2005 Annual Report 4d.Progress report. This report documents research conducted under a Specific Cooperative Agreement between ARS and the University of Hawai’i at Hilo. Additional details of research can be found in the report for the associated in-house project 5320-21000-010-00D, ‘Genomic and Biotechnological Approaches for Evaluating and Improving Tropical Crops’. Our work is focused on developing lettuce resistant to tomato spotted wilt virus (TSWV), as well as on developing a plant transformation vector for inducing gene silencing. In addition, we are developing a transient expression vector for inducing gene silencing. Gene silencing has been the focus of much recent biological work, and gene silencing targeted to plant viruses is an effective strategy for developing virus resistant plants. We cloned and sequenced the nonstructural protein genes from the small and medium RNAs (NSs and NSm) of TSWV. We are also in the process of cloning segments of the TSWV L gene. This virus isolate was obtained from a lettuce farm in Waimea, Hawaii, and we previously cloned and sequenced the nucleoprotein (N) gene from this isolate. These genes are now being transferred to the plant transformation vector pGA482G, for transformation of Nicotiana benthamiana and Lactuca sativa (lettuce). Our laboratory was equipped last year for plant transformation, and we are working towards transformation of lettuce (Lactuca sativa). Using published protocols, we are able to get this tissue to form callus rapidly and nicely, but are thus far unable to obtain viable shoots from this tissue. We continue to work on gene transfer to lettuce. We did, however, successfully transform N. benthamiana with the green fluorescent protein (GFP) gene, and took those lines through the R1 generation. Our aim is to develop virus-resistant lettuce specifically, and more broadly to examine the means for rapidly producing other virus resistant crops. Gene silencing can be triggered by transgenes of viral origin, and as a result confer virus resistance on a plant. We are developing novel vectors for triggering gene silencing, because in some cases transformation of a plant with a viral gene fails to confer resistance—this is in fact the case with the Nss and Nsm genes of TSWV. One of the physical triggers of gene silencing is known to be double stranded RNA (dsRNA), and we are developing plant transformation vectors for the efficient production of dsRNA from inserted DNA. Towards that end, we have cloned the tomato ubiquitin promoter, and we are constructing a plant transformation vector that will has a cloning site flanked by the cauliflower mosaic virus (CaMV) 35S promoter and the tomato ubiquitin promoter. These promoters will be oriented opposing each other, so together they will generate antiparallel transcripts and potentially dsRNA of the cloned DNA where these transcript sequences overlap. This is an alternative to using inverted repeated copies of the transforming DNA, which can be difficult to maintain because the inverted repeat can be lost to recombination during DNA replication, both in bacterial propagation and in the plant target. We are also investigating means for inducing gene silencing using transient expression vectors, with the aim of inducing virus-targeted silencing without the need to transform plants. We previously constructed a vector that contains the entire genome of tobacco mosaic virus (TMV) cloned into the mutagenesis vector pAlter. The TMV genome is cloned just downstream of the 35S promoter for transcription, and a ribozyme is inserted at the sequence corresponding to the TMV 3’ end. The resulting 35S-driven transcript is expected to have a capped 5’ end identical to the TMV genome, and a 3’ end that has four additional non-TMV nucleotides. We have shown this construct to be directly infectious, generating a systemic TMV infection in N. benthamiana and TMV-containing local lesions on Chenopodium quinoa. Since replication of the TMV genome is via a partially double-stranded RNA intermediate, insertion of DNAs into the non-replicase coding TMV section of this construct will produce dsRNAs from these DNAs during TMV replication. If the insertion of foreign genes into this TMV vector can induce silencing of those genes, we will subsequently inactivate the TMV movement and/or coat protein gene, so infection will be limited to just a few cells, and hopefully the silencing signal will move systemically in the plant. We continue to enjoy good collaborative relationships with the laboratories at USDA-PBARC. In particular, the laboratories of Dennis Gonsalves and Tracie Matsumoto have been instrumental in our progress. We also continue our collaboration with University of Hawaii extension agent Randy Hamasaki, who has facilitated our tissue collecting and information gathering trips to the Waimea farms.