2012 Annual Report
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, which is 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.
A chimeric construct comprising of 700 bases from the 3’-end of RNA-1 of Cherry leafroll virus (CLRV) strain W-8, cloned in sense and antisense orientation separated by an intron, has been made in pRNA69 for expression driven by 35S promoter. A NotI fragment comprising the chimeric construct was cloned into a binary vector pDU97.1005 and transformed into Agrobacterium tumefaciens strain EHA105. We have established somatic embryos from a male sterile walnut line to initiate transformation in the near future.
In addition to 35S promoter driven expression of the 3’-UTR of CLRV, we have cloned a phloem specific promoter rolC from Agrobacterium rhizogenes. A chimeric construct as described above has been constructed and is being cloned into pDU97.1005.
The complete sequence of genomic RNAs of CLRV strain w-8 was determined by deep sequencing of cDNA prepared using dsRNA extracted from virus inoculated Chenopodium quinoa. In addition to contigs generated by Illumina sequencing, several RT-PCR products spanning RNA-1 and RNA-2 were generated and sequenced. The sequences of RNA-1 and RNA-2 of CLRV were determined to be 7915 and 6359 nucleotides, respectively. Comparison of RNA-1 and RNA-2 sequences of W-8 with those of CLRV strains of Olm-1 and E-395, from cherry and rhubarb, respectively, indicated that W-8 was most closely related to the rhubarb strain. The identity scores for RNA-1 and RNA-2 were 85 and 83%, respectively, between W-8 and E-395.
We have designed primers to amplify the genes encoding polyproteins P1 and P2, encoded by RNA-1 and RNA-2, respectively, and are in the midst of cloning these two genes by RT-PCR.