Location: Subtropical Insects and Horticulture Research
Project Number: 6034-21000-018-020-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Dec 1, 2018
End Date: Nov 30, 2021
Objective:
This proposed project is to complete the evaluation of huanglongbing (HLB)-resistant transgenics already produced, continue development on improved construct variants, and provide the best material for release to the industry. Transgenics utilizing several different modes of action were selected through the use of novel methods of preliminary screening and have proven very promising in greenhouse and lab tests. Lines expressing citrus derived defensin-lipid binding protein chimeras, developed using biophysical modeling, have been shown to directly kill Candidatus Liberibacter asiaticus (CLas) through disrupting membranes. Other lines express antigen-binding fragments targeting CLas membrane proteins critical for virulence and avoiding plant defenses, and have shown an average 99% reduction in CLas titer compared to wild type controls in no-choice Asian citrus psyllid (ACP) inoculation. Resistance from these transgenics and verification of screening methodology will be confirmed using agreed common protocols and data collection; including greenhouse based no-choice ACP inoculation, field exposure at our Picos Farm and high throughput lab analysis. The availability of HLB resistant rootstock and scion varieties will offer a long term means of addressing HLB. While the first phases of this study are well-underway from progenitor projects, improved successive constructs will also be implemented using citrus phloem-specific promoters and enhanced transgenes. These constructs will follow our now streamlined and mature development and testing pipeline to speedily produce even more effective cultivars. Verified resistant materials have been and will continue to be sent to Department of Plant Industry (DPI) for shoot-tip-grafting and expected statewide trials comparing materials produced in diverse programs.
Approach:
Lab, greenhouse, and field studies of each transgenic construct will utilize the mature and efficient protocols in use at the U.S. Horticultural Research Laboratory by the Subtropical Insects and Horticulture Research Unit. In brief; 1) lab studies utilizing our high-throughput detached tissue assay measuring Candidatus Liberibacter Asiaticus (CLas) survival in leaves and Asian citrus psyllid (ACP), as well as ACP mortality; 2) greenhouse studies assessing CLas titer following no choice feeding of CLas positive ACP maintained at our facility; and 3) field studies conducted through graft inoculation and/or field exposure at our Picos Farm location. All greenhouse and field testing will be conducted in accordance with newly established common protocols. All analysis tools and required experience for these studies are available in house, including citrus transformation and regeneration, transgene and vector design, Real time quantitative polymerase chain reaction (RT-qPCR) analysis of transgene and CLas gene expression, protein isolation, plant maintenance in greenhouse and field conditions and disease symptom characterization. This is possible through the use of core facility equipment from the USDA-ARS and a diverse brain-trust of specialists in each aspect of the project. Expertise includes conventional breeding/screening, horticultural, genetics, proteomics, structural biology, entomology, and plant pathology. All contributors have strong familiarity with the needs of this research due to cooperative involvement on the preceding work to design, create and test anti-huanglongbing (HLB) constructs.
