Location: Subtropical Insects and Horticulture Research
Project Number: 6034-21000-020-003-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Oct 1, 2020
End Date: Aug 31, 2023
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. It may also identify peptides which can be applied exogenously to citrus trees to help sustain production. 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. 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 DPI for shoot-tip-grafting and expected statewide trials comparing materials produced in diverse programs. These constructs also induce ACP mortality when ACP feed on the transgenic leaves, so this component will also be investigated under the guidance of a Unit Entomologist.
Approach:
Lab, greenhouse, and field studies of each exogenous peptide and transgenic construct will utilize the mature and efficient protocols in use at our lab by our unit. In brief, with all studies replicated and using two sets of controls; 1) Candidatus Liberibacter asiaticus (CLas)-containing Asian citrus psyllid (ACP)-homogenate assays will be used to screen peptides (for use as transgenes for exoegenous application) for CLas-killing activity; 2) initial lab studies will screen transgenic lines with a high-throughput detached tissue assay measuring CLas survival in leaves and ACP, as well as ACP mortality; 3) greenhouse studies will assess CLas titer in transgenics following no choice feeding of CLas positive ACP maintained at our facility, and exogenous CLas-killing peptides will be assessed by application to small CLas infected trees ; and 4) field studies will be conducted through graft inoculation and/or field exposure at our 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, as well as Asian citrus psyllid (ACP) mortality studies. 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-HLB constructs.
