1a.Objectives (from AD-416)
1. Construct a chimeric gene to express a hairpin RNA (hRNA) corresponding to the 3'-end of RNA-1 and RNA-2.
2. Initiate agro-mediated transformation of a WIP clone 48-12 to express the hRNA.
3. Evaluate J. regia cv. Sinensis for resistance to CLRV infection.
4. Evaluate viral genes for induction of hypersensitive response by agroinfiltration.
1b.Approach (from AD-416)
1. Amplify about 700 bases and clone this gene in sense and antisense orientation separated by an intron. Upon expression of the transgene, the mRNA is processed and the intron will be cleaved to generate a hairpin RNA (hRNA) which will be targeted by the plant RNA-induced silencing complex to generate siRNA resulting systemic silencing of the cognate RNA.
2)Somatic Embryonic callus will be generated from WIP clone 48-12, a walnut selection identified by the UCD breeders as a male-sterile line and transformed by agroinfection. Transformed plants will be screened for the presence of transgene and grown to produce woody material to serve as interstocks.
3)wild leaf samples will be collected from walnut trees showing blackline disease and RT-PCR analysis will be performed on RNA extracted from the leaves.
4)Viral proteins that elicit HR will be determined and used subsequently to challenge test plants.
The agreement was established in support of objective #4 of the parent project, the goal being to examine the etiology, epidemiology, and control strategies for systemic/graft transmissible pathogens of fruit and nut trees. The goal of this project is to investigate genomic and horticultural approaches to manage blackline disease in walnuts. In the first few months of this project recruitment and hiring has been accomplished for support staff and purchases for necessary lab and field supplies to date have been processed. Preliminary field work has been accomplished. During the course of the year we plan to construct a initiate building plasmid constructs with Cherry leafroll virus sequences for RNAi-mediated silencing, initiate transformation of walnut lines and clone the viral genes to develop an inoculation assay.