Viral resistance using RNA homology-dependent silencing has been successfully engineered into many plant systems. Single or multiple viral-derived transgenes can be expressed in plants leading to RNA homology-dependent silencing and subsequent viral resistance. The use of this transgenic technology may be particularly effective in thwarting viral diseases where little or no genetic resistance has been identified. Resistance to rice yellow mottle virus is one example where traditional breeding cannot be used for improvement due to fertility barriers and genetic resistance being a poorly defined polygenic trait.The resultant RYMV resistant lines carried very low or non-detectable amounts of the ORF2 RNA transcript. Conversely,vertical grow towers transgenic lines that were susceptible had abundant amounts of the ORF2 transcript. Therefore the resistance phenotype was correlated with the loss of the viral transgene expression. This indicates that the mechanism of resistance was due to silencing of the ORF2 present as the transgene and in the RYMV RNA genome. The ORF2 sequence variation among different RYMV field isolates was found to be less than 10% at the nucleotide level suggesting that an RNA homology-dependent silencing approach may be effective in the field .
Viral resistance utilizing endogenous silencing mechanisms is not restricted to using a single open reading frame from one virus. Two ORF fragments from different viruses can be fused into a chimeric expression cassette to confer resistance to both viruses. One clear example was generated from using tomato spotted wilt virus and turnip mosaic virus . The open reading frame for the N gene encoding the nucleocapsid from TSWV was fused to the coat protein of TuMV and the resulting chimeric construct was used to transform tobacco. As with the example using RYMV,resistance of the transgenic plants to both viruses corresponded with the loss of transcript accumulation from both viruses as detected by northern analysis. Transgenic plants susceptible to both viruses showed accumulation of the gene fragment transcript for both viruses. These two examples have been evaluated in greenhouse experiments; however, a well-described example of RNA homology-dependent silencing for viral resistance is presently being utilized successfully in the field.One clear commercial success of generating enhanced resistance by stable expression of a viral gene is against the papaya ring spot virus . Papaya is grown throughout the tropics and subtropics and no natural resistance has been described for PRSV. A PRSV control strategy for the Hawaiian islands was developed using RNA homology-dependent silencing by expressing a mutated open reading frame for the coat protein from PRSV . Resistant transgenic plants were generated and were found to be devoid of the CP RNA indicating the RNA homology-dependent silencing of the plant-derived transgene and PRSV gene .
All PRSV strains present in Hawaii have been effectively controlled using silencing constructs derived from this mutant CP ORF. Sequence analysis demonstrated that these Hawaiian isolates had 97% or greater sequence homology to the mutant CP transgene. However, isolates of PRSV from outside of Hawaii can cause disease on the transgenic papaya lines. These geographically distinct isolates were found to have a lower sequence homology to the CP than the isolates from Hawaii. Thus, silencing of PRSV was contingent upon levels of sequence homology above 97% . Interestingly, PRSV and RYMV require different levels of homology between transgene and the endogenous gene to induce silencing. The silencing in RYMV was successful for all variations tested as compared with less than 3% divergence allowed for successful silencing in PRSV. Silencing is not only dependent upon the degree of homology but also the target sequence that is chosen. Much like the transgenic approach with R genes, each silencing construct must be carefully validated. Overall, RNA homology-dependant silencing has proven its utility in both the greenhouse and the field, and appears to be among the most versatile mechanisms currently available to engineer resistance to viruses.Crown-gall is a perennial problem in nurseries of fruit trees, nut trees and some bushy ornamental plants. Prevention of gall formation is a target for engineering resistance in these trees since breeding programs for resistance are not practical due to temporal considerations . When replanted, the trunks suffer cuts that are an entry point for the bacterium Agrobacterium tumefaciens, the causal agent of the disease, and infection becomes apparent with the formation of galls.
The bacterium causes disease by transforming the host cell with sets of oncogenes leading to uncontrolled cell division. These oncogenes encode biosynthetic genes for the production of plant hormones auxin and cytokinin. The endogenous plant genes and transferred oncogenes share no sequence homology making the bacterial genes an ideal target for RNA homology-dependent silencing . Arabidopsis and tomato plants were transformed with constructs containing direct inverted repeats of the auxin and cytokinin oncogenes . In planta, these tandem inverted repeats generate dsRNA molecules that in turn induce RNA homology-dependent silencing of the transformed Agrobacterium oncogenes. In the resulting transgenic tomato and Arabidopsis, Agrobacteriummediated transformation was not prevented but the formation of galls by oncogene expression was abrogated completely. This was confirmed by the lack of detectable RNA from the bacterial oncogenes. The transgenic plants did not show any developmental phenotypic variation indicating that endogenous hormone production was not altered by the presence of the silencing construct .Horticultural research is conducted primarily in the public sector, with research at private institutions playing a relatively minor role. As a result, research gaps naturally emerge between the basic research generated by public institutions and the research needs of industry. One approach for reducing this gap is to form public-private research partnerships that harness the complementary research and academic expertise of universities with the commercialization and marketing expertise found in industry. Such partnerships are proliferating, especially between universities and large life-sciences companies. Unfortunately, there are few concrete examples of such partnerships in agricultural biotechnology for the horticulture industry. The challenge is to adapt models of these partnerships to the research needs and structure of the horticulture industry, which produces crops such as fruits and vegetables, nuts, and nursery and ornamental crops. The traditional research paradigm posits a one-way flow from basic science conducted in public institutions to applied research and commercialization undertaken largely by private industry. This characterization does not accurately portray current trends in research and development .
Increasingly, public universities and private firms engage in joint research and establish interactive relationships. Several factors have contributed to this trend, including recent legislation , the restructuring of many of the larger life-sciences firms and an alignment of private and public incentives to pursue long-term R&D efforts . The potential benefits from university industry partnerships in the field of agricultural biotechnology are obvious. Scientific and practical knowledge can complement each other, leading to more rapid and far-reaching innovation. Universities need funding for their researchers, as well as intellectual property held by private companies and access to modern, commercially developed enabling technologies to ensure a first-rate graduate education for students. For its part, industry is interested in accessing new research and innovation, developing new products and hiring highly trained graduate students. However, obstacles to the formation of successful agreements are significant. Both parties in a research partnership face serious risks. These risks are rooted in the conflict between a university’s academic objectives and the private firm’s corporate incentives. One critical risk is the potential co-opting of the academic research agenda by private interests. University researchers risk the loss of academic freedom and integrity while industry risks the loss of investment capital,vertical growing racks privacy and proprietary information. Differences between the university’s educational objectives and corporate goals, as well as differences in the cultures, institutional incentives and time frames, can lead to a clash of cultures and values. Intellectual property rights issues are also a frequent source of contention. Given these risks, both parties need to enter into carefully structured research agreements.Most work examining research partnerships focuses either broadly, on such issues as the source of research funding, basic provisions of these agreements and associated problems and consequences , or narrowly, on specific aspects of a particular type of agreement . Although this literature is useful, it does not effectively address how to structure these public-private research partnerships. In response to this need, we have constructed templates based on the three stages of any university industry research partnerships, which provide a framework for characterizing their “front-end” and “back-end” options . University-industry research partnerships come in many forms. They may be targeted, with private firms designating specific research agendas, or they may be non-targeted. Research projects may have short or longer time horizons. Universities may enter agreements with a single private company or with groups of firms sharing a common interest . Collaborations may cover a single research project or be “megaagreements” covering a large range of interactions . Because of the inherent uncertainty in the research process, research partnerships can be structured in terms of ex ante decisions on the options embedded in the three stages of any agreement. These embedded options are specific decision points, such as determining which partner will control the research agenda.
Universities can define policies on this option ex ante, before potential partners are approached.Stage I: Setting the bargaining space. To start, potential research partners consider possible collaborations and associated trade offs. The vital aspect of this stage is determining exactly how partners will be identified and selected. Although deliberately seeking out partners rather than waiting to be approached with a proposal requires more effort upfront, it can substantially broaden the set of choices. For example, the public partner could elicit competitive bids from multiple private partners rather than just accepting or rejecting a single proposal. Stage II: Negotiating the agreement. Next, the agreement is negotiated and may or may not involve a formal contract. Front-end options determine the nature and scope of the research activities that the partnership will undertake, while back-end options determine how any benefits generated by the partnership will be distributed and how knowledge assets such as patents and commercial products are disseminated. Decisions in the front-end include specifying the research agenda, asset contributions, governance structures and scale of operations. Back-end options include designating patent filing responsibility, property and licensing rights, royalty rates and how research results will be disseminated. Stage III: Reviewing and renewing the partnership. Finally, the outcome of the partnership is assessed, with an eye toward whether to renew the agreement. Currently, there is no standard approach for formal review of large- or small-scale agreements. To assess whether a research partnership was successful or not, interested parties must rely on the informal reviews and vague impressions of both partners along with more tangible outcomes, such as the number of patents generated by the research. A key policy challenge is the development of concrete indicators or measures of productivity for public-private research partnerships.Based on these stages of forming agreements, we have designated four groups of templates. Strategic partnerships involve comprehensive, multiyear commitments between a university, or an academic department in a university, and a large company, with both partners dedicating significant assets. Formal procedures for determining research agendas and control of back-end assets are specified. Given their size, these agreements tend to come under significant scrutiny and often external review. One such agreement was the 5-year, $25 million research agreement between Novartis and UC Berkeley’s Department of Plant and Microbial Biology. The relationship, which generated approximately 20 innovations, was the subject of an internal campus review by the office of the Vice Chancellor for Research. The review found the research had not been skewed toward applied biotech research as feared and that graduate students were the primary beneficiaries.Research unit/center partnerships usually also involve the dedication of significant resources. Instead of involving existing academic departments, however, these research units are set up separately, allowing more distance between the partnership and the academic community at the university. Such partnerships may be linked to a single company, commodity group or companies that provide some or all of the financial resources for the research center. For example, the Seed Biotechnology Center at UC Davis is a partnership between the College of Agricultural and Environmental Sciences and the California seed industry.