Location: Crops Pathology and Genetics Research
Project Number: 2032-22000-016-33-R
Project Type: Reimbursable Cooperative Agreement
Start Date: May 1, 2018
End Date: Apr 30, 2019
1) Screen transgenic walnut lines expressing shRNA for resistance to CLRV. 2) Identify recessive alleles that can confer resistance to CLRV in J. regia. 3) Clone two eif4E genes identified in J. regia transcriptome and evaluate their interaction with VPg of CLRV. 4) Edit the eIF4E in walnuts using CRISPR system.
1) Screen transgenic walnut lines expressing shRNA for resistance to CLRV: We transformed somatic embryos of WIP clone 48-12 to express hairpin RNA corresponding to 700 nucleotides in the 3' end of CLRV genome using agrobacterium. So far, XX lines were regenerated from transformed embryos. These lines are in greenhouse in pots. We propose to screen these lines by grafting them onto CLRV-infected walnut trees. About 3 to 4 months after grafting, the developing shoots of transgenic lines will be examined by RT-PCR for CLRV infection. Chandler and Hartley grafts will be made similarly and used as controls. 2) Identify recessive allele(s) that can confer resistance to CLRV in J. regia. We have identified two eIF4E like genes in the transcriptome of J. regia. In addition to these, we will examine germplasm accessions by targeted sequencing of eIF4E-like genes to determine the diversity of these genes. 3) Clone two eif4E genes identified in J. regia transcriptome and evaluate their interaction with vPg of CLRV. We will custom order these two genes and clone them into a plasmid vector of yeast two hybrid system (Y2H) encoding the activation domain. Similarly, VPg also will be cloned into the binding domain of a vector in Y2H and its interaction with eIF4E will be evaluated. 4) Edit the eIF4E in walnuts using CRISPR system. Once the interacting eIF4E is identified we will develop guide RNAs for editing the gene or knockout. Edited walnut lines will be evaluated for resistance to CLRV. We intend to edit Chandler cultivar for walnut production and also a male sterile WIP clone 48- 12 for use as interstock.