One of the first major successes in the genetic engineering of useful traits into plants has been the engineering of virus resistance. The first example of genetically-engineered virus resistance was published in 1986, since then there have been more than 50 reports of genetically engineered plant virus resistance. These examples span a range of virus types, a variety of plant species, and have utilized several different types of genes. A unique feature of the genetically-engineered virus resistance is that the resistance genes came from the virus itself, rather than the host plant. Most examples have utilized coat protein genes, but more recently, replicase-derived genes have proved highly effective. Other strategies include the use of antisense or sense-defective sequences, and satellite or defective interfering RNAs. This talk will provide an overview of the different approaches, possible mechanisms, the crops and viruses to which they have been applied, and progress toward commercial applications.
Kaori Ando* and Rebecca Grumet
Phytophthora capsici fruit rot is an increasingly serious disease affecting cucumber production throughout the Eastern U.S. The absence of genetically resistant cultivars and rapid development of fungicide resistance makes it imperative to develop integrated disease management strategies. Cucumber fruits which come in direct contact with the soil-borne pathogen are usually located under the canopy where moist, warm conditions favor disease development. We sought to examine whether variations in plant architecture traits that influence canopy structure or fruit contact with the soil make conditions less favorable for disease development. As a `proof of concept' to test whether an altered canopy could facilitate P. capsici control, we tested the effect of increased row spacing and trellis culture on disease occurrence in the pickling cucumber `Vlaspik.' Trellis plots indicated that removal of fruit contact from soil reduced disease occurrence. Currently available variation in plant architecture was tested using nearly-isogenic genotypes varying for indeterminate (De), determinate (de), standard leaf (LL), and little leaf (ll) traits. Although differences were observed in peak mid-day temperatures under the different canopies, there were not differences in disease occurrence among the genotypes. A collection of 150 diverse cucumber accessions identified to serve as a representative sample of the germplasm, was observed for possible variation in plant architecture. Variation was observed for an array of traits including main stem length, internode length, leaf length and width, and number of branches. Interesting types that may allow for more open canopies include reduced branching habit and compact/bushy growth.
Kaori Ando and Rebecca Grumet
Fruit development proceeds from cell division to expansion, maturation, and ripening. Expansion is critical for size, yield, and quality; however, this period of development has received little attention. We used 454-pyrosequencing to develop a cucumber (Cucumis sativus) fruit transcriptome, identify highly expressed transcripts, and characterize key functions during exponential fruit growth. The resulting 187,406 expressed sequence tags (ESTs) were assembled into 13,878 contigs. Quantitative real-time polymerase chain reaction (qRT-PCR) verification of differentially expressed genes from fruit of different ages, and high correlation in transcript frequency between replicates, indicated that number of reads/contig reflects transcript abundance. Putative homologs were identified in Arabidopsis thaliana for 89% of the contigs represented by at least 10 ESTs; another 4% had homologs in other species. The remainder had homologs only in cucurbit species. The most highly expressed contigs were strongly enriched for growth (aquaporins, vacuolar ATPase, phloem proteins, tubulins, actins, cell wall-associated, and hormone-related), lipid, latex, and defense-related homologs. These results provide a resource for gene expression analysis in cucumber, profile gene expression in rapidly growing fruit, and shed insight into an important, but poorly characterized, developmental stage influencing fruit yield and quality.
Kaori Ando and Rebecca Grumet
Fruit rot induced by Phytophthora capsici Leonian is an increasingly serious disease affecting pickling cucumber (Cucumis sativus L.) production in many parts of the United States. The absence of genetically resistant cultivars and rapid development of fungicide resistance makes it imperative to develop integrated disease management strategies. Cucumber fruit which come in direct contact with the soil-borne pathogen are usually located under the canopy where moist and warm conditions favor disease development. We sought to examine whether variations in plant architecture traits that influence canopy structure or fruit contact with the soil could make conditions less favorable for disease development. As an extreme test for whether an altered canopy could facilitate P. capsici control, we tested the effect of increased row spacing and trellis culture on disease occurrence in the pickling cucumber `Vlaspik'. Temperature under the canopy was lowest in trellis plots, intermediate in increased spacing plots, and highest in control plots. Disease occurrence in the trellis plots was significantly lower than in other treatments, indicating that preventing fruit contact with the soil reduced disease occurrence. The effect of currently available variation in plant architecture was tested using nearly-isogenic genotypes varying for indeterminate (De), determinate (de), standard leaf (LL), and little leaf (ll) traits. Plants with standard architecture had higher peak mid-day temperatures under the canopy and greater levels of P. capsici infection; however, levels of disease occurrence were high for all genotypes. Screening a collection of ≈150 diverse cucumber accessions identified to serve as a representative sample of the germplasm, revealed variation for an array of architectural traits including main stem length, internode length, leaf length and width, and number of branches; values for `Vlaspik' were in the middle of the distribution. Plant architectures that may allow for more open canopies, including reduced branching habit and compact growth, were tested for disease incidence. One of the compact lines (PI 308916), which had a tendency to hold young fruit off the ground, exhibited lower disease occurrence. The reduced disease occurrence was not due to genetic resistance, suggesting that architecture which allows less contact of fruit with the soil could be useful for P. capsici control for pickling cucumber.
Guowei Fang and Rebecca Grumet
Zucchini yellow mosaic virus (ZYMV), a potyvirus, can cause major losses in cucurbit crops. With the goal of genetically engineering resistance to this disease we have engineered the ZYMV coat protein gene into a plant expression vector. The complete coat protein coding sequence, or the conserved core portion of the capsid gene, was attached to the 5' untranslated region of tobacco etch virus (TEV) in the pTL37 vector (Carrington et al., 1987, Nucl. Acid Res. 15:10066) The capsid constructs were successfully expressed by in vitro transcription and translation systems as verified by SDS-PAGE and ZYMV coat protein antibody. The constructs were then subcloned using polymerase chain reaction and attached to the CaMV 35 S transcriptional promoter on the CIBA-GEIGY pCIB710 plasmid. The constructs containing the CaMV 35S promoter, the 5' untranslated leader of TEV, and ZYMV coat protein sequences were then put between the Agrobacterium tumefaciens left and right borders in the pCIB10 vector and transferred to A. tumefaciens strain LBA4404 by triparental mating. These vectors are now being used to transform muskmelon and cucumber; resultant transgenic plants will be tested for ZYMV coat protein expression.
Rebecca Grumet and Robert Duvall
The determinate allele (de) of cucumber (Cucumis sativus L.) causes shorter vine length and fewer and shorter internodes and lateral branches than the indeterminate allele (De). Four sets of cucumber inbreds carrying determinate or indeterminate alleles were compared with respect to root growth rate in the field and greenhouse using an herbicide banding method. Although the lines exhibited the expected differences in shoot growth, differences in root growth were not correlated with the differences in shoot growth. These results indicate that root growth was independent of the determinate shoot growth allele.