Natural variation of potato allene oxide synthase 2 causes differential levels of jasmonates and pathogen resistance in Arabidopsis

Natural variation of plant pathogen resistance is often quantitative. This type of resistance can be genetically dissected in quantitative resistance loci (QRL). To unravel the molecular basis of QRL in potato (Solanum tuberosum), we employed the model plant Arabidopsis thaliana for functional analysis of natural variants of potato allene oxide synthase 2 (StAOS2). StAOS2 is a candidate gene for QRL on potato chromosome XI against the oömycete Phytophthora infestans causing late blight, and the bacterium Erwinia carotovora ssp. atroseptica causing stem black leg and tuber soft rot, both devastating diseases in potato cultivation. StAOS2 encodes a cytochrome P450 enzyme that is essential for biosynthesis of the defense signaling molecule jasmonic acid. Allele non-specific dsRNAi-mediated silencing of StAOS2 in potato drastically reduced jasmonic acid production and compromised quantitative late blight resistance. Five natural StAOS2 alleles were expressed in the null Arabidopsis aos mutant under control of the Arabidopsis AOS promoter and tested for differential complementation phenotypes. The aos mutant phenotypes evaluated were lack of jasmonates, male sterility and susceptibility to Erwinia carotovora ssp. carotovora. StAOS2 alleles that were associated with increased disease resistance in potato complemented all aos mutant phenotypes better than StAOS2 alleles associated with increased susceptibility. First structure models of ‘quantitative resistant’ versus ‘quantitative susceptible’ StAOS2 alleles suggested potential mechanisms for their differential activity. Our results demonstrate how a candidate gene approach in combination with using the homologous Arabidopsis mutant as functional reporter can help to dissect the molecular basis of complex traits in non model crop plants

Analysis of the Plant bos1 Mutant Highlights Necrosis as an Efficient Defence Mechanism during D. dadantii/Arabidospis thaliana Interaction

Dickeya dadantii is a broad host range phytopathogenic bacterium provoking soft rot disease on many plants including Arabidopsis. We showed that, after D. dadantii infection, the expression of the Arabidopsis BOS1 gene was specifically induced by the production of the bacterial PelB/C pectinases able to degrade pectin. This prompted us to analyze the interaction between the bos1 mutant and D. dadantii. The phenotype of the infected bos1 mutant is complex. Indeed, maceration symptoms occurred more rapidly in the bos1 mutant than in the wild type parent but at a later stage of infection, a necrosis developed around the inoculation site that provoked a halt in the progression of the maceration. This necrosis became systemic and spread throughout the whole plant, a phenotype reminiscent of that observed in some lesion mimic mutants. In accordance with the progression of maceration symptoms, bacterial population began to grow more rapidly in the bos1 mutant than in the wild type plant but, when necrosis appeared in the bos1 mutant, a reduction in bacterial population was observed. From the plant side, this complex interaction between D. dadantii and its host includes an early plant defence response that comprises reactive oxygen species (ROS) production accompanied by the reinforcement of the plant cell wall by protein cross-linking. At later timepoints, another plant defence is raised by the death of the plant cells surrounding the inoculation site. This plant cell death appears to constitute an efficient defence mechanism induced by D. dadantii during Arabidopsis infection

Regulation of CD1a Surface Expression and Antigen Presentation by Invariant Chain and Lipid Rafts

In immature dendritic cells (DCs), CD1a is almost exclusively expressed at the cell surface and its membrane organization is poorly understood. In this study, we report that MHC class II, invariant chain (Ii), and CD9 molecules are coimmunoprecipitated with CD1a in immature DCs, and that CD1a/Ii colocalization is dependent on lipid raft integrity. In HeLa-CIITA cells CD1a expression leads to increased Ii trafficking to the cell surface, confirming the relevance of this association. Furthermore, silencing of Ii in DCs induces significant CD1a accumulation on the plasma membrane whereas the total CD1a expression remains similar to that of control cells. These data suggest that CD1a recycling is facilitated by the association with the Ii. The CD1a localization in lipid rafts has functional relevance as demonstrated by inhibition of CD1a-restricted presentation following raft disruption. Overall, these findings identify Ii and lipid rafts as key regulators of CD1a organization on the surface of immature DCs and of its immunological function as Ag-presenting molecule

Extracorporeal photophoresis increases sensitivity of monocytes from patients with graft-versus-host disease to HLA-DR-mediated cell death

BACKGROUND: Graft-versus-host disease (GVHD) remains a cause of long-term morbidity after allogeneic hematopoietic stem cell transplantation, and recent studies indicate that extracorporeal photophoresis (ECP) is useful for treatment of steroid-refractory GVHD although the mechanisms are unclear. Antigen-presenting cells (APCs) such as dendritic cells have a central role in GVHD, and apoptosis of APCs by HLA-DR monoclonal antibody (MoAb) has been documented in vitro and in vivo. Monocytes have been identified as precursors of dendritic cells in vivo and particularly under conditions of inflammation. STUDY DESIGN AND METHODS: This study examined whether ECP altered the survival of peripheral blood monocytes from patients with GVHD, monocyte apoptosis after engagement of HLA-DR antigens with MoAb, and monocyte apoptosis after allointeraction with primary CD4+ T lymphocytes. Samples from patients from two centers were studied. RESULTS: It is reported here that ECP induced apoptosis of monocytes over a period of at least 48 hours. ECP also clearly increased cell death of monocytes after engagement of HLA-DR antigens with MoAb. In contrast, engagement of HLA-DR by allointeraction failed to induce significant cell death of monocytes, and this was unaltered by ECP treatment. CONCLUSION: These data reveal that monocytes from patients with GVHD are sensitive to HLA-DR-mediated apoptosis and that ECP treatment increases sensitivity to both spontaneous and HLA-DR-mediated apoptosis. Therefore, ECP treatment in combination with HLA-DR MoAbs could rapidly deplete monocytes and thereby reduce the contribution of monocyte-derived dendritic cells to GVHD

L'apoptose médiée par les CMH de classe II des cellules dendritiques matures agit par l'activation de l'iso-enzyme delta de la protéine kinase C

The mature dendritic cell (DC) is considered to be the most potent antigen-presenting cell. Regulation of the DC, particularly its survival, is therefore critical. Mature DC are markedly more sensitive to HLA-DR-mediated apoptosis than immature DC. To further characterize this key survival difference, we compared the intracellular signals initiated via HLA-DR in mature versus immature DC. Apoptosis was unchanged by inhibition of tyrosine kinases or phosphatases. HLA-DR-mediated re-localization of protein kinase C (PKC)-d to the nucleus was detected in mature DC by confocal microscopy and by immunoblotting. Activation of PKC-d in mature DC was revealed by the detection of the PKC-d catalytic fragment in the nuclear fraction isolated from mature DC which had been stimulated via HLA-DR. The broad-spectrum PKC inhibitor, Calphostin C, as well as the PKC-d-selective inhibitor, Rottlerin, inhibited HLA-DR-mediated apoptosis of mature cells. Taken together, these data reveal a role for the PKC-d isoenzyme in regulating HLA class II-mediated apoptosis of mature DC. Thus, the lifespan of the mature DC could be controlled by signals generated in the course of antigen presentation, and thereby prevent DC persistence and prolonged stimulation of T and B lymphocytes

An Orchestrated Intron Retention Program in Meiosis Controls Timely Usage of Transcripts during Germ Cell Differentiation

Global transcriptome reprogramming during spermatogenesis ensures timely expression of factors in each phase of male germ cell differentiation. Spermatocytes and spermatids require particularly extensive reprogramming of gene expression to switch from mitosis to meiosis and to support gamete morphogenesis. Here, we uncovered an extensive alternative splicing program during this transmeiotic differentiation. Notably, intron retention was largely the most enriched pattern, with spermatocytes showing generally higher levels of retention compared with spermatids. Retained introns are characterized by weak splice sites and are enriched in genes with strong relevance for gamete function. Meiotic intron-retaining transcripts (IRTs) were exclusively localized in the nucleus. However, differently from other developmentally regulated IRTs, they are stable RNAs, showing longer half-life than properly spliced transcripts. Strikingly, fate-mapping experiments revealed that IRTs are recruited onto polyribosomes days after synthesis. These studies reveal an unexpected function for regulated intron retention in modulation of the timely expression of select transcripts during spermatogenesis

Tax ubiquitylation and SUMOylation control the dynamic shuttling of Tax and NEMO between Ubc9 nuclear bodies and the centrosome

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BCL-2 and mutant NRAS interact physically and functionally in a mouse model of progressive myelodysplasia

Myelodysplastic syndromes (MDS) are clonal stem cell hematologic disorders that evolve to acute myeloid leukemia (AML) and thus model multistep leukemogenesis. Activating RAS mutations and overexpression of BCL-2 are prognostic features of MDS/AML transformation. Using NRASD12 and BCL-2, we created two distinct models of MDS and AML, where human (h)BCL-2 is conditionally or constitutively expressed. Our novel transplantable in vivo models show that expression of hBCL-2 in a primitive compartment by mouse mammary tumor virus–long terminal repeat results in a disease resembling human MDS, whereas the myeloid MRP8 promoter induces a disease with characteristics of human AML. Expanded leukemic stem cell (Lin−/Sca-1+/c-Kit+) populations and hBCL-2 in the increased RAS-GTP complex within the expanded Sca-1+ compartment are described in both MDS/AML–like diseases. Furthermore, the oncogenic compartmentalizations provide the proapoptotic versus antiapoptotic mechanisms, by activating extracellular signal-regulated kinase and AKT signaling, in determination of the neoplastic phenotype. When hBCL-2 is switched off with doxycycline in the MDS mice, partial reversal of the phenotype was observed with persistence of bone marrow blasts and tissue infiltration as RAS recruits endogenous mouse (m)BCL-2 to remain active, thus demonstrating the role of the complex in the disease. This represents the first in vivo progression model of MDS/AML dependent on the formation of a BCL-2:RAS-GTP complex. The colocalization of BCL-2 and RAS in the bone marrow of MDS/AML patients offers targeting either oncogene as a therapeutic strategy