An unexpected role for tomato threonine deaminase 2 in host defense against bacterial infection.

The hormones salicylic acid (SA) and jasmonic acid (JA) often act antagonistically in controlling plant defense pathways in response to hemibiotrophs/biotrophs (hemi/biotroph) and herbivores/necrotrophs, respectively. Threonine deaminase (TD) converts threonine to α-ketobutyrate and ammonia as the committed step in isoleucine (Ile) biosynthesis and contributes to JA responses by producing the Ile needed to make the bioactive JA-Ile conjugate. Tomato (Solanum lycopersicum) plants have two TD genes: TD1 and TD2. A defensive role for TD2 against herbivores has been characterized in relation to JA-Ile production. However, it remains unknown whether TD2 is also involved in host defense against bacterial hemi/biotrophic and necrotrophic pathogens. Here, we show that in response to the bacterial pathogen-associated molecular pattern (PAMP) flagellin flg22 peptide, an activator of SA-based defense responses, TD2 activity is compromised, possibly through carboxy-terminal cleavage. TD2 knockdown (KD) plants showed increased resistance to the hemibiotrophic bacterial pathogen Pseudomonas syringae but were more susceptible to the necrotrophic fungal pathogen Botrytis cinerea, suggesting TD2 plays opposite roles in response to hemibiotrophic and necrotrophic pathogens. This TD2 KD plant differential response to different pathogens is consistent with SA- and JA-regulated defense gene expression. flg22-treated TD2 KD plants showed high expression levels of SA-responsive genes, whereas TD2 KD plants treated with the fungal PAMP chitin showed low expression levels of JA-responsive genes. This study indicates TD2 acts negatively in defense against hemibiotrophs and positively against necrotrophs and provides insight into a new TD2 function in the elaborate crosstalk between SA and JA signaling induced by pathogen infection.

Pulsed FCAW of Martensitic Stainless Clads onto Mild Steel: Microstructure, Hardness, and Residual Stresses.

The low carbon martensitic stainless AWS 410NiMo steel has in its chemical composition 13% chromium, 4% nickel, and 0.4% molybdenum (wt.%) and is used in turbine recovery, rotors, and high-pressure steam pump housings due to its resistance to impact at low temperatures, as well as to corrosion and cavitation. Those applications of the AWS 410NiMo steel frequently demand repair, which is performed by welding or cladding. Arc welding is a well-established technique for joining materials and presents several parameters that influence the mechanical performance of the weld bead. Although numerous welding processes exist, optimizing welding parameters for specific applications and materials is always challenging. The present work deals with a systematic study to verify the correlation between the pulsed fluxed core arc welding (FCAW) parameters, namely pulse current and frequency, welding speed, and contact tip work distance (CTWD), and the bead morphology, microstructure formation, residual stress, and hardness of the martensitic clad. The substrate used was the AISI 1020 steel, and the AWS 410NiMo steel was the filler metal for clad deposition. From the initial nine (9) samples, three (3) were selected for in-depth characterization. Lower heat input resulted in lower dilution, more elevated hardness, and lower compressive residual stresses. Therefore, the results highlight the need for selecting the proper heat input, even when using a pulsed FCAW procedure, to achieve the desired performance of the clad. In the present case, a higher heat input appears to be more advantageous owing to the lower convexity index, smooth hardness transition between fusion and heat-affected zones in addition to more elevated compressive stresses.

Sandflies (Diptera: Psychodidae: Phlebotominae), present in an endemic area of cutaneous leishmaniasis in western Boyacá, Colombia / Flebótomos (Diptera: Psychodidae: Phlebotominae), presentes en una zona endémica de leishmaniasis cutánea en el occidente de Boyacá, Colombia

Abstract Introduction: Sandflies are known for having vector species of the tropical disease Leishmaniasis, a disease which is of an endemic nature in Western Boyacá, where the town of Otanche is one of the main source of Cutaneous Leishmaniasis. Objective: To identify the species of sandfly present in an endemic area of cutaneous leishmaniasis in West Boyacá. Methods: The search and collection of sandflies was carried out using CDC gravid traps, over a period of twelve hours (18:00- 06:00). Identification was carried out by revising the genitalia on both male and female samples under a microscope. The distribution took as reference households with a history of people infected with this disease, locating them intra, peri and extra domicile. Results: Were recollected 361 individuals (252 females and 109 male), belonging to 9 genres and 16 species. 60% of all recollected phlebotominae consists of Nyssomyia yuilli and Nyssomyia trapidoi. Other species recollected and relevant, due to vector precedent, are Lutzomyia hartmanni, Psychodopygus panamensis, Lutzomyia gomezi and Psychodopygus carrerai. Conclusion: It was established that, due to its abundance and vector precedent for the country and the area under study, Nyssomyia yuilli and Nyssomyia trapidoi constitute the species of phlebotominae which may be involved in the transmission of cutaneous Leishmaniasis in the region.

Resumen Introducción: Los flebótomos, son conocidos por tener especies vectoras de la enfermedad tropical Leishmaniasis, enfermedad que se presenta con carácter endémico en el occidente del departamento de Boyacá, donde el municipio de Otanche es uno de los principales focos de leishmaniasis cutánea. Objetivo: Identificar las especies de flebótomos presentes en una zona endémica de leishmaniasis cutánea en el occidente del Boyacá. Métodos: La búsqueda y recolección de los flebótomos se realizó con trampas CDC durante doce horas (18:00- 06:00), tomando como referencia viviendas con antecedentes de personas que hubieran tenido la enfermedad, ubicándolas en el intra, peri y extradomicilio. La identificación se realizó por medio de revisión del órgano genital de machos y hembras al microscopio. Resultados: Se colectaron 361 individuos (252 hembras y 109 machos), pertenecientes a 9 géneros y 16 especies, de las cuales, el 60% de toda la flebótomofauna recolectada está representada por Nyssomyia yuilli y Nyssomyia trapidoi. Otras especies colectadas y con importancia por antecedentes vectoriales son Lutzomyia hartmanni, Psychodopygus panamensis, Lutzomyia gomezi y Psychodopygus carrerai. Conclusión: Se estableció, que por sus altas abundancias y por sus antecedentes vectoriales para el país y para la zona de estudio, Nyssomyia yuilli y Nyssomyia trapidoi, constituyen las especies de flebótomos que pueden estar implicadas en la transmisión de leishmaniasis cutánea en la zona de estudio

Sandflies (Diptera: Psychodidae: Phlebotominae), present in an endemic area of cutaneous leishmaniasis in western Boyacá, Colombia.

INTRODUCTION: Sandflies are known for having vector species of the tropical disease Leishmaniasis, a disease which is of an endemic nature in Western Boyacá, where the town of Otanche is one of the main source of Cutaneous Leishmaniasis. OBJECTIVE: To identify the species of sandfly present in an endemic area of cutaneous leishmaniasis in West Boyacá. METHODS: The search and collection of sandflies was carried out using CDC gravid traps, over a period of twelve hours (18:00- 06:00). Identification was carried out by revising the genitalia on both male and female samples under a microscope. The distribution took as reference households with a history of people infected with this disease, locating them intra, peri and extra domicile. RESULTS: Were recollected 361 individuals (252 females and 109 male), belonging to 9 genres and 16 species. 60% of all recollected phlebotominae consists of Nyssomyia yuilli and Nyssomyia trapidoi. Other species recollected and relevant, due to vector precedent, are Lutzomyia hartmanni, Psychodopygus panamensis, Lutzomyia gomezi and Psychodopygus carrerai. CONCLUSION: It was established that, due to its abundance and vector precedent for the country and the area under study, Nyssomyia yuilli and Nyssomyia trapidoi constitute the species of phlebotominae which may be involved in the transmission of cutaneous Leishmaniasis in the region.

INTRODUCCIÓN: Los flebótomos, son conocidos por tener especies vectoras de la enfermedad tropical Leishmaniasis, enfermedad que se presenta con carácter endémico en el occidente del departamento de Boyacá, donde el municipio de Otanche es uno de los principales focos de leishmaniasis cutánea. OBJETIVO: Identificar las especies de flebótomos presentes en una zona endémica de leishmaniasis cutánea en el occidente del Boyacá. MÉTODOS: La búsqueda y recolección de los flebótomos se realizó con trampas CDC durante doce horas (18:00- 06:00), tomando como referencia viviendas con antecedentes de personas que hubieran tenido la enfermedad, ubicándolas en el intra, peri y extradomicilio. La identificación se realizó por medio de revisión del órgano genital de machos y hembras al microscopio. RESULTADOS: Se colectaron 361 individuos (252 hembras y 109 machos), pertenecientes a 9 géneros y 16 especies, de las cuales, el 60% de toda la flebótomofauna recolectada está representada por Nyssomyia yuilli y Nyssomyia trapidoi. Otras especies colectadas y con importancia por antecedentes vectoriales son Lutzomyia hartmanni, Psychodopygus panamensis, Lutzomyia gomezi y Psychodopygus carrerai. CONCLUSIÓN: Se estableció, que por sus altas abundancias y por sus antecedentes vectoriales para el país y para la zona de estudio, Nyssomyia yuilli y Nyssomyia trapidoi, constituyen las especies de flebótomos que pueden estar implicadas en la transmisión de leishmaniasis cutánea en la zona de estudio.

Variability of Two Metabolomic Platforms in CKD.

BACKGROUND AND OBJECTIVES: Nontargeted metabolomics can measure thousands of low-molecular-weight biochemicals, but important gaps limit its utility for biomarker discovery in CKD. These include the need to characterize technical and intraperson analyte variation, to pool data across platforms, and to outline analyte relationships with eGFR. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Plasma samples from 49 individuals with CKD (eGFR<60 ml/min per 1.73 m2 and/or ≥1 g proteinuria) were examined from two study visits; 20 samples were repeated as blind replicates. To enable comparison across two nontargeted platforms, samples were profiled at Metabolon and the Broad Institute. RESULTS: The Metabolon platform reported 837 known metabolites and 483 unnamed compounds (selected from 44,953 unknown ion features). The Broad Institute platform reported 594 known metabolites and 26,106 unknown ion features. Median coefficients of variation (CVs) across blind replicates were 14.6% (Metabolon) and 6.3% (Broad Institute) for known metabolites, and 18.9% for (Metabolon) unnamed compounds and 24.5% for (Broad Institute) unknown ion features. Median CVs for day-to-day variability were 29.0% (Metabolon) and 24.9% (Broad Institute) for known metabolites, and 41.8% for (Metabolon) unnamed compounds and 40.9% for (Broad Institute) unknown ion features. A total of 381 known metabolites were shared across platforms (median correlation 0.89). Many metabolites were negatively correlated with eGFR at P<0.05, including 35.7% (Metabolon) and 18.9% (Broad Institute) of known metabolites. CONCLUSIONS: Nontargeted metabolomics quantifies >1000 analytes with low technical CVs, and agreement for overlapping metabolites across two leading platforms is excellent. Many metabolites demonstrate substantial intraperson variation and correlation with eGFR.

Author Correction: Phosphocode-dependent functional dichotomy of a common co-receptor in plant signalling.

In Extended Data Fig. 5d of this Letter, the blots for anti-pS612 and anti-BAK1 were inadvertently duplicated. This figure has been corrected online.

Phosphocode-dependent functional dichotomy of a common co-receptor in plant signalling.

Multicellular organisms use cell-surface receptor kinases to sense and process extracellular signals. Many plant receptor kinases are activated by the formation of ligand-induced complexes with shape-complementary co-receptors1. The best-characterized co-receptor is BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1), which associates with numerous leucine-rich repeat receptor kinases (LRR-RKs) to control immunity, growth and development2. Here we report key regulatory events that control the function of BAK1 and, more generally, LRR-RKs. Through a combination of phosphoproteomics and targeted mutagenesis, we identified conserved phosphosites that are required for the immune function of BAK1 in Arabidopsis thaliana. Notably, these phosphosites are not required for BAK1-dependent brassinosteroid-regulated growth. In addition to revealing a critical role for the phosphorylation of the BAK1 C-terminal tail, we identified a conserved tyrosine phosphosite that may be required for the function of the majority of Arabidopsis LRR-RKs, and which separates them into two distinct functional classes based on the presence or absence of this tyrosine. Our results suggest a phosphocode-based dichotomy of BAK1 function in plant signalling, and provide insights into receptor kinase activation that have broad implications for our understanding of how plants respond to their changing environment.

Critical roles of mTORC1 signaling and metabolic reprogramming for M-CSF-mediated myelopoiesis.

Myelopoiesis is necessary for the generation of mature myeloid cells during homeostatic turnover and immunological insults; however, the metabolic requirements for this process remain poorly defined. Here, we demonstrate that myelopoiesis, including monocyte and macrophage differentiation, requires mechanistic target of rapamycin complex 1 (mTORC1) signaling and anabolic metabolism. Loss of mTORC1 impaired myelopoiesis under steady state and dampened innate immune responses against Listeria monocytogenes infection. Stimulation of hematopoietic progenitors with macrophage colony-stimulating factor (M-CSF) resulted in mTORC1-dependent anabolic metabolism, which in turn promoted expression of M-CSF receptor and transcription factors PU.1 and IRF8, thereby constituting a feed-forward loop for myelopoiesis. Mechanistically, mTORC1 engaged glucose metabolism and initiated a transcriptional program involving Myc activation and sterol biosynthesis after M-CSF stimulation. Perturbation of glucose metabolism or disruption of Myc function or sterol biosynthesis impaired myeloid differentiation. Integrative metabolomic and genomic profiling further identified one-carbon metabolism as a central node in mTORC1-dependent myelopoiesis. Therefore, the interplay between mTORC1 signaling and metabolic reprogramming underlies M-CSF-induced myelopoiesis.

Homeostatic control of metabolic and functional fitness of Treg cells by LKB1 signalling.

Regulatory T cells (Treg cells) have a pivotal role in the establishment and maintenance of immunological self-tolerance and homeostasis. Transcriptional programming of regulatory mechanisms facilitates the functional activation of Treg cells in the prevention of diverse types of inflammatory responses. It remains unclear how Treg cells orchestrate their homeostasis and interplay with environmental signals. Here we show that liver kinase B1 (LKB1) programs the metabolic and functional fitness of Treg cells in the control of immune tolerance and homeostasis. Mice with a Treg-specific deletion of LKB1 developed a fatal inflammatory disease characterized by excessive TH2-type-dominant responses. LKB1 deficiency disrupted Treg cell survival and mitochondrial fitness and metabolism, but also induced aberrant expression of immune regulatory molecules including the negative co-receptor PD-1 and the TNF receptor superfamily proteins GITR and OX40. Unexpectedly, LKB1 function in Treg cells was independent of conventional AMPK signalling or the mTORC1-HIF-1α axis, but contributed to the activation of ß-catenin signalling for the control of PD-1 and TNF receptor proteins. Blockade of PD-1 activity reinvigorated the ability of LKB1-deficient Treg cells to suppress TH2 responses and the interplay with dendritic cells primed by thymic stromal lymphopoietin. Thus, Treg cells use LKB1 signalling to coordinate their metabolic and immunological homeostasis and to prevent apoptotic and functional exhaustion, thereby orchestrating the balance between immunity and tolerance.

The ubiquitin ligase SEVEN IN ABSENTIA (SINA) ubiquitinates a defense-related NAC transcription factor and is involved in defense signaling.

We recently identified a defense-related tomato (Solanum lycopersicum) NAC (NAM, ATAF1,2, CUC2) transcription factor, NAC1, that is subjected to ubiquitin-proteasome system-dependent degradation in plant cells. In this study, we identified a tomato ubiquitin ligase (termed SEVEN IN ABSENTIA3; SINA3) that ubiquitinates NAC1, promoting its degradation. We conducted coimmunoprecipitation and bimolecular fluorescence complementation to determine that SINA3 specifically interacts with the NAC1 transcription factor in the nucleus. Moreover, we found that SINA3 ubiquitinates NAC1 in vitro and promotes NAC1 degradation via polyubiquitination in vivo, indicating that SINA3 is a ubiquitin ligase that ubiquitinates NAC1, promoting its degradation. Our real-time PCR analysis indicated that, in contrast to our previous finding that NAC1 mRNA abundance increases upon Pseudomonas infection, the SINA3 mRNA abundance decreases in response to Pseudomonas infection. Moreover, using Agrobacterium-mediated transient expression, we found that overexpression of SINA3 interferes with the hypersensitive response cell death triggered by multiple plant resistance proteins. These results suggest that SINA3 ubiquitinates a defense-related NAC transcription factor for degradation and plays a negative role in defense signaling.

Competitive binding of antagonistic peptides fine-tunes stomatal patterning.

During development, cells interpret complex and often conflicting signals to make optimal decisions. Plant stomata, the cellular interface between a plant and the atmosphere, develop according to positional cues, which include a family of secreted peptides called epidermal patterning factors (EPFs). How these signalling peptides orchestrate pattern formation at a molecular level remains unclear. Here we report in Arabidopsis that Stomagen (also called EPF-LIKE9) peptide, which promotes stomatal development, requires ERECTA (ER)-family receptor kinases and interferes with the inhibition of stomatal development by the EPIDERMAL PATTERNING FACTOR 2 (EPF2)-ER module. Both EPF2 and Stomagen directly bind to ER and its co-receptor TOO MANY MOUTHS. Stomagen peptide competitively replaced EPF2 binding to ER. Furthermore, application of EPF2, but not Stomagen, elicited rapid phosphorylation of downstream signalling components in vivo. Our findings demonstrate how a plant receptor agonist and antagonist define inhibitory and inductive cues to fine-tune tissue patterning on the plant epidermis.

Co-Immunoprecipitation of Membrane-Bound Receptors.

The study of cell-surface receptor dynamics is critical for understanding how cells sense and respond to changing environments. Therefore, elucidating the mechanisms by which signals are perceived and communicated into the cell is necessary to understand immunity, development, and stress. Challenges in testing interactions of membrane-bound proteins include their dynamic nature, their abundance, and the complex dual environment (lipid/soluble) in which they reside. Co-Immunoprecipitation (Co-IP) of tagged membrane proteins is a widely used approach to test protein-protein interaction in vivo. In this protocol we present a method to perform Co-IP using enriched membrane proteins in isolated microsomal fractions. The different variations of this protocol are highlighted, including recommendations and troubleshooting guides in order to optimize its application. This Co-IP protocol has been developed to test the interaction of receptor-like kinases, their interacting partners, and peptide ligands in stable Arabidopsis thaliana lines, but can be modified to test interactions in transiently expressed proteins in tobacco, and potentially in other plant models, or scaled for large-scale protein-protein interactions at the membrane.

The tomato cell death suppressor Adi3 is restricted to the endosomal system in response to the Pseudomonas syringae effector protein AvrPto.

The tomato (Solanum lycopersicum) AGC protein kinase Adi3 functions as a suppressor of cell death and was first identified as an interactor with the tomato resistance protein Pto and the Pseudomonas syringae effector protein AvrPto. Models predict that loss of Adi3 cell death suppression (CDS) activity during Pto/AvrPto interaction leads to the cell death associated with the resistance response initiated from this interaction. Nuclear localization is required for Adi3 CDS. Prevention of nuclear accumulation eliminates Adi3 CDS and induces cell death by localizing Adi3 to intracellular punctate membrane structures. Here we use several markers of the endomembrane system to show that the punctate membrane structures to which non-nuclear Adi3 is localized are endosomal in nature. Wild-type Adi3 also localizes in these punctate endosomal structures. This was confirmed by the use of endosomal trafficking inhibitors, which were capable of trapping wild-type Adi3 in endosomal-like structures similar to the non-nuclear Adi3. This suggests Adi3 may traffic through the cell using the endomembrane system. Additionally, Adi3 was no longer found in the nucleus but was visualized in these punctate endosomal-like membranes during the cell death induced by the Pto/AvrPto interaction. Therefore we propose that inhibiting nuclear import and constraining Adi3 to the endosomal system in response to AvrPto is a mechanism to initiate the cell death associated with resistance.

Two Pdk1 phosphorylation sites on the plant cell death suppressor Adi3 contribute to substrate phosphorylation.

The tomato AGC kinase Adi3 is phosphorylated by Pdk1 for activation of its cell death suppression activity. The Pdk1 phosphorylation site for activation of Adi3 is at Ser539. However, there is at least one additional Pdk1 phosphorylation site on Adi3 that has an unknown function. Here we identify an Arabidopsis thaliana sequence homologue of Adi3 termed AGC1-3. Two Pdk1 phosphorylation sites were identified on AGC1-3, activation site Ser596 and Ser269, and by homology Ser212 on Adi3 was identified as a second Pdk1 phosphorylation site. While Ser212 is not required for Adi3 autophosphorylation, Ser212 was shown to be required for full phosphorylation of the Adi3 substrate Gal83.

Ubiquitination of the tomato cell death suppressor Adi3 by the RING E3 ubiquitin ligase AdBiL.

Programmed cell death (PCD) is an organized process by which organisms selectively remove cells according to developmental needs or in response to biotic or abiotic stress. Despite recent efforts to understand mechanisms by which cell death takes place in plants, several gaps remain in our understanding of the molecular elements involved. The tomato PCD suppressor Adi3 is an AGC kinase that shares functional homology with the mammalian inhibitor of apoptosis PKB. Regulation of PKB stability, cell localization, and activation state is achieved through post-translational modifications such as ubiquitination. In an effort to understand the regulation of Adi3 function, we studied its interaction with the E3 ubiquitin ligase AdBiL. Using in vitro ubiquitination assays we show that AdBiL is an active E3 ubiquitin ligase using the E2 ubiquitin ligase UBC8 to ubiquitinate Adi3. Adi3 is also degraded in a proteasome-dependent manner. Our data draws additional parallels between Adi3 and PKB to support the functional relationship between these two PCD regulators.

The ß-subunit of the SnRK1 complex is phosphorylated by the plant cell death suppressor Adi3.

The protein kinase AvrPto-dependent Pto-interacting protein3 (Adi3) is a known suppressor of cell death, and loss of its function has been correlated with cell death induction during the tomato (Solanum lycopersicum) resistance response to its pathogen Pseudomonas syringae pv tomato. However, Adi3 downstream interactors that may play a role in cell death regulation have not been identified. We used a yeast two-hybrid screen to identify the plant SnRK1 (for Sucrose non-Fermenting-1-Related Protein Kinase1) protein as an Adi3-interacting protein. SnRK1 functions as a regulator of carbon metabolism and responses to biotic and abiotic stresses. SnRK1 exists in a heterotrimeric complex with a catalytic α-subunit (SnRK1), a substrate-interacting ß-subunit, and a regulatory γ-subunit. Here, we show that Adi3 interacts with, but does not phosphorylate, the SnRK1 α-subunit. The ability of Adi3 to phosphorylate the four identified tomato ß-subunits was also examined, and it was found that only the Galactose Metabolism83 (Gal83) ß-subunit was phosphorylated by Adi3. This phosphorylation site on Gal83 was identified as serine-26 using a mutational approach and mass spectrometry. In vivo expression of Gal83 indicates that it contains multiple phosphorylation sites, one of which is serine-26. An active SnRK1 complex containing Gal83 as the ß-subunit and sucrose nonfermenting4 as the γ-subunit was constructed to examine functional aspects of the Adi3 interaction with SnRK1 and Gal83. These assays revealed that Adi3 is capable of suppressing the kinase activity of the SnRK1 complex through Gal83 phosphorylation plus the interaction with SnRK1 and suggested that this function may be related to the cell death suppression activity of Adi3.

Direct ubiquitination of pattern recognition receptor FLS2 attenuates plant innate immunity.

Innate immune responses are triggered by the activation of pattern-recognition receptors (PRRs). The Arabidopsis PRR FLAGELLIN-SENSING 2 (FLS2) senses bacterial flagellin and initiates immune signaling through association with BAK1. The molecular mechanisms underlying the attenuation of FLS2 activation are largely unknown. We report that flagellin induces recruitment of two closely related U-box E3 ubiquitin ligases, PUB12 and PUB13, to FLS2 receptor complex in Arabidopsis. BAK1 phosphorylates PUB12 and PUB13 and is required for FLS2-PUB12/13 association. PUB12 and PUB13 polyubiquitinate FLS2 and promote flagellin-induced FLS2 degradation, and the pub12 and pub13 mutants displayed elevated immune responses to flagellin treatment. Our study has revealed a unique regulatory circuit of direct ubiquitination and turnover of FLS2 by BAK1-mediated phosphorylation and recruitment of specific E3 ligases for attenuation of immune signaling.

The T-loop extension of the tomato protein kinase AvrPto-dependent Pto-interacting protein 3 (Adi3) directs nuclear localization for suppression of plant cell death.

In tomato (Solanum lycopersicum), resistance to Pseudomonas syringae pv. tomato is elicited by the interaction of the host Pto kinase with the pathogen effector protein AvrPto, which leads to various immune responses including localized cell death termed the hypersensitive response. The AGC kinase Adi3 functions to suppress host cell death and interacts with Pto only in the presence of AvrPto. The cell death suppression (CDS) activity of Adi3 requires phosphorylation by 3-phosphoinositide-dependent protein kinase 1 (Pdk1) and loss of Adi3 function is associated with the hypersensitive response cell death initiated by the Pto/AvrPto interaction. Here we studied the relationship between Adi3 cellular localization and its CDS activity. Adi3 is a nuclear-localized protein, and this localization is dictated by a nuclear localization signal found in the Adi3 T-loop extension, an approximately 80 amino acid insertion into the T-loop, or activation loop, which is phosphorylated for kinase activation. Nuclear localization of Adi3 is required for its CDS activity and loss of nuclear localization causes elimination of Adi3 CDS activity and induction of cell death. This nuclear localization of Adi3 is dependent on Ser-539 phosphorylation by Pdk1 and non-nuclear Adi3 is found in punctate structures throughout the cell. Our data support a model in which Pdk1 phosphorylation of Adi3 directs nuclear localization for CDS and that disruption of Adi3 nuclear localization may be a mechanism for induction of cell death such as that during the Pto/AvrPto interaction.