Genome editing of the disease susceptibility gene CsLOB1 in citrus confers resistance to citrus canker.
Author information: Jia H1, Zhang Y1, Orbovic V1, Xu J1, White F2, Jones J2, Wang N1.
1Citrus Research and Education Center, Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences (IFAS), University of Florida, Lake Alfred, Fl, U.S.A.
2Department of Plant Pathology, IFAS, University of Florida, Gainesville, FL, U. S. A.
Date of e-pub: December 2016
Abstract: Citrus is a highly valued tree crop worldwide, while, at the same time, citrus production faces many biotic challenges, including bacterial canker and Huanglongbing (HLB). Breeding for disease resistant varieties is the most efficient and sustainable approach to control plant diseases. Traditional breeding of citrus varieties is challenging due to multiple limitations, including polyploidy, polyembryony, extended juvenility, and long crossing cycles. Targeted genome editing technology has the potential to shorten varietal development for some traits, including disease resistance. Here, we used CRISPR/Cas9/sgRNA technology to modify the canker susceptibility gene CsLOB1 in Duncan grapefruit. Six independent lines, DLOB 2, DLOB 3, DLOB 9, DLOB 10, DLOB 11 and DLOB 12, were generated. Targeted next-generation sequencing of the six lines showed the mutation rate was 31.58%, 23.80%, 89.36%, 88.79%, 46.91% and 51.12% for DLOB 2, DLOB 3, DLOB9, DLOB 10, DLOB 11, and DLOB 12, respectively, of the cells in each line. DLOB 2 and DLOB 3 showed canker symptoms similar to wild type grapefruit, when inoculated with the pathogen Xanthomonas citri subsp. citri (Xcc). No canker symptoms were observed on DLOB 9, DLOB 10, DLOB 11 and DLOB 12 at 4 days post inoculation (DPI) with Xcc. Pustules caused by Xcc were observed on DLOB 9, DLOB 10, DLOB 11 and DLOB 12 in later stages, which were much reduced compared to that on wild type grapefruit. The pustules on DLOB 9 and DLOB 10 did not develop into typical canker symptoms. No side effects and off-target mutations were detected in the mutated plants. This study indicates that genome editing using CRISPR technology will provide a promising pathway to generate disease resistant citrus varieties. This article is protected by copyright. All rights reserved.
Dynamics of antigen presentation to transgene product-specific CD4+ T cells and of Treg induction upon hepatic AAV gene transfer.
Author information: Perrin GQ1, Zolotukhin I1, Sherman A1, Biswas M1, de Jong YP2, Terhorst C3, Davidoff AM4, Herzog RW1.
1Department of Pediatrics, University of Florida , Gainesville, Florida, USA.
2Division of Gastroenterology and Hepatology, Weill Cornell Medicine , New York, New York, USA.
3Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School , Boston, Massachusetts, USA.
4Department of Surgery, St. Jude Children’s Research Hospital , Memphis, Tennessee, USA.
Date of e-pub: December 2016
Abstract: The tolerogenic hepatic microenvironment impedes clearance of viral infections but is an advantage in viral vector gene transfer, which often results in immune tolerance induction to transgene products. Although the underlying tolerance mechanism has been extensively studied, our understanding of antigen presentation to transgene product-specific CD4+ T cells remains limited. To address this, we administered hepatotropic adeno-associated virus (AAV8) vector expressing cytoplasmic ovalbumin (OVA) into wt mice followed by adoptive transfer of transgenic OVA-specific T cells. We find that that the liver-draining lymph nodes (celiac and portal) are the major sites of MHC II presentation of the virally encoded antigen, as judged by in vivo proliferation of DO11.10 CD4+ T cells (requiring professional antigen-presenting cells, e.g., macrophages) and CD4+CD25+FoxP3+ Treg induction. Antigen presentation in the liver itself contributes to activation of CD4+ T cells egressing from the liver. Hepatic-induced Treg rapidly disseminate through the systemic circulation. By contrast, a secreted OVA transgene product is presented in multiple organs, and OVA-specific Treg emerge in both the thymus and periphery. In summary, liver draining lymph nodes play an integral role in hepatic antigen presentation and peripheral Treg induction, which results in systemic regulation of the response to viral gene products.
Author information: Michels AW1, Landry LG1, McDaniel KA1, Yu L1, Campbell-Thompson M2, Kwok WW3, Jones KL4, Gottlieb PA1, Kappler JW1,5,6, Tang Q7, Roep BO8, Atkinson MA2, Mathews CE2, Nakayama M9,5.
1Barbara Davis Center for Childhood Diabetes, University of Colorado School of Medicine, Aurora, CO, USA.
2Department of Pathology, Immunology, and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, FL, USA.
3Benaroya Research Institute at Virginia Mason, Seattle, WA, USA; Department of Medicine, University of Washington, Seattle, WA, USA.
4Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO, USA.
5Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, CO, USA.
6Howard Hughes Medical Institute, Denver, CO, USA; Department of Biomedical Research, National Jewish Health, Denver, CO, USA; Program in Structural Biology and Biochemistry, University of Colorado School of Medicine, Aurora, CO, USA.
7Department of Surgery, University of California, San Francisco, San Francisco, CA, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA.
8Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, 2300 RC Leiden, the Netherlands; Department of Diabetes Immunology, Diabetes and Metabolism Research Institute, Beckman Research Institute of City of Hope, Duarte, CA, USA.
9Barbara Davis Center for Childhood Diabetes, University of Colorado School of Medicine, Aurora, CO, USA Maki.Nakayama@ucdenver.edu.
Date of e-pub: December 2016
Abstract: Type 1 diabetes results from chronic autoimmune destruction of insulin-producing beta cells within pancreatic islets. While insulin is a critical self-antigen in animal models of autoimmune diabetes, due to extremely limited access to pancreas samples, little is known about human antigenic targets for islet-infiltrating T-cells. Here we show that proinsulin peptides are targeted by islet-infiltrating T-cells from type 1 diabetes patients. We identified hundreds of T-cells from inflamed pancreatic islets of three young type 1 diabetes organ donors with a short disease duration with high risk HLA genes using a direct T-cell receptor (TCR) sequencing approach without long-term cell culture. Among 85 selected CD4 TCRs tested for reactivity to preproinsulin peptides presented by diabetes-susceptible HLA-DQ and -DR molecules, one T-cell recognized C-peptide amino acids 19-35, and two clones from separate donors responded to insulin B chain amino acids 9-23 (B:9-23) which is known to be a critical self-antigen driving disease progress in animal models of autoimmune diabetes. These B:9-23-specific T-cells from islets responded to whole proinsulin and islets, while previously identified B:9-23 responsive clones from peripheral blood did not, highlighting the importance of proinsulin-specific T-cells in the islet microenvironment.
NOTE: These abstracts were retrieved from the U.S. National Library of Medicine website managed in collaboration with the U.S. National Library of Medicine