1a. Objectives (from AD-416):
The objectives of this cooperative research project are to: -Develop virus-resistant plants using transgenic plant technology. -Evaluate lines of transgenic lettuce and tomato for tomato spotted wilt virus (TSWV) resistance. -Develop orchid plants with resistance to cymbidium mosaic virus (CymMV).
1b. Approach (from AD-416):
We will work towards developing virus-resistant plants, using transgenic plant technology. Tomato spotted wilt virus (TSWV) has worldwide impact, and particularly in the tropics and subtropics. We have cloned and sequenced several TSWV genes, and transferred them to lettuce plants. We will evaluate these lines for TSWV resistance. In addition, we will evaluate lines of transgenic tomato for TSWV resistance. These tomato plants were developed by the PBARC research group, and contain a gene for TSWV resistance bred in using conventional means, in addition to a TSWV transgene. After initial greenhouse evaluations, we will move the most promising lines on to field trials. TSWV is an extremely important plant virus in Hawaii and elsewhere, and the development of TSWV-resistant lettuce and tomato will greatly benefit agriculture in Hawaii and abroad. Work will also begin towards developing orchid plants with resistance to cymbidium mosaic virus (CymMV), the most important orchid virus worldwide. Most Hawaiian orchid growers that sell cut flowers specialize in a just a few Dendrobium varieties, and experience CymMV infection rates of over 90%. These growers have selected cultivars with good tolerance to virus infection, but they are now severely limited with regard to bringing in new cultivars, as well as selling potted plants. In addition, shipments of orchids from overseas are often infected with CymMV, so there is an influx of new CymMV strains to Hawaii. Recent techniques for engineering virus resistance in plants can provide resistance to multiple strains of a plant virus, and we have cloned and sequenced several of the Hawaiian isolates of CymMV. The availability of CymMV-resistant dendrobiums will benefit the orchid cut-flower growers directly, and will also aid the entire orchid industry by reducing the overall numbers of virus-infected plants.
3. Progress Report:
Tomato spotted wilt virus (TSWV) is the most important viral disease on lettuce (Lactuca sativa) in Hawaii, and we have developed transgenic lettuce with resistance to TSWV; this understanding directly contributes to objective 3 of the in-house project. We transferred the TSWV nucleocapsid (N) protein gene to lettuce cv. Grand Rapids using Agrobacterium-mediated transformation. While the primary transformants exhibited early bolting and poor seed set (perhaps due to environmental conditions), the R1 and R2 generations exhibit normal morphology and seed set. The lines we have challenged thus far are of the R2 generation, they have the N gene in a translation-competent context and they are all exhibiting good resistance. We are using a locally-obtained TSWV isolate to conduct the challenge experiments. We are currently determining copy number and zygosity of the resistant transgenic plants. TSWV infection on susceptible lettuce (including our nontransgenic controls) normally produces numerous necrotic lesions at 4 or 5 days post-inoculation, followed by a systemic infection evidenced by systemic veinal necrosis, and finally collapse and death of the infected plant. Transgenic resistance conferred by the TSWV N gene in our challenges resulted in a >90% reduction in lesion numbers, lack of systemic infection, and a recovery with subsequent normal plant development. While the plants described above have a translatable N gene expressed via the CaMV 35S promoter, we also have lettuce containing the N gene expressed in both sense and antisense contexts, and expressed via the tomato ubiquitin promoter, but those plants have not yet been challenged. We also have transgenic Nicotiana benthamiana plants with these constructs, as well as constructs containing the TSWV nonstructural proteins Nss and Nsm. Romaine lettuce production in Hawaii is also affected by TSWV, so we are attempting to develop TSWV-resistant Romaine lettuce using the TSWV N gene constructs that were effective in lettuce cv. Grand Rapids. We have evaluated three Romaine transformation protocols thus far and have not yet developed an efficient protocol for transforming Romaine lettuce. We have a single small plant that has survived antibiotic selection and we are currently determining whether this plant is transgenic. Orchid production is another significant Hawaiian crop greatly affected by plant viruses. The most important orchid viruses are cymbidium mosaic virus (CymMV) and odontoglossum ringspot virus (ORSV), with CymMV being more prevalent in Hawaii. We recently conducted a survey and found that cut-flower farms have a very high (>85% of plants) rate of virus infection while potted plant farms have much lower infection rates, (<5%). Both CymMV and ORSV are mechanically-transmitted viruses and cut-flower farms tend to have much older plants and flower-harvesting activity greatly facilitates virus spread. At the request of local orchid growers, we are examining protocols for disinfesting harvesting tools between plants for two reasons. Firstly, because a widely-used disinfectant is being discontinued, and second, that same disinfectant hasn’t been very effective in mitigating virus spread in cut-flower farms. A relatively common practice in Hawaii is hybridizing and selecting orchids in Hawaii, followed by tissue-culture multiplication in overseas labs. The resulting imported tissue-cultured plants sometimes arrive back in Hawaii infected by viruses acquired overseas. By cloning and sequencing some of these isolates, we have determined that these are exotic strains introduced to Hawaii as a result of tissue-cultured propagation in overseas laboratories. Some of these isolates carry coat protein gene mutations that render them undetectable with common serology-based virus detection kits. We have advised Hawaiian orchid growers to incorporate PCR-based testing in addition to serology-based testing when receiving material propagated overseas. Importantly, we have advised growers to perform comprehensive testing (>5 tissue samples per plant, tested with serological assay in addition to PCR) when selecting material for mass propagation by tissue culture.