The NLR immune receptor ADR1 and lipase-like proteins EDS1 and PAD4 mediate stomatal immunity in Nicotiana benthamiana and Arabidopsis.

In the presence of pathogenic bacteria, plants close their stomata to prevent pathogen entry. Intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors recognize pathogenic effectors and activate effector-triggered immune responses. However, the regulatory and molecular mechanisms of stomatal immunity involving NLR immune receptors are unknown. Here, we show that the Nicotiana benthamiana RPW8-NLR central immune receptor ACTIVATED DISEASE RESISTANCE 1 (NbADR1), together with the key immune proteins ENHANCED DISEASE SUSCEPTIBILITY 1 (NbEDS1) and PHYTOALEXIN DEFICIENT 4 (NbPAD4), plays an essential role in bacterial pathogen- and flg22-induced stomatal immunity by regulating the expression of salicylic acid (SA) and abscisic acid (ABA) biosynthesis or response-related genes. NbADR1 recruits NbEDS1 and NbPAD4 in stomata to form a stomatal immune response complex. The transcription factor NbWRKY40e, in association with NbEDS1 and NbPAD4, modulates the expression of SA and ABA biosynthesis or response-related genes to influence stomatal immunity. NbADR1, NbEDS1, and NbPAD4 are required for the pathogen infection-enhanced binding of NbWRKY40e to the ISOCHORISMATE SYNTHASE 1 promoter. Moreover, the ADR1-EDS1-PAD4 module regulates stomatal immunity in Arabidopsis (Arabidopsis thaliana). Collectively, our findings show the pivotal role of the core intracellular immune receptor module ADR1-EDS1-PAD4 in stomatal immunity, which enables plants to limit pathogen entry.

Ethylene signaling modulates air humidity responses in plants.

Air humidity significantly impacts plant physiology. However, the upstream elements that mediate humidity sensing and adaptive responses in plants remain largely unexplored. In this study, we define high humidity-induced cellular features of Arabidopsis plants and take a quantitative phosphoproteomics approach to obtain a high humidity-responsive landscape of membrane proteins, which we reason are likely the early checkpoints of humidity signaling. We found that a brief high humidity exposure (i.e., 0.5 h) is sufficient to trigger extensive changes in membrane protein abundance and phosphorylation. Enrichment analysis of differentially regulated proteins reveals high humidity-sensitive processes such as 'transmembrane transport', 'response to abscisic acid', and 'stomatal movement'. We further performed a targeted screen of mutants, in which high humidity-responsive pathways/proteins are disabled, to uncover genes mediating high humidity sensitivity. Interestingly, ethylene pathway mutants (i.e., ein2 and ein3eil1) display a range of altered responses, including hyponasty, reactive oxygen species level, and responsive gene expression, to high humidity. Furthermore, we observed a rapid induction of ethylene biosynthesis genes and ethylene evolution after high humidity treatment. Our study sheds light on the potential early signaling events in humidity perception, a fundamental but understudied question in plant biology, and reveals ethylene as a key modulator of high humidity responses in plants.

A sweet story from Phytophthora-soybean interaction.

Phytopathogenic microbes obtain nutrients from host plants to support their growth and metabolism. A recent study by Zhu et al. revealed that the oomycete pathogen Phytophthora sojae upregulates the activity of soybean trehalose 6-phosphate synthase 6 (GmTPS6) and increases trehalose accumulation (through an effector PsAvh413) to promote nutritional gain.

High air humidity dampens salicylic acid pathway and NPR1 function to promote plant disease.

The occurrence of plant disease is determined by interactions among host, pathogen, and environment. Air humidity shapes various aspects of plant physiology and high humidity has long been known to promote numerous phyllosphere diseases. However, the molecular basis of how high humidity interferes with plant immunity to favor disease has remained elusive. Here we show that high humidity is associated with an "immuno-compromised" status in Arabidopsis plants. Furthermore, accumulation and signaling of salicylic acid (SA), an important defense hormone, are significantly inhibited under high humidity. NPR1, an SA receptor and central transcriptional co-activator of SA-responsive genes, is less ubiquitinated and displays a lower promoter binding affinity under high humidity. The cellular ubiquitination machinery, particularly the Cullin 3-based E3 ubiquitin ligase mediating NPR1 protein ubiquitination, is downregulated under high humidity. Importantly, under low humidity the Cullin 3a/b mutant plants phenocopy the low SA gene expression and disease susceptibility that is normally observed under high humidity. Our study uncovers a mechanism by which high humidity dampens a major plant defense pathway and provides new insights into the long-observed air humidity influence on diseases.

CURLY LEAF modulates apoplast liquid water status in Arabidopsis leaves.

The apoplast of plant leaves, the intercellular space between mesophyll cells, is normally largely filled with air with a minimal amount of liquid water in it, which is essential for key physiological processes such as gas exchange to occur. Phytopathogens exploit virulence factors to induce a water-rich environment, or "water-soaked" area, in the apoplast of the infected leaf tissue to promote disease. We propose that plants evolved a "water soaking" pathway, which normally keeps a nonflooded leaf apoplast for plant growth but is disturbed by microbial pathogens to facilitate infection. Investigation of the "water soaking" pathway and leaf water control mechanisms is a fundamental, yet previously overlooked, aspect of plant physiology. To identify key components in the "water soaking" pathway, we performed a genetic screen to isolate Arabidopsis (Arabidopsis thaliana) severe water soaking (sws) mutants that show liquid water overaccumulation in the leaf under high air humidity, a condition required for visible water soaking. Here, we report the sws1 mutant, which displays rapid water soaking upon high humidity treatment due to a loss-of-function mutation in CURLY LEAF (CLF), encoding a histone methyltransferase in the POLYCOMB REPRESSIVE COMPLEX 2 (PRC2). We found that the sws1 (clf) mutant exhibits enhanced abscisic acid (ABA) levels and stomatal closure, which are indispensable for its water soaking phenotype and mediated by CLF's epigenetic regulation of a group of ABA-associated NAM, ATAF, and CUC (NAC) transcription factor genes, NAC019/055/072. The clf mutant showed weakened immunity, which likely also contributes to the water soaking phenotype. In addition, the clf plant supports a substantially higher level of Pseudomonas syringae pathogen-induced water soaking and bacterial multiplication, in an ABA pathway and NAC019/055/072-dependent manner. Collectively, our study sheds light on an important question in plant biology and demonstrates CLF as a key modulator of leaf liquid water status via epigenetic regulation of the ABA pathway and stomatal movement.

Glutathione and neodiosmin feedback sustain plant immunity.

Plants have evolved a two-layer immune system comprising pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) that is activated in response to pathogen invasion. Microbial patterns and pathogen effectors can be recognized by surface-localized pattern-recognition receptors (PRRs) and intracellularly localized nucleotide-binding leucine-rich repeat receptors (NLRs) to trigger PTI and ETI responses, respectively. At present, the metabolites activated by PTI and ETI and their roles and signalling pathways in plant immunity are not well understood. In this study, metabolomic analysis showed that ETI and PTI induced various flavonoids and amino acids and their derivatives in plants. Interestingly, both glutathione and neodiosmin content were specifically up-regulated by ETI and PTI, respectively, which significantly enhanced plant immunity. Further studies showed that glutathione and neodiosmin failed to induce a plant immune response in which PRRs/co-receptors were mutated. In addition, glutathione-reduced mutant gsh1 analysis showed that GSH1 is also required for PTI and ETI. Finally, we propose a model in which glutathione and neodiosmin are considered signature metabolites induced in the process of ETI and PTI activation in plants and further continuous enhancement of plant immunity in which PRRs/co-receptors are needed. This model is beneficial for an in-depth understanding of the closed-loop mode of the positive feedback regulation of PTI and ETI signals at the metabolic level.

Ascorbate peroxidase 1 allows monitoring of cytosolic accumulation of effector-triggered reactive oxygen species using a luminol-based assay.

Biphasic production of reactive oxygen species (ROS) has been observed in plants treated with avirulent bacterial strains. The first transient peak corresponds to pattern-triggered immunity (PTI)-ROS, whereas the second long-lasting peak corresponds to effector-triggered immunity (ETI)-ROS. PTI-ROS are produced in the apoplast by plasma membrane-localized NADPH oxidases, and the recognition of an avirulent effector increases the PTI-ROS regulatory module, leading to ETI-ROS accumulation in the apoplast. However, how apoplastic ETI-ROS signaling is relayed to the cytosol is still unknown. Here, we found that in the absence of cytosolic ascorbate peroxidase 1 (APX1), the second phase of ETI-ROS accumulation was undetectable in Arabidopsis (Arabidopsis thaliana) using luminol-based assays. In addition to being a scavenger of cytosolic H2O2, we discovered that APX1 served as a catalyst in this chemiluminescence ROS assay by employing luminol as an electron donor. A horseradish peroxidase (HRP)-mimicking APX1 mutation (APX1W41F) further enhanced its catalytic activity toward luminol, whereas an HRP-dead APX1 mutation (APX1R38H) reduced its luminol oxidation activity. The cytosolic localization of APX1 implies that ETI-ROS might accumulate in the cytosol. When ROS were detected using a fluorescent dye, green fluorescence was observed in the cytosol 6 h after infiltration with an avirulent bacterial strain. Collectively, these results indicate that ETI-ROS eventually accumulate in the cytosol, and cytosolic APX1 catalyzes luminol oxidation and allows monitoring of the kinetics of ETI-ROS in the cytosol. Our study provides important insights into the spatial dynamics of ROS accumulation in plant immunity.

Promotion of Arabidopsis immune responses by a rhizosphere fungus via supply of pipecolic acid to plants and selective augment of phytoalexins.

The ascomycete insect pathogenic fungi such as Metarhizium species have been demonstrated with the abilities to form the rhizosphere or endophytic relationships with different plants for nutrient exchanges. In this study, after the evident infeasibility of bacterial disease development in the boxed sterile soils, we established a hydroponic system for the gnotobiotic growth of Arabidopsis thaliana with the wild-type and transgenic strain of Metarhizium robertsii. The transgenic fungus could produce a high amount of pipecolic acid (PIP), a pivotal plant-immune-stimulating metabolite. Fungal inoculation experiments showed that M. robertsii could form a non-selective rhizosphere relationship with Arabidopsis. Similar to the PIP uptake by plants after exogenous application, PIP level increased in Col-0 and could be detected in the PIP-non-producing Arabidopsis mutant (ald1) after fungal inoculations, indicating that plants can absorb the PIP produced by fungi. The transgenic fungal strain had a better efficacy than the wild type to defend plants against the bacterial pathogen and aphid attacks. Contrary to ald1, fmo1 plants could not be boosted to resist bacterial infection after treatments. After fungal inoculations, the phytoalexins camalexin and aliphatic glucosinolate were selectively increased in Arabidopsis via both PIP-dependent and -independent ways. This study unveils the potential mechanism of the fungus-mediated beneficial promotion of plant immunity against biological stresses. The data also highlight the added values of M. robertsii to plants beyond the direct suppression of insect pest populations.

Regulation and integration of plant jasmonate signaling: a comparative view of monocot and dicot.

The phytohormone jasmonate plays a pivotal role in various aspects of plant life, including developmental programs and defense against pests and pathogens. A large body of knowledge on jasmonate biosynthesis, signal transduction as well as its functions in diverse plant processes has been gained in the past two decades. In addition, there exists extensive crosstalk between jasmonate pathway and other phytohormone pathways, such as salicylic acid (SA) and gibberellin (GA), in co-regulation of plant immune status, fine-tuning the balance of plant growth and defense, and so on, which were mostly learned from studies in the dicotyledonous model plants Arabidopsis thaliana and tomato but much less in monocot. Interestingly, existing evidence suggests both conservation and functional divergence in terms of core components of jasmonate pathway, its biological functions and signal integration with other phytohormones, between monocot and dicot. In this review, we summarize the current understanding on JA signal initiation, perception and regulation, and highlight the distinctive characteristics in different lineages of plants.

Bacterial effectors manipulate plant abscisic acid signaling for creation of an aqueous apoplast.

Phytopathogens like Pseudomonas syringae induce "water soaking" in the apoplastic space of plant leaf tissue as a key virulence mechanism. Water soaking is commonly observed in diverse pathosystems, yet the underlying physiological basis remains largely elusive. Here, we show that one of the strong P. syringae water-soaking inducers, AvrE, alters the regulation of abscisic acid (ABA) to induce ABA signaling, stomatal closure, and, thus, water soaking. AvrE binds and inhibits the function of Arabidopsis type one protein phosphatases (TOPPs), which negatively regulate ABA by suppressing SnRK2s, a key node of the ABA signaling pathway. The topp12537 quintuple mutants display significantly enhanced water soaking after P. syringae inoculation, whereas the loss of the ABA pathway dampens P. syringae-induced water soaking and disease. Our study uncovers the hijacking of ABA signaling and stomatal closure by P. syringae effectors as key mechanisms of disease susceptibility.

A bacterial kinase phosphorylates OSK1 to suppress stomatal immunity in rice.

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.

Synergistically promoting plant health by harnessing synthetic microbial communities and prebiotics.

Soil-borne diseases cause serious economic losses in agriculture. Managing diseases with microbial preparations is an excellent approach to soil-borne disease prevention. However, microbial preparations often exhibit unstable effects, limiting their large-scale application. This review introduces and summarizes disease-suppressive soils, the relationship between carbon sources and the microbial community, and the application of human microbial preparation concepts to plant microbial preparations. We also propose an innovative synthetic microbial community assembly strategy with synergistic prebiotics to promote healthy plant growth and resistance to disease. In this review, a new approach is proposed to improve traditional microbial preparations; provide a better understanding of the relationships among carbon sources, beneficial microorganisms, and plants; and lay a theoretical foundation for developing new microbial preparations.

Pattern-recognition receptors are required for NLR-mediated plant immunity.

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.

PTI-ETI crosstalk: an integrative view of plant immunity.

Plants resist attacks by pathogens via innate immune responses, which are initiated by cell surface-localized pattern-recognition receptors (PRRs) and intracellular nucleotide-binding domain leucine-rich repeat containing receptors (NLRs) leading to pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), respectively. Although the two classes of immune receptors involve different activation mechanisms and appear to require different early signalling components, PTI and ETI eventually converge into many similar downstream responses, albeit with distinct amplitudes and dynamics. Increasing evidence suggests the existence of intricate interactions between PRR-mediated and NLR-mediated signalling cascades as well as common signalling components shared by both. Future investigation of the mechanisms underlying signal collaboration between PRR-initiated and NLR-initiated immunity will enable a more complete understanding of the plant immune system. This review discusses recent advances in our understanding of the relationship between the two layers of plant innate immunity.

Bacterial Infection and Hypersensitive Response Assays in Arabidopsis-Pseudomonas syringae Pathosystem.

Arabidopsis thaliana-Pseudomonas syringae pathosystem has been used as an important model system for studying plant-microbe interactions, leading to many milestones and breakthroughs in the understanding of plant immune system and pathogenesis mechanisms. Bacterial infection and plant disease assessment are key experiments in the studies of plant-pathogen interactions. The hypersensitive response (HR), which is characterized by rapid cell death and tissue collapse after inoculation with a high dose of bacteria, is a hallmark response of plant effector-triggered immunity (ETI), one layer of plant immunity triggered by recognition of pathogen-derived effector proteins. Here, we present a detailed protocol for bacterial disease and hypersensitive response assays applicable to studies of Pseudomonas syringae interaction with various plant species such as Arabidopsis, Nicotiana benthamiana, and tomato.

Phyllosphere microbiota: Community dynamics and its interaction with plant hosts.

Plants are colonized by various microorganisms in natural environments. While many studies have demonstrated key roles of the rhizosphere microbiota in regulating biological processes such as nutrient acquisition and resistance against abiotic and biotic challenges, less is known about the role of the phyllosphere microbiota and how it is established and maintained. This review provides an update on current understanding of phyllosphere community assembly and the mechanisms by which plants and microbes establish the phyllosphere microbiota for plant health.

A plant genetic network for preventing dysbiosis in the phyllosphere.

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.

Pseudomonas syringae: what it takes to be a pathogen.

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.

Regulation of growth-defense balance by the JASMONATE ZIM-DOMAIN (JAZ)-MYC transcriptional module.

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.