Optimization of transgene-mediated silencing in Phytophthora infestans and its association with small-interfering RNAs

Methods for silencing genes in Phytophthora transformants have been demonstrated previously, but wide variation in effectiveness was reported in different studies. To optimize this important tool for functional genomics, we compared the abilities of sense, antisense, and hairpin transgenes introduced by protoplast, electroporation, and bombardment methods to silence the inf1 elicitin gene in Phytophthora infestans. A hairpin construct induced silencing three times more often than sense or antisense vectors, and protoplast transformation twice as much as electroporation. Using hairpins introduced into protoplasts, 61% of strains were silenced, and transgene copy number was positively correlated with silencing. The utility of bombardment was reduced by the occurrence of heterokaryons containing silenced and non-silenced nuclei, but silenced strains were obtainable from about 20% of primary transformants by single-nuclear purification. Most inf1-deficient strains were fully silenced, however some exhibited partial suppression. These produced inf1-derived RNAs of about 21-nt which correspond to both the sense and antisense strands of inf1, implicating an RNAi-like mechanism in silencing.     

RNA Silencing Mediated by Direct Repeats in Maize: A Potential Tool for Functional Genomics

It has been shown in tobacco and Arabidopsis that transgenes with multiple direct repeats induce RNA silencing at high frequency. In this study, we tried to establish a direct repeat-induced RNA silencing system in maize and evaluate whether it can be developed as a high throughput tool for functional genomics. Our results showed that the construct phC4, which carries four direct repeats of a chloramphenicol acetyl-transferase (CAT) gene, was able to induce silencing of itself with high efficiency in maize. Using a transient expression system, we further demonstrated that construct phC3G with a β-glucuronidase (GUS) gene located downstream of three direct repeats of CAT gene silenced not only itself in maize calli but also an “endogenous” GUS gene, which was stably expressed in maize calli. Most importantly, when constructs with the maize iojap (ij) gene inserted in either sense or antisense orientation into the downstream of four direct repeats of CAT gene were transformed into maize plants, co-suppression of endogenous and transgenic ij genes was detected in majority of transgenic maize plants. Our co-suppression results suggest that with improvements, this new approach has the potential to become an efficient research tool for high throughput functional genomics.     

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.     

Adenovirus-delivered antisense RNA and shRNA exhibit different silencing efficiencies for the endogenous transforming growth factor-beta (TGF-beta) type II receptor

Gene silencing is an essential tool in gene discovery and gene therapy. Traditionally, viral delivery of antisense RNA and, more recently, small interfering RNA (siRNA) molecules in the form of small hairpin RNAs (shRNA) has been used as a strategy to achieve gene silencing. Nevertheless, the enduring challenge is to identify molecules that specifically and optimally silence a given target gene. In this study, we tested a set of adenovirus-delivered antisense RNA fragments and adenovirus-delivered shRNA molecules for their ability to target human transforming growth factor-beta type II receptor (TGFbetaRII). We used a dicistronic reporter, consisting of the coding sequences for TGFbetaRII and green fluorescent protein (GFP) to screen for optimal silencing agents targeting TGFbetaRII. Our results show, for both antisense RNA and shRNA molecules, that their effectiveness in the GFP screen correlated directly with their ability to reduce exogenously expressed TGFbetaRII. Unexpectedly, the antisense RNAs were unable to silence endogenous TGFbetaRII. In contrast, the shRNAs were able to silence endogenous TGFbetaRII. The shRNA that demonstrated the most pronounced effect on the dicistronic TGFbetaRII/GFP reporter reduced endogenous TGFbetaRII protein expression by 70% in A549 cells and reduced TGFbeta signaling by >80% in HeLa cells.     

Engineering tomato for resistance to tomato leaf curl disease using viral <Emphasis Type="Italic">rep</Emphasis> gene sequences

Transgenic tomato resistant to tomato leaf curl disease (ToLCD) using replicase (rep) gene sequences of Tomato leaf curl virus in antisense orientation were developed via Agrobacterium-mediated transformation. A binary vector carrying the antisense rep gene (untranslatable full length sequence, 1086 bp) along with the npt II gene was used for transformation. High level of resistance and inheritability of the transgene was observed up to T₂ stage following challenge inoculation with the virus. The mechanism of resistance appears RNA-mediated, since the plants carried the untranslatable antisense rep gene. Progeny analysis of these plants showed classical Mendelian pattern of inheritance in two of the six transgenic lines having single transgene insertion.     

RNA silencing in the phytopathogenic fungus Magnaporthe oryzae

Systematic analysis of RNA silencing was carried out in the blast fungus Magnaporthe oryzae (formerly Magnaporthe grisea) using the enhanced green fluorescence protein (eGFP) gene as a model. To assess the ability of RNA species to induce RNA silencing in the fungus, plasmid constructs expressing sense, antisense, and hairpin RNAs were introduced into an eGFP-expressing transformant. The fluorescence of eGFP in the transformant was silenced much more efficiently by hairpin RNA of eGFP than by other RNA species. In the silenced transformants, the accumulation of eGFP mRNA was drastically reduced, but no methylation of the promoter or coding region was involved in it. In addition, we found small interfering RNAs (siRNAs) only in the silenced transformants. Interestingly, the siRNAs consisted of RNA molecules with at least three different sizes ranging from 19 to 23 nucleotides, and all of them contained both sense and antisense strands of the eGFP gene. To our knowledge, this is the first demonstration in which different molecular sizes of siRNAs have been found in filamentous fungi. Overall, these results indicate that RNA silencing operates in M. oryzae, which gives us a new tool for genome-wide gene analysis in this fungus.     

Simultaneous suppression of multiple genes by single transgenes. Down-regulation of three unrelated lignin biosynthetic genes in tobacco

Many reports now describe the manipulation of plant metabolism by suppressing the expression of single genes. The potential of such work could be greatly expanded if multiple genes could be coordinately suppressed. In the work presented here, we test a novel method for achieving this by using single chimeric constructs incorporating partial sense sequences for multiple genes to target suppression of two or three lignin biosynthetic enzymes. We compare this method with a more conventional approach to achieving the same end by crossing plants harboring different antisense transgenes. Our results indicate that crossing antisense plants is less straightforward and predictable in outcome than anticipated. Most progeny had higher levels of target enzyme activity than predicted and had lost the expected modifications to lignin structure. In comparison, plants transformed with the chimeric partial sense constructs had more consistent high level suppression of target enzymes and had significant changes to lignin content, structure, and composition. It was possible to suppress three target genes coordinately using a single chimeric construct. Our results indicate that chimeric silencing constructs offer great potential for the rapid and coordinate suppression of multiple genes on diverse biochemical pathways and that the technique therefore deserves to be adopted by other researchers.     

Barley stripe mosaic virus-induced gene silencing in a monocot plant

RNA silencing of endogenous plant genes can be achieved by virus-mediated, transient expression of homologous gene fragments. This powerful, reverse genetic approach, known as virus-induced gene silencing (VIGS), has been demonstrated only in dicot plant species, where it has become an important tool for functional genomics. Barley stripe mosaic virus (BSMV) is a tripartite, positive-sense RNA virus that infects many agriculturally important monocot species including barley, oats, wheat and maize. To demonstrate VIGS in a monocot host, we modified BSMV to express untranslatable foreign inserts downstream of the gammab gene, in either sense or antisense orientations. Phytoene desaturase (PDS) is required for synthesizing carotenoids, compounds that protect chlorophyll from photo-bleaching. A partial PDS cDNA amplified from barley was 90, 88 and 74% identical to PDS cDNAs from rice, maize and Nicotiana benthamiana, respectively. Barley infected with BSMV expressing barley, rice or maize PDS fragments became photo-bleached and accumulated phytoene (the substrate for PDS) in a manner similar to plants treated with the chemical inhibitor of PDS, norflurazon. In contrast, barley infected with wild-type BSMV, or BSMV expressing either N. benthamiana PDS or antisense green fluorescent protein (GFP), did not photo-bleach or accumulate phytoene. Thus BSMV silencing of the endogenous PDS was homology-dependent. Deletion of the coat protein enhanced the ability of BSMV to silence PDS. This is the first demonstration of VIGS in a monocot, and suggests that BSMV can be used for functional genomics and studies of RNA-silencing mechanisms in monocot plant species.