Rice Genetics IV - IRRI books - International Rice Research Institute

Carbofuran (trade name, Furadan) is a highly toxic, systemic carbamate (cholinesteraseinhibitor) insecticide. It is effective in protecting the crop from substantialyield losses, but its toxicity to nontarget organisms in the rice ecosystem is a seriousproblem and its use has been discontinued in the U.S. Although alternative effectivechemical insecticides are now approved, their efficacy against RWW is still beingevaluated and this, combined with a lack of viable traditional sources of resistance tothe RWW, makes biotechnology an attractive option for combating this pest. In collaborationwith the group of John Kemp (New Mexico State University, Las Cruces),we are exploring the use of a codon-optimized version of the Btt CryIIIA crystal toxingene, which is effective against coleopteran insects (Sutton et al 1992).Because of the 4-wk feeding period during which larvae feed on the roots, weinitially biolistically cotransformed rice (Buchholz et al 1998) with p35S-CryIIIA,which uses a 35S promoter to drive expression of the Btt CryIIIA coding region (Fig.1A), and mUbi1/bar (Fig. 1B) as a selectable marker. Resulting plants were designatedJKA lines. Unfortunately, an extensive investigation in our laboratory of foreigngene expression in several hundred transgenic rice plants generated byelectroporation or biolistics revealed that transformants bearing multicopy inserts frequentlydisplayed non-Mendelian segregation (Kumpatla and Hall 1999, Kumpatla etal 1997). For example, characterization of several R 1 progeny revealed that mUbi1/bar and 35S/Btt were extensively methylated and transcriptionally inactivated in thesilenced lines (Fig. 2). The epigenetic modification of the transgene sequences wasfurther confirmed by the reactivation of the bar gene expression in R 2 seedlings (fromthe silenced lines) germinated on medium containing 5-azacytidine (AzaC) (Kumpatlaet al 1997).It appears that, once a locus is sensitized to methylation, it is very prone to methylation.Thus, in an investigation of 34 R 2 plants in which bialaphos resistance wasreactivated by seedling exposure to AzaC, all except one were re-silenced by 50 dafter germination. The single line that retained resistance to maturity is male sterileand yields no transgenic seed when crossed with wild-type pollen. Reestablishmentof silencing was found to correlate in each case with methylation of the ubiquitinpromoter (Kumpatla and Hall 1998a). When following JKA52 progeny of a homozygous,nonsilenced R 1 line, we found that promoter methylation and silencing mayarise in the R 3 generation (Kumpatla and Hall 1998b). The stochastic nature of initiationof methylation is intriguing: in plants grown under identical conditions, all progenyof two lines remained nonmethylated and unsilenced, whereas three other linesgave progeny that were 5% to 40% silenced. It is important to recognize that chromosomallocation does not participate in these differences since all lines studied areprogeny of a single ancestral plant.Gene-specific silencingAre some promoters especially prone to silencing?In early investigations of monocot transformation, comparison was made betweenthe efficacy of using circular and linearized forms of plasmid (Battraw and Hall 1991).Gene silencing and its reactivation in transgenic rice 467