Over 225,000 independent Agrobacterium transferred DNA (T-DNA) insertion events in the genome of the reference plant Arabidopsis thaliana have been created that represent near saturation of the gene space. The precise locations were determined for more than 88,000 T-DNA insertions, which resulted in the identification of mutations in more than 21,700 of the approximately 29,454 predicted Arabidopsis genes. Genome-wide analysis of the distribution of integration events revealed the existence of a large integration site bias at both the chromosome and gene levels. Insertion mutations were identified in genes that are regulated in response to the plant hormone ethylene.

https://science.sciencemag.org/content/sci/301/5633/653.full.pdf

Genome-Wide Insertional Mutagenesis of Arabidopsis thaliana

https://www.cell.com/trends/plant-science/pdf/S1360-1385(02)02251-3.pdf

GATEWAY vectors for Agrobacterium-mediated plant transformation.

*Summary Gateway cloning technology facilitates high-throughput cloning of target sequences by making use of the bacteriophage lambda site-specific recombination system. Target sequences are first captured in a commercially available ‘entry vector’ and are then recombined into various ‘destination vectors’ for expression in different experimental organisms. Gateway technology has been embraced by a number of plant laboratories that have engineered destination vectors for promoter specificity analyses, protein localization studies, protein/protein interaction studies, constitutive or inducible protein expression studies, gene knockdown by RNA interference, or affinity purification experiments. We review the various types of Gateway destination vectors that are currently available to the plant research community and provide links and references to enable additional information to be obtained concerning these vectors. We also describe a set of ‘pEarleyGate’ plasmid vectors for Agrobacterium-mediated plant transformation that translationally fuse FLAG, HA, cMyc, AcV5 or tandem affinity purification epitope tags onto target proteins, with or without an adjacent fluorescent protein. The oligopeptide epitope tags allow the affinity purification, immunolocalization or immunoprecipitation of recombinant proteins expressed in vivo. We demonstrate the utility of pEarleyGate destination vectors for the expression of epitope-tagged proteins that can be affinity captured or localized by immunofluorescence microscopy. Antibodies detecting the FLAG, HA, cMyc and AcV5 tags show relatively little cross-reaction with endogenous proteins in a variety of monocotyledonous and dicotyledonous plants, suggesting broad utility for the tags and vectors.

/pdf/gateway-compatible-vectors-for-plant-functional-genomics-and-1jp90m60g7.pdf

Gateway-compatible vectors for plant functional genomics and proteomics.

Silicon is beneficial to plant growth and helps plants to overcome abiotic and biotic stresses by preventing lodging (falling over) and increasing resistance to pests and diseases, as well as other stresses. Silicon is essential for high and sustainable production of rice, but the molecular mechanism responsible for the uptake of silicon is unknown. Here we describe the Low silicon rice 1 (Lsi1) gene, which controls silicon accumulation in rice, a typical silicon-accumulating plant. This gene belongs to the aquaporin family and is constitutively expressed in the roots. Lsi1 is localized on the plasma membrane of the distal side of both exodermis and endodermis cells, where casparian strips are located. Suppression of Lsi1 expression resulted in reduced silicon uptake. Furthermore, expression of Lsi1 in Xenopus oocytes showed transport activity for silicon only. The identification of a silicon transporter provides both an insight into the silicon uptake system in plants, and a new strategy for producing crops with high resistance to multiple stresses by genetic modification of the root's silicon uptake capacity.

A silicon transporter in rice

Plant microRNAs (miRNAs) affect only a small number of targets with high sequence complementarity, while animal miRNAs usually have hundreds of targets with limited complementarity. We used artificial miRNAs (amiRNAs) to determine whether the narrow action spectrum of natural plant miRNAs reflects only intrinsic properties of the plant miRNA machinery or whether it is also due to past selection against natural miRNAs with broader specificity. amiRNAs were designed to target individual genes or groups of endogenous genes. Like natural miRNAs, they had varying numbers of target mismatches. Previously determined parameters of target selection for natural miRNAs could accurately predict direct targets of amiRNAs. The specificity of amiRNAs, as deduced from genome-wide expression profiling, was as high as that of natural plant miRNAs, supporting the notion that extensive base pairing with targets is required for plant miRNA function. amiRNAs make an effective tool for specific gene silencing in plants, especially when several related, but not identical, target genes need to be downregulated. We demonstrate that amiRNAs are also active when expressed under tissue-specific or inducible promoters, with limited nonautonomous effects. The design principles for amiRNAs have been generalized and integrated into a Web-based tool (http://wmd.weigelworld.org).

/pdf/highly-specific-gene-silencing-by-artificial-micrornas-in-46188e4r0f.pdf

Highly specific gene silencing by artificial microRNAs in Arabidopsis.

Post-transcriptional silencing of plant genes using anti-sense or co-suppression constructs usually results in only a modest proportion of silenced individuals. Recent work has demonstrated the potential for constructs encoding self-complementary 'hairpin' RNA (hpRNA) to efficiently silence genes. In this study we examine design rules for efficient gene silencing, in terms of both the proportion of independent transgenic plants showing silencing, and the degree of silencing. Using hpRNA constructs containing sense/anti-sense arms ranging from 98 to 853 nt gave efficient silencing in a wide range of plant species, and inclusion of an intron in these constructs had a consistently enhancing effect. Intron-containing constructs (ihpRNA) generally gave 90-100% of independent transgenic plants showing silencing. The degree of silencing with these constructs was much greater than that obtained using either co-suppression or anti-sense constructs. We have made a generic vector, pHANNIBAL, that allows a simple, single PCR product from a gene of interest to be easily converted into a highly effective ihpRNA silencing construct. We have also created a high-throughput vector, pHELLSGATE, that should facilitate the cloning of gene libraries or large numbers of defined genes, such as those in EST collections, using an in vitro recombinase system. This system may facilitate the large-scale determination and discovery of plant gene functions in the same way as RNAi is being used to examine gene function in Caenorhabditis elegans.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-313X.2001.01105.x

Construct design for efficient, effective and high-throughput gene silencing in plants.

Construct design for efficient, effective and high-throughput gene silencing in plants

A major challenge in the post-genome era of plant biology is to determine the functions of all genes in the plant genome. A straightforward approach to this problem is to reduce or knockout expression of a gene with the hope of seeing a phenotype that is suggestive of its function. Insertional mutagenesis is a useful tool for this type of study but is limited by gene redundancy, lethal knockouts, non-tagged mutants, and the inability to target the inserted element to a specific gene. The efficacy of gene silencing in plants using inverted-repeat transgene constructs that encode a hairpin RNA (hpRNA) has been demonstrated by a number of groups, and has several advantages over insertional mutagenesis. In this paper we describe two improved pHellsgate vectors that facilitate rapid generation of hpRNA-encoding constructs. pHellsgate 4 allows the production of an hpRNA construct in a single step from a single polymerase chain reaction product, while pHellsgate 8 requires a two-step process via an intermediate vector. We show that these vectors are effective at silencing three endogenous genes in Arabidopsis, FLOWERING LOCUS C, PHYTOENE DESATURASE and ETHYLENE INSENSITIVE 2. We also show that a construct of sequences from two genes silences both genes.

High-throughput vectors for efficient gene silencing in plants

A major challenge in the post-genome era of plant biology is to determine the functions of all genes in the plant genome. A straightforward approach to this problem is to reduce or knockout expression of a gene with the hope of seeing a phenotype that is suggestive of its function. Insertional mutagenesis is a useful tool for this type of study but is limited by gene redundancy, lethal knockouts, non-tagged mutants, and the inability to target the inserted element to a specific gene. The efficacy of gene silencing in plants using inverted-repeat transgene constructs that encode a hairpin RNA (hpRNA) has been demonstrated by a number of groups, and has several advantages over insertional mutagenesis. In this paper we describe two improved pHellsgate vectors that facilitate rapid generation of hpRNA-encoding constructs, pHellsgate 4 allows the production of an hpRNA construct in a single step from a single polymerase chain reaction product, while pHellsgate 8 requires a two-step process via an intermediate vector. We show that these vectors are effective at silencing three endogenous genes in Arabidopsis, FLOWERING LOCUS C, PHYTOENE DESATURASE and ETHYLENE INSENSITIVE 2. We also show that a construct of sequences from two genes silences both genes.

S. Varsha Wesley

Papers

Construct design for efficient, effective and high-throughput gene silencing in plants.

Construct design for efficient, effective and high-throughput gene silencing in plants

High-throughput vectors for efficient gene silencing in plants

High-throughput vectors for efficient gene silencing in plants