM ABA was lower than that in the ABA-free control, mainly due to ABA-induced stomatal closure. The difference in Ag NP sorption with and without ABA was less pronounced for Col-7 than for Ler, suggesting different sorption behaviors between these two ecotypes. In contrast, there was no significant difference in foliar sorption of Ag NPs by the ost1-2 and scord7 mutants with and without ABA treatment. Ag NPs were widely attached to the Arabidopsis leaf surface, and found at cell membrane, cytoplasm, and plasmodesmata, as revealed by scanning electron microscopy and transmission electron microscopy, respectively. These results highlight the important role of stomata in the internationalization of ENPs in plants and may have broad implications in foliar application of nanopesticides and minimizing contamination of food crops by ENPs.

Pseudomonas syringaeuses a multitude of virulence factors to infect plants. In this Review, Xin and colleagues highlight key virulence strategies — immune suppression and water soaking — that allow this bacterium to become a successful pathogen under the right environmental conditions. Pseudomonas syringae is one of the best-studied plant pathogens and serves as a model for understanding host–microorganism interactions, bacterial virulence mechanisms and host adaptation of pathogens as well as microbial evolution, ecology and epidemiology. Comparative genomic studies have identified key genomic features that contribute to P. syringae virulence. P. syringae has evolved two main virulence strategies: suppression of host immunity and creation of an aqueous apoplast to form its niche in the phyllosphere. In addition, external environmental conditions such as humidity profoundly influence infection. P. syringae may serve as an excellent model to understand virulence and also of how pathogenic microorganisms integrate environmental conditions and plant microbiota to become ecologically robust and diverse pathogens of the plant kingdom.

Burkholderia anthina XXVI is a rhizosphere bacterium isolated from a mango orchard in Mexico. This strain has a significant biological control activity against the causal agent of mango anthracnose, Colletotrichum gloeosporioides, likely through the production of siderophores and other secondary metabolites. Here, we present a draft genome sequence of B. anthina XXVI (approximately 7.7 Mb; and G + C content of 67.0%), with the aim of gaining insight into the genomic basis of antifungal modes of action, ecological success as a biological control agent, and full biosynthetic potential.

Throughout their life plants are associated with various microorganisms, including commensal, symbiotic and pathogenic microorganisms. Pathogens are genetically adapted to aggressively colonize and proliferate in host plants to cause disease. However, disease outbreaks occur only under permissive environmental conditions. The interplay between host, pathogen and environment is famously known as the 'disease triangle'. Among the environmental factors, rainfall events, which often create a period of high atmospheric humidity, have repeatedly been shown to promote disease outbreaks in plants, suggesting that the availability of water is crucial for pathogenesis. During pathogen infection, water-soaking spots are frequently observed on infected leaves as an early symptom of disease. Recent studies have shown that pathogenic bacteria dedicate specialized virulence proteins to create an aqueous habitat inside the leaf apoplast under high humidity. Water availability in the apoplastic environment, and probably other associated changes, can determine the success of potentially pathogenic microbes. These new findings reinforce the notion that the fight over water may be a major battleground between plants and pathogens. In this article, we will discuss the role of water availability in host-microbe interactions, with a focus on plant-bacterial interactions.

Summary The plant hormone jasmonate (JA) promotes the degradation of JASMONATE ZIM‐DOMAIN (JAZ) proteins to relieve repression on diverse transcription factors (TFs) that execute JA responses. However, little is known about how combinatorial complexity among JAZ–TF interactions maintains control over myriad aspects of growth, development, reproduction, and immunity.We used loss‐of‐function mutations to define epistatic interactions within the core JA signaling pathway and to investigate the contribution of MYC TFs to JA responses in Arabidopsis thaliana.Constitutive JA signaling in a jaz quintuple mutant (jazQ) was largely eliminated by mutations that block JA synthesis or perception. Comparison of jazQ and a jazQ myc2 myc3 myc4 octuple mutant validated known functions of MYC2/3/4 in root growth, chlorophyll degradation, and susceptibility to the pathogen Pseudomonas syringae. We found that MYC TFs also control both the enhanced resistance of jazQ leaves to insect herbivory and restricted leaf growth of jazQ. Epistatic transcriptional profiles mirrored these phenotypes and further showed that triterpenoid biosynthetic and glucosinolate catabolic genes are up‐regulated in jazQ independently of MYC TFs.Our study highlights the utility of genetic epistasis to unravel the complexities of JAZ–TF interactions and demonstrates that MYC TFs exert master control over a JAZ‐repressible transcriptional hierarchy that governs growth–defense balance., See also the Commentary on this article by Wasternack, 215: 1291–1294.