Plant innate immunity relies on the recognition of pathogen effector molecules by nucleotide-binding-leucine-rich repeat (NB-LRR) immune receptor families. Previously we have shown the N immune receptor, a member of TIR-NB-LRR family, indirectly recognizes the 50 kDa helicase (p50) domain of Tobacco mosaic virus (TMV) through its TIR domain. We have identified an N receptor-interacting protein, NRIP1, that directly interacts with both N's TIR domain and p50. NRIP1 is a functional rhodanese sulfurtransferase and is required for N to provide complete resistance to TMV. Interestingly, NRIP1 that normally localizes to the chloroplasts is recruited to the cytoplasm and nucleus by the p50 effector. As a consequence, NRIP1 interacts with N only in the presence of the p50 effector. Our findings show that a chloroplastic protein is intimately involved in pathogen recognition. We propose that N's activation requires a prerecognition complex containing the p50 effector and NRIP1.

Virus-induced gene silencing (VIGS) is a widely used, powerful technique for reverse genetics. VIGS vectors derived from the Tobacco rattle virus (TRV) are among the most popular for VIGS. We have developed a TRV RNA2 vector that allows the insertion of gene silencing fragments by ligation-independent cloning (LIC). This new vector has several advantages over previous vectors, particularly for applications involving the analysis of large numbers of sequences, since TRV-LIC vectors containing the desired insert are obtained with 100% efficiency. Importantly, this vector allows the high-throughput cloning of silencing fragments without the use of costly enzymes required for recombination, as is the case with GATEWAY-based vectors. We generated a collection of silencing vectors based on 400 tomato (Solanum lycopersicum) expressed sequence tags in this TRV-LIC background. We have used this vector to identify roles for SlMADS1 and its Nicotiana benthamiana homologs, NbMADS4-1 and -2 in flowering. We find that NbMADS4-1 and -2 act nonredundantly in floral development and silencing of either gene results in loss of organ identity. This TRV-LIC vector should be a valuable resource to the plant community.The last decade has seen an explosion in the availability of plant gene sequences. The genomes of the model species Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) have both been sequenced, while those of tomato (Solanum lycopersicum) and maize (Zea mays) are currently being sequenced (Mueller et al., 2005; http://www.sgn.cornell.edu/about/tomato_project/; http://www.maizesequence.org/index.html). Large collections of ESTs have also been generated for a variety of species that are widely used for research purposes. Concomitant with the availability of this sequence information, many important aspects of plant growth and development have been analyzed by DNA microarrays, leading to the identification of numerous genes potentially involved in these processes. At this time then, the challenge to most plant biologists is to effectively mine this data to identify and characterize the genes and gene products that are critical to the crucial processes that have been investigated. This calls for techniques that start with a known DNA sequence and allow the determination of biological function. This approach is called reverse genetics and some of the most common methods for performing reverse genetic studies are based on RNA silencing.Although recently discovered, RNA silencing is a wellcharacterized, endogenous system for monitoring RNA inside a cell and eliminating foreign molecules or inhibiting mRNA translation (for review, see Brodersen and Voinnet, 2006). It is a homology-based process that uses small RNA fragments to identify targets for destruction or inhibition. RNA silencing is also indispensable for normal plant growth and development, regulating the expression of central genes in flowering, meristem identity, and other processes (Meins et al., 2005). In plants, RNA silencing plays critical roles in viral ...

Programmed cell death (PCD) initiated at the pathogeninfected sites during the plant innate immune response is thought to prevent the development of disease. Here, we describe the identification and characterization of an ER-localized type IIB Ca 2 þ -ATPase (NbCA1) that function as a regulator of PCD. Silencing of NbCA1 accelerates viral immune receptor N-and fungal-immune receptor Cf9-mediated PCD, as well as non-host pathogen Pseudomonas syringae pv. tomato DC3000 and the general elicitor cryptogein-induced cell death. The accelerated PCD rescues loss-of-resistance phenotype of Rar1, HSP90-silenced plants, but not SGT1-silenced plants. Using a genetically encoded calcium sensor, we show that downregulation of NbCA1 results in the modulation of intracellular calcium signalling in response to cryptogein elicitor. We further show that NbCAM1 and NbrbohB function as downstream calcium decoders in N-immune receptormediated PCD. Our results indicate that ER-Ca 2 þ -ATPase is a component of the calcium efflux pathway that controls PCD during an innate immune response.

Summary The plant innate immune response requires a rapid, global reprogramming of cellular processes. Here we employed two complementary proteomic methods, two-dimensional differential in-gel electrophoresis (2D-DIGE) and iTRAQ, to identify differentially regulated proteins early during a defense response. Besides defense-related proteins, the constituents of the largest category of up-regulated proteins were cytoplasmic- and endoplasmic reticulum (ER)-residing molecular chaperones. Silencing of ER-resident protein disulfide isomerases, NbERp57 and NbP5, and the calreticulins, NbCRT2 and NbCRT3, lead to a partial loss of N immune receptor-mediated defense against Tobacco mosaic virus (TMV). Furthermore, NbCRT2 and NbCRT3 are required for the expression of a novel induced receptor-like kinase (IRK). IRK is a plasma membrane-localized protein required for the N-mediated hypersensitive response programmed cell death (HR-PCD) and resistance to TMV. These data support a model in which ER-resident chaperones are required for the accumulation of membrane bound or secreted proteins that are necessary for innate immunity.

In vivo models that recapitulate human erythropoiesis with persistence of circulating red blood cells (RBCs) have remained elusive. We report an immunodeficient murine model in which combined human liver and cytokine humanization confer enhanced human erythropoiesis and RBC survival in the circulation. We deleted the fumarylacetoacetate hydrolase (Fah) gene in MISTRG mice expressing several human cytokines in place of their murine counterparts. Liver humanization by intrasplenic injection of human hepatocytes (huHep) eliminated murine complement C3 and reduced murine Kupffer cell density. Engraftment of human sickle cell disease (SCD)–derived hematopoietic stem cells in huHepMISTRGFah−/− mice resulted in vaso-occlusion that replicated acute SCD pathology. Combined liver–cytokine–humanized mice will facilitate the study of diseases afflicting RBCs, including bone marrow failure, hemoglobinopathies, and malaria, and also preclinical testing of therapies.

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Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are heterogeneous clonal disorders. Isocitrate dehydrogenase-1 and -2 (IDH1/2) mutations are detected in ~20% of AML and ~5% of MDS, in which they confer gain of a neomorphic function that leads to the production of (R)-2-hydroxyglutarate (2HG). Targeted inhibition of mutant IDH1/2 has resulted in significant responses in IDH1/2 mutant MDS and AML but is not curative and patients relapse (Stein et al. Blood 2016, DiNardo et al. N Engl J Med 2018). 2HG accumulation inhibits the function of histone demethylases (KDM4A and KDM4B) that are critical for the homologous recombination (HR) DNA repair pathway and consequently for the repair of DNA double strand breaks (DSBs) (Mallette et al. EMBO J 2012, Sulkowski et al. Sci Transl Med 2017). In HR deficient tumors, Poly-ADP-Ribose Polymerase (PARP) is essential for DNA single strand break (SSB) repair. In IDH mutant tumors PARP inhibitors induce synthetic lethality by suppressing the repair of SSBs, which eventually get converted into DSBs (Javle and Curtin Br J Cancer 2011). We previously demonstrated that in AML, IDH1/2 mutations impair DNA damage response by inducing a defect in HR, and that this renders leukemia cells susceptible to PARP inhibitors in vitro. We hypothesized that this vulnerability would also exist in IDH mutant MDS and more importantly, that this vulnerability would persist in MDS/AML resistant to IDH1/2 inhibitors. To determine whether PARP inhibition targets IDH mutant MDS/AML in vivo, we took advantage of 2 syngeneic mouse models of MDS and AML relying on co-mutation of SRSF2/IDH2 and FLT3/IDH2, respectively. Olaparib (PARP inhibitor) effectively targeted IDH2 mutant but not IDH2 wild type MDS/AML (Fig. 1A). We next sought to determine whether PARP inhibition mediated synthetic lethality persists in MDS/AML resistant to targeted IDH inhibition. We transduced IDH2 mutant murine cells with IDH2 WT or IDH2 MUT lentiviral vectors carrying one of two published resistance mutations. While these resistance mutations conferred resistance to the targeted IDH2 inhibitor Enasidenib, cells remained sensitive to Olaparib (Fig. 1B). Patient MDS/AML is heterogeneous and in general carries additional genetic mutations and epigenetic alterations. We therefore engrafted IDH1/2 WT and MUT MDS/AML patient samples in cytokine humanized immunodeficient mice and treated with vehicle of Olaparib. Engrafted mice were assigned to vehicle or Olaparib 8 weeks after transplantation based on equal engraftment levels determined by BM aspiration. Mice were treated with vehicle or Olaparib via IP injection for 21 days. Human engraftment levels and plasma 2-HG levels were significantly reduced in Olaparib treated animals when compared to pre-treatment and vehicle-treated mice (Fig. 1C). Of note, when equal numbers of huCD34+ cells from vehicle or Olaparib treated mice were transplanted into next generation mice, engraftment was significantly higher for recipients of human cells from vehicle than Olaparib treated mice, suggesting that Olaparib is toxic to leukemia initiating cells. In contrast, IDH WT MDS/AML was insensitive to Olaparib treatment (Fig. 1C). In conclusion, PARP inhibition is effective in vivo against IDH mutant MDS/AML and can overcome targeted IDH inhibitor resistance. Disclosures Flavell: Zai labs: Consultancy; SMOC: Equity Ownership; Troy: Equity Ownership; Artizan Biosciences: Equity Ownership; GSK: Consultancy; Rheos Biomedicines: Equity Ownership. Prebet:Boehringer Ingelheim: Research Funding; Boehringer Ingelheim: Research Funding; novartis: Honoraria; pfizer: Honoraria; Boehringer Ingelheim: Research Funding; Agios: Consultancy, Research Funding; novartis: Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; pfizer: Honoraria; Genentech: Consultancy; pfizer: Honoraria; novartis: Honoraria; Tetraphase: Consultancy; novartis: Honoraria; pfizer: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; novartis: Honoraria; pfizer: Honoraria. Bindra:Cybrexa: Consultancy, Equity Ownership.

In vivo models of human erythropoiesis with generation of circulating mature human red blood cells (huRBC) have remained elusive, limiting studies of primary human red cell disorders. In our prior study, we have generated the first combined cytokine-liver humanized immunodeficient mouse model (huHepMISTRG-Fah) with fully mature, circulating huRBC when engrafted with human CD34+ hematopoietic stem and progenitor cells (HSPCs)1. Here we present for the first time a humanized mouse model of human sickle cell disease (SCD) which replicates the hallmark pathophysiologic finding of vaso-occlusion in mice engrafted with primary patient-derived SCD HSPCs. SCD is an inherited blood disorder caused by a single point mutation in the beta-globin gene. Murine models of SCD exclusively express human globins in mouse red blood cells in the background of murine globin knockouts2 which exclusively contain murine erythropoiesis and red cells and thus fail to capture the heterogeneity encountered in patients. To determine whether enhanced erythropoiesis and most importantly circulating huRBC in engrafted huHepMISTRG-Fah mice would be sufficient to replicate the pathophysiology of SCD, we engrafted it with adult SCD BM CD34+ cells as well as age-matched control BM CD34+ cells. Overall huCD45+ and erythroid engraftment in BM (Fig. a, b) and PB (Fig. c, d) were similar between control or SCD. Using multispectral imaging flow cytometry, we observed sickling huRBCs (7-11 sickling huRBCs/ 100 huRBCs) in the PB of SCD (Fig. e) but not in control CD34+ (Fig. f) engrafted mice. To determine whether circulating huRBC would result in vaso-occlusion and associated findings in SCD engrafted huHepMISTRG-Fah mice, we evaluated histological sections of lung, liver, spleen, and kidney from control and SCD CD34+ engrafted mice. SCD CD34+ engrafted mice lungs showed an increase in alveolar macrophages (arrowheads) associated with alveolar hemorrhage and thrombosis (arrows) but not observed control engrafted mice (Fig. g). Spleens of SCD engrafted mice showed erythroid precursor expansion, sickled erythrocytes in the sinusoids (arrowheads), and vascular occlusion and thrombosis (arrows) (Fig. h). Liver architecture was disrupted in SCD engrafted mice with RBCs in sinusoids and microvascular thromboses (Fig. i). Congestion of capillary loops and peritubular capillaries and glomeruli engorged with sickled RBCs was evident in kidneys (Fig. j) of SCD but not control CD34+ engrafted mice. SCD is characterized by ineffective erythropoiesis due to structural abnormalities in erythroid precursors3. As a functional structural unit, erythroblastic islands (EBIs) represent a specialized niche for erythropoiesis, where a central macrophage is surrounded by developing erythroblasts of varying differentiation states4. In our study, both SCD (Fig. k) and control (Fig. l) CD34+ engrafted mice exhibited EBIs with huCD169+ huCD14+ central macrophages surrounded by varying stages of huCD235a+ erythroid progenitors, including enucleated huRBCs (arrows). This implies that huHepMISTRG-Fah mice have the capability to generate human EBIs in vivo and thus represent a valuable tool to not only study the effects of mature RBC but also to elucidate mechanisms of ineffective erythropoiesis in SCD and other red cell disorders. In conclusion, we successfully engrafted adult SCD patient BM derived CD34+ cells in huHepMISTRG-Fah mice and detected circulating, sickling huRBCs in the mouse PB. We observed pathological changes in the lung, spleen, liver and kidney, which are comparable to what is seen in the established SCD mouse models and in patients. In addition, huHepMISTRG-Fah mice offer the opportunity to study the role of the central macrophage in human erythropoiesis in health and disease in an immunologically advantageous context. This novel mouse model could therefore serve to open novel avenues for therapeutic advances in SCD. Reference 1. Song Y, Shan L, Gybli R, et. al. In Vivo reconstruction of Human Erythropoiesis with Circulating Mature Human RBCs in Humanized Liver Mistrg Mice. Blood. 2019;134:338. 2. Ryan TM, Ciavatta DJ, Townes TM. Knockout-transgenic mouse model of sickle cell disease. Science. 1997;278(5339):873-876. 3. Blouin MJ, De Paepe ME, Trudel M. Altered hematopoiesis in murine sickle cell disease. Blood. 1999;94(4):1451-1459. 4. Manwani D, Bieker JJ. The erythroblastic island. Curr Top Dev Biol. 2008;82:23-53. Disclosures Xu: Seattle Genetics: Membership on an entity's Board of Directors or advisory committees. Flavell:Zai labs: Consultancy; GSK: Consultancy.