We have shown that high-level resistance to plum pox virus (PPV) in transgenic plum clone C5 is based on post-transcriptional gene silencing (PTGS), otherwise termed RNA silencing (Scorza et al. Transgenic Res. 10:201-209, 2001). In order to more fully characterize RNA silencing in woody perennial crops, we investigated the production of short interfering RNA (siRNA) in transgenic plum clones C3 and C5, both of which harbor the capsid protein (CP) gene of PPV. We used as a control, plum PT-23, a clone only transformed with the two marker genes, NPTII and GUS. We show in the current report that C5 constitutively produces two classes of siRNA, the short (21-22 nucleotides) and long (≈27 nucleotides) species in the absence of PPV inoculation. Transgenic susceptible clone C3 and the control clone PT-23, when healthy, produce no siRNA. Upon infection, these clones produce only the short siRNA (21-22 nt). This siRNA production suggests that plum trees naturally respond to virus infection by initiating PTGS or PTGS-like mechanisms. This study also suggests that high-level virus resistance in woody perennials may require the production of both the short and long size classes of siRNA, as are produced by the resistant C5 plum clone.
Jean-Michel Hily, Ralph Scorza*, and Michel Ravelonandro
William R. Woodson
The target of many genetic engineering experiments is to inhibit the expression of an endogenous gene. For example, research in my laboratory attempts to suppress the expression of ethylene biosynthetic pathway genes to inhibit the production of ethylene and delay flower senescence. The silencing of endogenous genes is generally accomplished by engineering plants to express either antisense or sense RNAs homologous to the target sequence. The mechanism by which gene silencing occurs is not clearly understood. Genetic and molecular analyses of transgene-induced silencing has revealed both meiotically reversible and fully stable phenotypes resulting from the expression of the transgene. In several cases, the mechanisms potentially involved in the silencing of the transgene and concomitant reversion of phenotype have been studied. These include transgene copy number, configuration of the integrated DNA, level of transgene RNA, and environmental factors. In many cases the silencing of transgenes was correlated with DNA methylation. These phenomena and the implications for engineering horticultural crops to express transgenes will be discussed in this workshop.
Hyo-Won Seo, Jung-Yoon Yi, Young-Il Hahm, Hyun-Mook Cho, and Kuen Woo Park
Three potato (Solanum tuberosum L.) cultivars `Superior', `Irish Cobbler', and `Jopung' were transformed by co-cultivation with tuber discs and disarmed Agrobacterium tumefaciens LBA4404 carrying modified vector pBI121, that contained PLRV coat protein (CP) gene and controlled by CaMV35S promoter. Putative transformants were selected and their genomic DNA and RNA transcripts were analyzed for the confirmation of genetic stability by RT-PCR, PCR, southern, and northern blot. The growth characteristics and viral resistance of progenies of transgenic potato plants were investigated. Twelve lines among the different seven-times manipulated transgenic lines were grown in greenhouse and isolates trial field. PLRV coat protein gene was stably inherited in `Superior', but not in `Jopung'. `Jopung' was less stable than `Irish Cobbler' and `Superior' at genetic stability of PLRV CP gene. And some of these transgenic lines were highly resisted in PLRV multiplication. The yield of transformants was reduced in `Irish Cobbler' but not in `Superior'. Possible explanations for these types of resistance are gene silencing and positional effects of transformed PLRV CP genes and that had cultivar specificity. We consider the appearance of escaped transformants in `Jopung' for emergence of chimeric explants from early selection stage.
Gojko Jelenkovic, Sharon Billings, Qi Chen, James Lashomb, George Hamilton, and Gerald Ghidiu
A population of 300 putative transgenic eggplants (Solanum melongena L.) carrying the syn cryIIIA gene was produced and tested for resistance to the Colorado potato beetle [CPB; Leptinotarsa decemlineata (Say)]. Toxicity tests in planta and in vitro demonstrated that 69% of the transformed plants were resistant to neonate larvae and adult CPB. Transgenicity of the plants was confirmed by studies of GUS expression and Southern and northern analysis. Primary transformants, having a single insert of the construct, upon selfing, produced progenies cosegregating for the uidA and syn cryIIIA genes at the expected 3:1 ratios with a few exceptions in which only one of the genes was expressed. The latter was attributed to the gene silencing phenomenon. The segregating resistant R1 seedlings showed the same level of resistance as the parental genotypes in growth chamber tests and under field conditions. One genotype carrying two copies of the construct, upon selfing, segregated at a 15:1 ratio for GUS expression and resistance to CPB, while Southern analysis revealed a 9:3:3:1 genotypic segregation ratio for individual copies of the construct.
Kullanart Obsuwan, Wayne B. Borth, John Hu, and Adelheid R. Kuehnle
A Cymbidium mosaic virus movement protein gene with a site-specific mutation (mut11) under control of a ubiquitin promoter was inserted using biolistics into two Dendrobium varieties with the intention of creating CymMV-resistant orchids. Presence of the transgene in regenerated plants of D. × Jaquelyn Thomas `Uniwai Mist' and D. x Jaq–Hawaii `Uniwai Pearl' was confirmed by PCR using genomic DNA, and mut11-positive plants were potted ex vitro. Forty-two transgenic plants and four non-transgenic control plants at the 4- to 6-leaf stage were inoculated with a 1:1000 dilution of CymMV obtained from infected orchids. Plants were analyzed for systemic infection using tissue blot immunoassay (TBIA). Seventeen plants from at least six independent transformations remained virus-free, whereas all control plants were infected with CymMV within 1 month. Further analysis by RT-PCR showed that the mut-11 mRNA was detectable in only two of these 17 plants. All plants were challenged again with a second CymMV inoculation as above, followed by TBIA analysis after 1 month. Thirteen of 17 plants remained free from virus. A third challenge of these plants with CymMV as above was followed by TBIA analysis at 1 week, 2 weeks, 1 month, 3 months, 6 months, and 12 months after challenge. Results at 2 weeks post-inoculation showed that all six controls and four individual transgenic plants, including the RT-PCR-positive plants, became systemically infected. Nine transgenic plants from both varieties remained free from CymMV 12 months after the third challenge. Lack of detectable mut11 mRNA in these resistant lines suggests that a post-transcriptional gene silencing (PTGS) mechanism may be conferring resistance to CymMV.
Elysia K. Krieger, Edwards Allen, Larry A. Gilbertson, James K. Roberts, William Hiatt, and Rick A. Sanders
an antisense transgene can trigger RNAi ( Fagard and Vaucheret, 2000 ; Stam et al., 1997 , 2000 ; Watson et al., 2005 ), and there are cases where cassettes lacking promoters induce gene silencing in plants with T-DNAs linked the same way we show
Hiroko Sato, Tadashi Takamizo, Tsutomu Shimizu, Kiyoshi Kawai, and Koichiro Kaku
plants, low copy of transgene was integrated, whereas more than four integrated genes were observed in the susceptible plants ( Fig. 2 ). All resistant plants with a single transgene in T 1 progenies did not exhibit apparent gene silencing ( Fig. 6
Christopher C. Dickinson, Alexandra J. Weisberg, and John G. Jelesko
viral induced gene silencing) and genome-editing methods (e.g., zinc finger, TALEN, or CRISPR-CAS9) to enable molecular genetic investigations of poison ivy ecophysiology and metabolism. Literature Cited Abe, I. Utsumi, Y. Oguro, S. Noguchi, H. 2004 The
Hai-nan Liu, Jian-rong Feng, Xiao-fang Liu, Wen-hui Li, Wen-juan Lv, and Ming Luo
( Freiman et al., 2012 ; Jiang et al., 2013 ; Liu et al., 2010 ). The efficacy of gene silencing is highest when the RNAi expression vectors contain ihpRNA ( Wesley et al., 2001 ). However, the traditional process for constructing ihpRNA is complicated and
Ralph Scorza, Michel Ravelonandro, Ann Callahan, Ioan Zagrai, Jaroslav Polak, Tadeuz Malinowski, Mariano Cambra, Laurene Levy, Vern Damsteegt, Boris Krška, John Cordts, Dennis Gonsalves, and Chris Dardick
, M. 2004 Stability of gene silencing-based resistance to Plum pox virus in transgenic plum ( Prunus domestica L.) under field conditions Transgenic Res. 13 427 436 Jefferson, R.A. 1987 Assaying chimeric genes in plants: The GUS gene fusion system