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Rebecca Grumet

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.

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Daniel Keifenheim and Cindy Tong*

Anthocyanins are a class of flavonoids that are responsible for pigments in flowers, fruit, and potato periderm. Developing `Norland' potatoes synthesize anthocyanins in periderm tissue when the tubers are mere swollen stolon tips. As the tubers enlarge, anthocyanin accumulation seems to stop, and anthocyanins synthesized early in development seem to be diluted as the tubers enlarge. Expression of dihydroflavonol reductase (DFR) limits anthocyanin synthesis in grape and maize fruit, and in petunia and snapdragon flowers. However, DFR expression in periderm tissue occurred throughout tuber development (Hung et al., 1999). To determine if expression of late anthocyanin pathway genes limit anthocyanin synthesis in developing potato tubers, we performed RNA gel blot analyses. Expression of leucoanthocyanidin dioxygenase and UDP glucose: flavonoid 3-O-glucosyl transferase was observed in swollen stolon tips but not in periderm of later tuber development stages. Surprisingly, expression was also observed in cortex tissue, although that tissue remained white throughout tuber growth.

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Rasika G. Mudalige-Jayawickrama, Michele M. Champagne, A. David Hieber, and Adelheid R. Kuehnle

Two full-length cDNA clones, Den-CHS-4 and Den-DFR-1, encoding chalcone synthase (CHS) and dihydroflavonol 4-reductase (DFR) were obtained from flower bud RNA of a lavender cyanidin-accumulating Dendrobium Sw. hybrid using reverse transcription-polymerase chain reaction (RT-PCR). Northern analyses indicated that both genes are expressed in all developmental stages of buds, with highest expression in the medium-sized buds. RT-PCR analyses showed that DFR expression was confined to floral tissue while CHS was expressed in floral and vegetative tissues but not in pseudobulbs. The nucleotide sequence of a DFR clone isolated from a pale orange pelargonidin-accumulating Dendrobium hybrid was exactly the same as Den-DFR-1, ruling out the substrate specificity of DFR as a possible cause of the color difference.

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K.S. Ling, C.A. Clark, C. Kokkinos, J. R. Bohac, S.S. Hurtt, R. L. Jarret, and A. G. Gillaspie

Sweet potato virus disease (SPVD) is the most devastating virus disease on sweetpotato [Ipomoea batatas (L.) Lam] world wide, especially in East Africa. However, weather it is present in the U.S. is unknown. SPVD is caused by co-infection of sweetpotato feathery mottle virus (SPFMV) and sweetpotato chlorotic stunt virus (SPCSV). Presence of two other potyviruses, sweetpotato virus G (SPVG) and Ipomoea vein mosaic virus (IVMV) has also been confirmed in the U.S. Sweet potato leaf curl virus (SPLCV), a whitefly (Bemisia tabaci) transmitted Begomovirus, also has the potential to spread to commercial sweetpotato fields and poses a great threat to the sweetpotato industry. The U.S. collection of sweetpotato germplasm contains about 700 genotypes or breeding lines introduced from over 20 different countries. Newly introduced sweetpotato germplasm from foreign sources are routinely screened for major viruses with serology and graft-transmission onto indicator plants (Ipomoea setosa). However, a large portion of this collection including heirloom cultivars or old breeding materials has not been systemically screened for these major sweetpotato viruses. In this study, a total of 69 so-called heirloom sweetpotato PI accessions were evaluated for their virus status. We used Real-time PCR to detect five sweetpotato viruses, including four RNA viruses (SPCSV, SPFMV, SPVG, and IVMV) and one DNA virus (SPLCV). A multiplex Real-time RT-PCR system was developed to detect three RNA viruses (SPFMV, SPVG, and IVMV). Preliminary data indicated that about 15% of these heirloom sweetpotato germplasm carried at least one of these viruses tested. Details on virus infection status will be presented.

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A.Q. Villordon, C.A. Clark, R.A. Valverde, R.L. Jarret, and D.R. LaBonte

Previous work by our group has detected the presence of a heterogeneous population of Ty1-copia-like reverse transcriptase retrotransposon sequences in the sweetpotato genome. Recently, we detected the presence of putatively active Ty1-copia-like reverse transcriptase sequences from a virus-infected `Beauregard' sweetpotato clone. In the current study, we report the differential detection of putatively stress-activated sequences in clones from seedling 91-189. The clones were infected with different combinations of virus isolates followed by extraction of leaf RNA samples at three sampling dates (weeks 2, 4, and 6) after inoculation. After repeated DNAse treatments to eliminate contaminating DNA, the RNA samples were subjected to first strand cDNA synthesis using random decamer primers followed by PCR analysis utilizing Ty1-copia reverse transcriptase-specific primers. Through this approach, we detected amplified fragments within the expected size range (280-300 bp) from clones infected with isolates of sweetpotato leaf curl (SPLC) and feathery mottle viruses (FMV) (week 2 and 6) and FMV (week 4). We were unable to detect PCR products from the noninfected clones or the other infected samples. The data suggests that specific viruses may be involved in the expression of these Ty1-copia-related reverse transcriptase sequences. It also appears that sampling at various dates is necessary to detect putative activity over time. This preliminary information is essential before proceeding to the construction and screening of cDNA libraries to isolate and fully characterize the putatively active sweetpotato Ty1-copia-like retrotransposon sequences. Through the partial or complete characterization of sweetpotato Ty1-copia elements, sequences that correspond to cis-regulatory element(s) can be identified and further studied for their roles in responding to specific stress factors.

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Keizo Yonemori, Junya Yoshida, Ayako Ikegami, Akihiko Sato, Masahiko Yamada, and Akira Kitajima

Pollination-constant and non-astringent (PCNA)-type persimmon has probably originated from astringent (non-PCNA)-type as a mutant that terminates condensed tannin accumulation at an early stage of fruit development. This trait is confirmed to be recessive and is controlled by a single locus. Since PCNA-type fruit stops tannin accumulation at an early stage, comparison of the gene expressions between PCNA- and non-PCNA-type will reveal the genes conferring condensed tannin accumulation in persimmon fruit. We performed suppression subtractive hybridization (SSH) analysis for detecting differentially expressed genes in non-PCNA-type fruit using BC1 offspring from a cross between PCNA `Fuyu' and non-PCNA “275-13” (F1 progeny derived from non-PCNA `Aizumishirazu' × PCNA `Taishu'). Fruits from seven individuals of PCNA or non-PCNA offspring in BC1 were sampled at early two stages of fruit development and total RNA was extracted by hot borate method from each fruit of different stage. Then, RNA was pooled as PCNA or non-PCNA bulk at two stages and cDNA was synthesized from each bulk for SSH analysis. A total of 5000 clones expressed differentially in non-PCNA-type fruit were picked from SSH library of two stages and 198 positive clones confirmed by differential screening were sequenced. The homologous sequences for the genes involved in flavonoid biosynthesis (CHS, CHI, F3H, F3'5'H, DFR, UFGT, and ANS) were obtained from the clones. The genes that are not considered to be involved in flavonoid biosynthesis so far (SCPL and DHQ) were also detected with high frequencies. We will discuss the role of these genes for condensed tannin accumulation in persimmon fruit.

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Carol M. Foster and William R. Graves

Desiccation damage in ornamental plant species is of particular concern to the nursery and landscape industry. Species in two genera, Acer and Alnus, display fundamental differences in how drought affects leaves. The same soil moisture content that causes foliar desiccation and abscission in Alnus maritima (Marsh.) Nutt. (seaside alder) causes neither response in Acer rubrum L. (red maple). Understanding molecular mechanisms associated with plant response to drought stress can be an im portant factor in developing strategies for improved sustainability in urban landscapes. Our objective was to characterize expression of drought-induced dehydrin genes in leaves of `Red Sunset' red maple (desiccation-resistant) and seaside alder (desiccation-sensitive) in response to dehydration and rehydration. Potted cuttings grown in a glasshouse were subjected to four cycles of drought and rehydration. Stomatal conductance and volumetric moisture content of rooting medium were used to determine when drought cycles ended. During the second and fourth cycles, leaves were sampled for RNA and protein extraction. Dehydrin probes were generated from genomic DNA of both species by using PCR with primers designed from conserved regions in dehydrin genes. Southern blot analyses revealed the presence of dehydrin genes in seaside alder and red maple genomes. Reverse transcriptase (RT)-PCR was used to isolate desiccation-induced dehydrin cDNAs from total RNA extracted from drought-stressed leaves. The cDNA clones show 61% to 66% identity at the nucleic acid level with dehydrin genes of soybean, sunflower, radish, and potato. Accumulation of dehydrin transcripts and proteins in leaves in response to dehydration and rehydration are being studied through northern and western blot analyses, respectively. Our results may lead to a rapid screening technique for seedlings with improved mechanisms of drought resistance.

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Binoy Babu, Gary Knox, Mathews L. Paret, and Francisco M. Ochoa-Corona

single-stranded negative-sense RNA virus ( Laney et al., 2011 ). The virus is transmitted by the eriophyid mite species Phyllocoptes fructiphilus ( Amrine et al., 1988 ; Laney et al., 2011 ) and by grafting ( Amrine et al., 1988 ). The mites do not fly

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Carole Bassett, Ann Callahan, and Linda Dunn

sequence information in our comparisons and Richard Meagher for kindly providing the soybean 18S RNA probe. Use of a trade name or trade mark does not imply endorsement to the exclusion of other, similar products. The cost of publishing this paper was

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M. Pilar Bañados, Gary D. Coleman, and Tony H. H. Chen

In poplar (Populus deltoides) a 32kDa bark storage protein (BSP) accumulates during the fall, and is a major form of stored nitrogen during overwintering. This protein is induced by short-day (SD) photoperiod and may play an important role in nitrogen cycling in the plant. To determine the effect of plant nitrogen status upon BSP gene expression, poplar plants were grown in controlled environmental chambers under either SD or long-day (LD) photoperiods and watered with either 5, 10, 50, and 100 mM NH4NO3 for four weeks. [15N]-NH4NO3 was applied during the first and third weeks. SDS-PAGE and western blot analysis were used to detect the relative amounts of BSP. RNA gel blot analysis was used to determine the changes in BSP gene expression. BSP accumulation was enhanced by increasing levels of nitrogen under both photoperiods, however, SD photoperiod appears to moderate the response. These results indicate that BSP gene expression is dependant upon the nutritional status of the plant. [15N] analysis will also be presented.