Post-transcriptional Gene Silencing of GBSSI in Potato: Effects of Size and Sequence of the Inverted Repeats

In the past, silencing of granule-bound starch synthase (GBSSI) in potato was achieved by antisense technology, where it was observed that inclusion of the 3' end of the GBSSI coding region increased silencing efficiency. Since higher silencing efficiencies were desired, GBSSI inverted repeat constructs were designed and tested in potato. First, large inverted repeats comprising the 5' and the 3' half of the GBSSI cDNA were tested. The 5' IR construct gave a significantly higher silencing efficiency than the 3' IR construct. Since it was not known whether the observed difference was due to the sequence or the orientation of the inverted repeat, the GBSSI cDNA was divided into three regions, after which each region was tested in small inverted repeats in two orientations. To this end large numbers of independent transformants were produced for each construct. The results suggested that there was no effect of inverted repeat orientation on silencing efficiency. The percentage of transformants showing strong inhibition varied from 48% for a 3'-derived construct to 87% for a 5' as well as a middle region-derived construct. Similar to the large inverted repeats, the 3' sequences induced the least efficient silencing implying that the observed differences in silencing efficiency are caused by sequence differences. The small inverted repeat constructs with a repeat size of 500-600 bp and a spacer of about 150 bp were more efficient silencing inducers than the large inverted repeat constructs where the size of the repeat was 1.1 or 1.3 kb whilst the size of spacer was 1.3 or 1.1 kb. The results presented here show that size and sequence of the inverted repeat influenced silencing efficiency.     

Composition of endogenous alleles can influence the level of antisense inhibition of granule-bound starch synthase gene expression in tetraploid potato plants

The T-DNA composition was analysed of twelve potato genotypes obtained after transforming a tetraploid cultivar with an antisense granule-bound starch synthase (GBSSI) gene. In five transformants (labelled TB50 nos.) the antisense GBSSI gene was driven by the CaMV 35S promoter, while in the remaining seven (labelled TBK50 nos.) the GBSSI promoter was used. In these twelve transformants the antisense effect on amylose production in potato tuber starch ranged from complete suppression to no discernible inhibition, and the number of T-DNA insertions ranged from one to at least fifteen. The antisense effect of individual T-DNA loci in progeny of these transformants was studied. Progeny containing a single T-DNA showed no inhibition of GBSSI activity. Only multiple, linked T-DNA insertions resulted in substantial antisense inhibition. T-DNA fragments present in duplex in selfed progeny resulted in a larger antisense effect than that in the parent (which contained the T-DNA insertions in simplex). Furthermore, the antisense effects of some T-DNA-containing linkage groups were influenced by the composition of endogenous GBSSI alleles. For practical breeding this implies that (1) the efficiency of obtaining primary potato transformants showing complete inhibition of GBSSI gene expression by antisense RNA is genotype-dependent, and (2) many transformants have to be produced per genotype to be able to select plants with maximum suppression of GBSSI and a minimum number of T-DNA loci.     

Simultaneous silencing of multiple genes in the apple scab fungus, Venturia inaequalis, by expression of RNA with chimeric inverted repeats

RNA-mediated gene silencing has been demonstrated in plants, animals, and more recently in filamentous fungi. Here, we report high frequency, RNA-mediated gene silencing in the apple scab fungus, Venturia inaequalis. The green fluorescent protein (GFP) transgene was silenced in a GFP-expressing transformant. An endogenous gene, trihydroxynaphthalene reductase (THN), involved in melanin biosynthesis, was also silenced. Silencing of these two genes resulted in obvious phenotypes in vitro. High frequency gene silencing was achieved using hairpin constructs for the GFP or the THN genes transferred by Agrobacterium (71 and 61%, respectively). THN-silenced transformants exhibited a distinctive light brown phenotype and maintained the ability to infect apple. Of significance was the simultaneous silencing of the two genes from a single chimeric, inverted repeat hairpin construct. Silencing of both genes with this construct occurred at a frequency of 51% of all the transformants. All 125 colonies silenced for the GFP gene were also silenced for THN. As THN and GFP silenced transformants have readily detectable phenotypes, the genes have utility as markers for gene silencing. Simultaneous, multiple gene silencing, utilising such marker genes, will enable the development of high through-put screening for functional genomics. This chimeric technology will be particularly valuable when linked with silenced genes that have no obvious phenotype in vitro.     

Inhibition of the gene expression for granule-bound starch synthase I by RNA interference in sweet potato plants

Granule-bound starch synthase I (GBSSI) is one of the key enzymes catalyzing the formation of amylose, a linear α(1,4)D-glucan polymer, from ADP-glucose. Amylose-free transgenic sweet potato plants were produced by inhibiting sweet potato GBSSI gene expression through RNA interference. The gene construct consisting of an inverted repeat of the first exon separated by intron 1 of GBSSI driven by the CaMV 35S promoter was integrated into the sweet potato genome by Agrobacterium tumefaciens-mediated transformation. In over 70% of the regenerated transgenic plants, the expression of GBSSI was inactivated giving rise to storage roots containing amylopectin but not amylose. Electrophoresis analysis failed to detect the GBSSI protein, suggesting that gene silencing of the GBSSI gene had occurred. These results clearly demonstrate that amylose synthesis is completely inhibited in storage roots of sweet potato plants by the constitutive production of the double-stranded RNA of GBSSI fragments. We conclude that RNA interference is an effective method for inhibiting gene expression in the starch metabolic pathway.