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.
Eileen Kabelka and Rebecca Grumet
Potyviruses cause severe loss in cucurbit crops. Inbred lines derived from the Chinese cucumber cultivar, Taichung Mau Gua (TMG), have been identified to be resistant to several potyviruses including zucchini yellow mosaic virus (ZYMV), zucchini yellow fleck virus, watermelon mosaic virus, and the watermelon strain of papaya ringspot virus. Recently, an additional virus that infects cucurbits, the Moroccan watermelon mosaic virus (MWMV), has been identified to be a distinct member of the potyvirus group. In this study, we sought to determine if TMG-1 is resistant to MWMV and, if so, examine whether a relationship exists between resistance to MWMV and resistance to ZYMV. Progeny analyses show that TMG-1 is resistant to MWMV and, like resistance to ZYMV, MWMV resistance is conferred by a single recessive gene. Sequential inoculation of progeny possessing resistance to ZYMV followed by MWMV (or MWMV followed by ZYMV) suggests that both resistances are conferred by the same gene, or two tightly linked genes. Additionally, all F3 families derived from F2 individuals selected for resistance to ZYMV, were resistant to MWMV. A second source of resistance to ZYMV, allelic to the TMG-1 source, has been incorporated into the Dutch hybrid Dina. Progeny analyses show Dina to posses a single recessive gene for MWMV resistance. As with TMG-1, no segregation of resistances was observed when ZYMV resistant progeny were inoculated with MWMV (or MWMV followed by ZYMV). Collectively, these results suggest that a single gene, or two tightly linked genes, control resistance to the potyviruses ZYMV and MWMV.
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.
Kaori Ando and Rebecca Grumet
Phytophthoracapsici, a soil-borne oomycete pathogen causing fruit rot in cucumber, has become a limiting factor for cucumber production in the Midwest. In the process of screening plant introductions (PIs) for resistance to P. capsici, it appeared that degree of susceptibility might decrease as fruits develop. To examine this more carefully, detached, greenhouse-grown, hand-pollinated `Vlaspik' fruits aged 2–18 days post-pollination (dpp) were inoculated with P. capsici mycelium and evaluated for symptoms. There was a reproducible decrease in susceptibility with increasing fruit age. The fruits that sporulated were usually younger and smaller (2–9 dpp), 10-3 dpp fruit tended to develop water-soaked symptoms, while the fruits that remained symptom-free were usually older (>14 dpp) and oversized for pickling cucumber. The transition from susceptible to more resistant appeared to occur at the end of the period of rapid fruit elongation. Detached field-grown `Straight Eight' fruits showed similar size-related trends. Candidate resistant genotypes identified from the PI screening were re-screened using 7 and 14 dpp fruits. Again an age-dependent difference in response was observed, indicating that the increase in resistance is not genotype-specific. Furthermore, field observations suggest a gradation of susceptibility within the fruits as the blossom end was most frequently infected. Preliminary tests of detached greenhouse-grown, hand-pollinated fruits suggested that as the fruits grew older, the blossom end remained susceptible longer than the stem end. These findings could have implications for appropriate screening methods, the stage of fruit likely to become infected in the field, and appropriate spray practices.
Eileen Kabelka and Rebecca Grumet
Cucumbers and other cucurbit crops are subject to severe losses due to an array of potyviruses, including zucchini yellow mosaic virus (ZYMV), watermelon mosaic virus (WMV), and the watermelon strain of papaya ringspot virus (PRSV-W). Sources of resistance to these viruses have been identified within the cucumber germplasm, including resistance to ZYMV, WMV, and PRSV in `TMG-l'; resistance to PRSV in `Surinam'; and to ZYMV in `Dina'. In this study, we sought to determine the allelic relationships between resistance to PRSV in `Surinam' and `TMG' and resistance to ZYMV in `Dina' and `TMG'. Segregation ratios among F1, F2, and backcross progeny of `Surinam' and `TMG' indicated that the alleles are at the same locus. Similarly, progeny analysis indicates that the alleles for ZYMV resistance in `Dina' and `TMG' are at the same locus. In each case, however, the alleles appear to differ from one another with respect to dominance relationships, symptom expression, and/or response to different viral strains. We are further characterizing these differences by screening progeny of crosses to a common susceptible parent.