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To be successful plant pathogens, microbes use \"effector proteins\" to manipulate host functions to their benefit. Identifying host targets of effector proteins and characterizing their role in the infection process allow us to better understand plant–pathogen interactions and the plant immune system. Yeast two-hybrid analysis and coimmunoprecipitation were used to demonstrate that the Phytophthora infestans effector AVIRULENCE 2 (PiAVR2) interacts with all three BRI1-SUPPRESSOR1-like (BSL) family members from potato (Solanum tuberosum). Transient expression of BSL1, BSL2, and BSL3 enhanced P. infestans leaf infection. BSL1 and BSL3 suppressed INFESTIN 1 elicitin-triggered cell death, showing that they negatively regulate immunity. Virus-induced gene silencing studies revealed that BSL2 and BSL3 are required for BSL1 stability and show that basal levels of immunity are increased in BSL-silenced plants. Immune suppression by BSL family members is dependent on the brassinosteroid-responsive host transcription factor CIB1/HBI1-like 1. The P. infestans effector PiAVR2 targets all three BSL family members in the crop plant S. tuberosum. These phosphatases, known for their role in growth-promoting brassinosteroid signaling, all support P. infestans virulence and thus can be regarded as susceptibility factors in late blight infection.

Plants have evolved effective strategies to defend themselves against pathogen invasion. Starting from the plasma membrane with the recognition of microbe-associated molecular patterns (MAMPs) via pattern recognition receptors, internal cellular signaling pathways are induced to ultimately fend off the attack. Phospholipase D (PLD) hydrolyzes membrane phospholipids to produce phosphatidic acid (PA), which has been proposed to play a second messenger role in immunity. The Arabidopsis (Arabidopsis thaliana) PLD family consists of 12 members, and for some of these, a specific function in resistance toward a subset of pathogens has been shown. We demonstrate here that Arabidopsis PLD𝛾1, but not its close homologs PLD𝛾2 and PLD𝛾3, is specifically involved in plant immunity. Genetic inactivation of PLD𝛾1 resulted in increased resistance toward the virulent bacterium Pseudomonas syringae pv. tomato DC3000 and the necrotrophic fungus Botrytis cinerea. As pld𝛾1 mutant plants responded with elevated levels of reactive oxygen species to MAMP treatment, a negative regulatory function for this PLD isoform is proposed. Importantly, PA levels in pld𝛾1 mutants were not affected compared to stressed wild-type plants, suggesting that alterations in PA levels are not likely the cause for the enhanced immunity in the pld𝛾1 line. Instead, the plasma-membrane-attached PLD𝛾1 protein colocalized and associated with the BAK1-INTERACTING RECEPTOR-LIKE KINASES BIR2 and BIR3, which are known negative regulators of pattern-triggered immunity. Moreover, complex formation of PLD𝛾1 and BIR2 was further promoted upon MAMP treatment. Hence, we propose that PLD𝛾1 acts as a negative regulator of plant immune responses in complex with immunity-related proteins BIR2 and BIR3.

Plants optimize their growth and survival through highly integrated regulatory networks that coordinate defensive measures and developmental transitions in response to environmental cues. Protein phosphatase 2A (PP2A) is a key signaling component that controls stress reactions and growth at different stages of plant development, and the PP2A regulatory subunit PP2A‐Bʹ𝛾 is required for negative regulation of pathogenesis responses and for maintenance of cell homeostasis in short-day conditions. Here, we report molecular mechanisms by which PP2A‐Bʹ𝛾 regulates Botrytis cinerea resistance and leaf senescence in Arabidopsis (Arabidopsis thaliana). We extend the molecular functionality of PP2A‐Bʹ𝛾 to a protein kinase–phosphatase interaction with the defense-associated calcium-dependent protein kinase CPK1 and present indications this interaction may function to control CPK1 activity. In presenescent leaf tissues, PP2A-Bʹ𝛾 is also required to negatively control the expression of salicylic acid-related defense genes, which have recently proven vital in plant resistance to necrotrophic fungal pathogens. In addition, we find the premature leaf yellowing of pp2a-bʹ𝛾 depends on salicylic acid biosynthesis via SALICYLIC ACID INDUCTION DEFICIENT2 and bears the hallmarks of developmental leaf senescence. We propose PP2A-Bʹ𝛾 agedependently controls salicylic acid-related signaling in plant immunity and developmental leaf senescence.

During plant growth and development, ethylene and abscisic acid (ABA) play important roles and exert synergistic or antagonistic effects on various biological processes, but the detailed mechanism underlying the interaction of the two phytohormones, especially in the regulation of the accumulation of reactive oxygen species (ROS), is largely unclear. Here, we report that ethylene inhibits but ABA promotes the accumulation of ROS in Arabidopsis (Arabidopsis thaliana) seedlings. Furthermore, changes in the biosynthesis of ascorbic acid (AsA) act as a key factor in integrating the interaction of ethylene and ABA in the regulation of ROS levels. We found that ethylene and ABA antagonistically regulate AsA biosynthesis via ETHYLENE-INSENSITIVE3 (EIN3) and ABA INSENSITIVE4 (ABI4), which are key factors in the ethylene and ABA signaling pathways, respectively. In addition, ABI4 is transcriptionally repressed by EIN3 in ethylene-regulated AsA biosynthesis. Via transcriptome analysis and molecular and genetic experiments, we identified VITAMIN C DEFECTIVE2as the direct target of ABI4 in the regulation of AsA biosynthesis and ROS accumulation. Thus, the EIN3-ABI4- VITAMIN C DEFECTIVE2 transcriptional cascade involves a mechanism by which ethylene and ABA antagonistically regulate AsA biosynthesis and ROS accumulation in response to complex environmental stimuli.

Heterotrimeric G-proteins influence almost all aspects of plant growth, development, and responses to biotic and abiotic stresses in plants, likely via their interaction with specific effectors. However, the identity of such effectors and their mechanism of action are mostly unknown. While investigating the roles of different G-protein subunits in modulating the oil content in Camelina (Camelina sativa), an oil seed crop, we uncovered a role of G𝛽 proteins in controlling anisotropic cell expansion. Knockdown of G𝛽 genes causes reduced longitudinal and enhanced transverse expansion, resulting in altered cell, tissue, and organ shapes in transgenic plants during vegetative and reproductive development. These plants also exhibited substantial changes in their fatty acid and phospholipid profiles, which possibly leads to the increased oil content of the transgenic seeds. This increase is potentially caused by the direct interaction of G𝛽 proteins with a specific patatin-like phospholipase, pPLAIII𝛿. Camelina plants with suppressed G𝛽 expression exhibit higher lipase activity, and show phenotypes similar to plants overexpressing pPLAIII𝛿, suggesting that the G𝛽 proteins are negative regulators of pPLAIII𝛿. These results reveal interactions between the G-protein–mediated and lipid signaling/metabolic pathways, where specific phospholipases may act as effectors that control key developmental and environmental responses of plants.

During the transition from warm to cool seasons, plants experience decreased temperatures, shortened days, and decreased red/far-red (R/FR) ratios of light. The mechanism by which plants integrate these environmental cues to maintain plant growth and adaptation remains poorly understood. Here, we report that low temperature induced the transcription of PHYTOCHROME A and accumulation of LONG HYPOCOTYL5 (SlHY5, a basic Leu zipper transcription factor) in tomato (Solanum lycopersicum) plants, especially under short day conditions with low R/FR light ratios. Reverse genetic approaches and physiological analyses revealed that silencing of SlHY5 increased cold susceptibility in tomato plants, whereas overexpression of SlHY5 enhanced cold tolerance. SlHY5 directly bound to and activated the transcription of genes encoding a gibberellin-inactivation enzyme, namely GIBBERELLIN2-OXIDASE4, and an abscisic acid biosynthetic enzyme, namely 9-CIS-EPOXYCAROTENOID DIOXYGENASE6 (SlNCED6). Thus, phytochrome A-dependent SlHY5 accumulation resulted in an increased abscisic acid/gibberellin ratio, which was accompanied by growth cessation and induction of cold response. Furthermore, silencing of SlNCED6 compromises short day- and low R/FR-induced tomato resistance to cold stress. These findings provide insight into the molecular genetic mechanisms by which plants integrate environmental stimuli with hormones to coordinate their growth with impending cold temperatures. Moreover, this work reveals a molecular mechanism that plants have evolved for growth and survival in response to seasonal changes.

Arabidopsis (Arabidopsis thaliana) GARP (Golden2, ARR-B, Psr1) family transcription factors, GOLDEN2-LIKE1 and -2 (GLK1/2), function in different biological processes; however, whether and how these transcription factors modulate the response to abscisic acid (ABA) remain unknown. In this study, we used a glk1 glk2 double mutant to examine the role of GLK1/2 in the ABA response. The glk1 glk2 double mutant displayed ABA-hypersensitive phenotypes during seed germination and seedling development and an osmotic stress-resistant phenotype during seedling development. Genome-wide RNA sequencing analysis of the glk1 glk2 double mutant revealed that GLK1/2 regulate several ABA-responsive genes, including WRKY40, in the presence of ABA. Chromatin immunoprecipitation and gel retardation assays showed that GLK1/2 directly associate with the WRKY40 promoter via the recognition of a consensus sequence. Additionally, RNA sequencing analysis of the glk1 glk2 double mutant and wrky40 single mutant revealed that GLK1/2 and WRKY40 control a common set of downstream target genes in response to ABA. Furthermore, results of a genetic interaction test showed that the glk1 glk2 wrky40 triple mutant displayed similar ABA hypersensitivity to the wrky40 single mutant and the glk1 glk2 double mutant, while the glk1 glk2 wrky40 abi5-c (ABI5 CRISPR/Cas9 mutant) quadruple mutant displayed similar ABA hyposensitivity to the abi5-7 single mutant. Based on these results, we propose that the GLK1/2-WRKY40 transcription module plays a negative regulatory role in the ABA response.

Plants exhibit diverse polar behaviors in response to directional and nondirectional environmental signals, termed tropic and nastic movements, respectively. The ways in which plants incorporate directional information into tropic behaviors is well understood, but it is less well understood how nondirectional stimuli, such as ambient temperatures, specify the polarity of nastic behaviors. Here, we demonstrate that a developmentally programmed polarity of auxin flow underlies thermo-induced leaf hyponasty in Arabidopsis (Arabidopsis thaliana). In warm environments, PHYTOCHROME-INTERACTING FACTOR4 (PIF4) stimulates auxin production in the leaf. This results in the accumulation of auxin in leaf petioles, where PIF4 directly activates a gene encoding the PINOID (PID) protein kinase. PID is involved in polarization of the auxin transporter PIN-FORMED3 to the outer membranes of petiole cells. Notably, the leaf polarity determining ASYMMETRIC LEAVES1 (AS1) directs the induction of PID to occur predominantly in the abaxial petiole region. These observations indicate that the integration of PIF4-mediated auxin biosynthesis and polar transport, and the AS1-mediated developmental shaping of polar auxin flow, coordinate leaf thermonasty, which facilitates leaf cooling in warm environments. We believe that leaf thermonasty is a suitable model system for studying the developmental programming of environmental adaptation in plants.

Salinity impairs seed germination and seedling establishment. We investigated the role of Arabidopsis (Arabidopsis thaliana) CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR 6 (CAMTA6) in salinity stress responses during early germination. Compared with the wild type, the camta6-4 and camta6-5 mutants were more tolerant to NaCl and abscisic acid (ABA) and accumulated less Na⁺. In contrast, 4- to 11-d-old camta6 seedlings were more sensitive to NaCl. In camta6, expression of HIGH-AFFINITY K⁺ TRANSPORTER1 (AtHKT1;1), encoding an Na⁺/K⁺ transporter, was restricted to the radicles and was not enhanced by NaCl or ABA. During germination, the camta6 hkt1 double mutant was as sensitive as the wild type and hkt1 to NaCl, suggesting that HKT1;1 is crucial for the salt tolerance of camta6. An ABA response element in the HKT1;1 promoter was found to be indispensable for the enhanced expression of the gene in response to NaCl and to ABA. Transcriptome analysis of the wild type and camta6-5 with and without salt treatment revealed 1,020 up-regulated and 1,467 down-regulated saltresponsive genes in the wild type. Among these, 638 up-regulated and 1,242 down-regulated genes were classified as CAMTA6-dependent. Expression of several known salt stress-associated genes, including SALT OVERLY SENSITIVE1 and Na⁺/H⁺ ANTIPORTER, was impaired in camta6 mutants. Bioinformatics analysis of the 5' upstream sequences of the salt-responsive CAMTA6-dependent up-regulated genes revealed the CACGTGTC motif as the most prominent element, representing an ABA response element and a potential CAMTA-binding site. We suggest that CAMTA6 regulates, directly or indirectly, the expression of most of the salt-responsive genes in germinating seeds, including genes that are crucial for Na⁺ homeostasis and salt stress tolerance.

DNA methylation plays an important role in diverse developmental processes in many eukaryotes, including the response to environmental stress. Abscisic acid (ABA) is a plant hormone that is up-regulated under stress. The involvement of DNA methylation in the ABA response has been reported but is poorly understood. DNA demethylation is a reverse process of DNA methylation and often induces structural changes of chromatin leading to transcriptional activation. In Arabidopsis (Arabidopsis thaliana), active DNA demethylation depends on the activity of REPRESSOR OF SILENCING 1 (ROS1), which directly excises 5-methylcytosine from DNA. Here we showed that ros1 mutants were hypersensitive to ABA during early seedling development and root elongation. Expression levels of some ABA-inducible genes were decreased in ros1 mutants, and more than 60% of their proximal regions became hypermethylated, indicating that a subset of ABA-inducible genes are under the regulation of ROS1-dependent DNA demethylation. Notable among them is NICOTINAMIDASE 3 (NIC3) that encodes an enzyme that converts nicotinamide to nicotinic acid in the NAD⁺ salvage pathway. Many enzymes in this pathway are known to be involved in stress responses. The nic3 mutants display hypersensitivity to ABA, whereas overexpression of NIC3 restores normal ABA responses. Our data suggest that NIC3 is responsive to ABA but requires ROS1-mediated DNA demethylation at the promoter as a prerequisite to transcriptional activation. These findings suggest that ROS1-induced active DNA demethylation maintains the active state of NIC3 transcription in response to ABA.