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.

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.

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.

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.

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.

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.

DGK5-mediated phosphatidic acid homeostasis interplays with reactive oxygen species in plant immune signaling.

Reactive oxygen species (ROS) and phosphatidic acid (PA) are important second messengers in plant immunity. PA binding to RBOHD, an NADPH oxidase responsible for ROS production, enhances RBOHD stability and promotes ROS production. Distinct phosphorylation of the lipid kinase DGK5 optimizes the PA burst in regulating ROS production.

Bacteria establish an aqueous living space in plants crucial for virulence.

High humidity has a strong influence on the development of numerous diseases affecting the above-ground parts of plants (the phyllosphere) in crop fields and natural ecosystems, but the molecular basis of this humidity effect is not understood. Previous studies have emphasized immune suppression as a key step in bacterial pathogenesis. Here we show that humidity-dependent, pathogen-driven establishment of an aqueous intercellular space (apoplast) is another important step in bacterial infection of the phyllosphere. Bacterial effectors, such as Pseudomonas syringae HopM1, induce establishment of the aqueous apoplast and are sufficient to transform non-pathogenic P. syringae strains into virulent pathogens in immunodeficient Arabidopsis thaliana under high humidity. Arabidopsis quadruple mutants simultaneously defective in a host target (AtMIN7) of HopM1 and in pattern-triggered immunity could not only be used to reconstitute the basic features of bacterial infection, but also exhibited humidity-dependent dyshomeostasis of the endophytic commensal bacterial community in the phyllosphere. These results highlight a new conceptual framework for understanding diverse phyllosphere-bacterial interactions.

Structural basis of JAZ repression of MYC transcription factors in jasmonate signalling.

The plant hormone jasmonate plays crucial roles in regulating plant responses to herbivorous insects and microbial pathogens and is an important regulator of plant growth and development. Key mediators of jasmonate signalling include MYC transcription factors, which are repressed by jasmonate ZIM-domain (JAZ) transcriptional repressors in the resting state. In the presence of active jasmonate, JAZ proteins function as jasmonate co-receptors by forming a hormone-dependent complex with COI1, the F-box subunit of an SCF-type ubiquitin E3 ligase. The hormone-dependent formation of the COI1-JAZ co-receptor complex leads to ubiquitination and proteasome-dependent degradation of JAZ repressors and release of MYC proteins from transcriptional repression. The mechanism by which JAZ proteins repress MYC transcription factors and how JAZ proteins switch between the repressor function in the absence of hormone and the co-receptor function in the presence of hormone remain enigmatic. Here we show that Arabidopsis MYC3 undergoes pronounced conformational changes when bound to the conserved Jas motif of the JAZ9 repressor. The Jas motif, previously shown to bind to hormone as a partly unwound helix, forms a complete α-helix that displaces the amino (N)-terminal helix of MYC3 and becomes an integral part of the MYC N-terminal fold. In this position, the Jas helix competitively inhibits MYC3 interaction with the MED25 subunit of the transcriptional Mediator complex. Our structural and functional studies elucidate a dynamic molecular switch mechanism that governs the repression and activation of a major plant hormone pathway.

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.

Allelic diversity in an NLR gene BPH9 enables rice to combat planthopper variation.

Brown planthopper (BPH), Nilaparvata lugens Stål, is one of the most devastating insect pests of rice (Oryza sativa L.). Currently, 30 BPH-resistance genes have been genetically defined, most of which are clustered on specific chromosome regions. Here, we describe molecular cloning and characterization of a BPH-resistance gene, BPH9, mapped on the long arm of rice chromosome 12 (12L). BPH9 encodes a rare type of nucleotide-binding and leucine-rich repeat (NLR)-containing protein that localizes to the endomembrane system and causes a cell death phenotype. BPH9 activates salicylic acid- and jasmonic acid-signaling pathways in rice plants and confers both antixenosis and antibiosis to BPH. We further demonstrated that the eight BPH-resistance genes that are clustered on chromosome 12L, including the widely used BPH1, are allelic with each other. To honor the priority in the literature, we thus designated this locus as BPH1/9 These eight genes can be classified into four allelotypes, BPH1/9-1, -2, -7, and -9 These allelotypes confer varying levels of resistance to different biotypes of BPH. The coding region of BPH1/9 shows a high level of diversity in rice germplasm. Homologous fragments of the nucleotide-binding (NB) and leucine-rich repeat (LRR) domains exist, which might have served as a repository for generating allele diversity. Our findings reveal a rice plant strategy for modifying the genetic information to gain the upper hand in the struggle against insect herbivores. Further exploration of natural allelic variation and artificial shuffling within this gene may allow breeding to be tailored to control emerging biotypes of BPH.

Host target modification as a strategy to counter pathogen hijacking of the jasmonate hormone receptor.

In the past decade, characterization of the host targets of pathogen virulence factors took a center stage in the study of pathogenesis and disease susceptibility in plants and humans. However, the impressive knowledge of host targets has not been broadly exploited to inhibit pathogen infection. Here, we show that host target modification could be a promising new approach to "protect" the disease-vulnerable components of plants. In particular, recent studies have identified the plant hormone jasmonate (JA) receptor as one of the common targets of virulence factors from highly evolved biotrophic/hemibiotrophic pathogens. Strains of the bacterial pathogen Pseudomonas syringae, for example, produce proteinaceous effectors, as well as a JA-mimicking toxin, coronatine (COR), to activate JA signaling as a mechanism to promote disease susceptibility. Guided by the crystal structure of the JA receptor and evolutionary clues, we succeeded in modifying the JA receptor to allow for sufficient endogenous JA signaling but greatly reduced sensitivity to COR. Transgenic Arabidopsis expressing this modified receptor not only are fertile and maintain a high level of insect defense, but also gain the ability to resist COR-producing pathogens Pseudomonas syringae pv. tomato and P. syringae pv. maculicola. Our results provide a proof-of-concept demonstration that host target modification can be a promising new approach to prevent the virulence action of highly evolved pathogens.

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.

Pseudomonas syringae Effector Avirulence Protein E Localizes to the Host Plasma Membrane and Down-Regulates the Expression of the NONRACE-SPECIFIC DISEASE RESISTANCE1/HARPIN-INDUCED1-LIKE13 Gene Required for Antibacterial Immunity in Arabidopsis.

Many bacterial pathogens of plants and animals deliver effector proteins into host cells to promote infection. Elucidation of how pathogen effector proteins function not only is critical for understanding bacterial pathogenesis but also provides a useful tool in discovering the functions of host genes. In this study, we characterized the Pseudomonas syringae pv tomato DC3000 effector protein Avirulence Protein E (AvrE), the founding member of a widely distributed, yet functionally enigmatic, bacterial effector family. We show that AvrE is localized in the plasma membrane (PM) and PM-associated vesicle-like structures in the plant cell. AvrE contains two physically interacting domains, and the amino-terminal portion contains a PM-localization signal. Genome-wide microarray analysis indicates that AvrE, as well as the functionally redundant effector Hypersensitive response and pathogenicity-dependent Outer Protein M1, down-regulates the expression of the NONRACE-SPECIFIC DISEASE RESISTANCE1/HARPIN-INDUCED1-LIKE13 (NHL13) gene in Arabidopsis (Arabidopsis thaliana). Mutational analysis shows that NHL13 is required for plant immunity, as the nhl13 mutant plant displayed enhanced disease susceptibility. Our results defined the action site of one of the most important bacterial virulence proteins in plants and the antibacterial immunity function of the NHL13 gene.

Induction and suppression of PEN3 focal accumulation during Pseudomonas syringae pv. tomato DC3000 infection of Arabidopsis.

The pleiotropic drug resistance (PDR) proteins belong to the super-family of ATP-binding cassette (ABC) transporters. AtPDR8, also called PEN3, is required for penetration resistance of Arabidopsis to nonadapted powdery mildew fungi. During fungal infection, plasma-membrane-localized PEN3 is concentrated at fungal entry sites, as part of the plant's focal immune response. Here, we show that the pen3 mutant is compromised in resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. P. syringae pv. tomato DC3000 infection or treatment with a flagellin-derived peptide, flg22, induced strong focal accumulation of PEN3-green fluorescent protein. Interestingly, after an initial induction of PEN3 accumulation, P. syringae pv. tomato DC3000 but not the type-III-secretion-deficient mutant hrcC could suppress PEN3 accumulation. Moreover, transgenic overexpression of the P. syringae pv. tomato DC3000 effector AvrPto was sufficient to suppress PEN3 focal accumulation in response to flg22. Analyses of P. syringae pv. tomato DC3000 effector deletion mutants showed that individual effectors, including AvrPto, appear to be insufficient to suppress PEN3 accumulation when delivered by bacteria, suggesting a requirement for a combined action of multiple effectors. Collectively, our results indicate that PEN3 plays a positive role in plant resistance to a bacterial pathogen and show that focal accumulation of PEN3 protein may be a useful cellular response marker for the Arabidopsis-P. syringae interaction.

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.

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.