The utility of transposable elements as tools for the isolation of plant genes

Transposable elements are segments of DNA which have the unique capability of being able to excise from one site in the genome and reintegrate into new, different sites elsewhere in the genome. When transposition takes place and integration occurs within a gene locus, mutations are frequently generated producing variegated or recessive phenotypes. This ability of transposable elements to act as mutagenic agents through their association with particular gene sequences has lead to the development of the procedure of transposon tagging or gene tagging in higher plants. Through this technique, transposable elements can be used to clone and isolate genes of interest for which little or nothing is known about the final product (i.e., polypeptide). This offers tremendous potential for the isolation of a variety of agronomically important genes, which are virtually impossible to recover by other currently available gene cloning methodologies. To date, the technique has been used successfully to isolate genes from corn and snapdragon. Using gene transfer technologies, the potential now exists to extend this approach to clone genes from other plant species. Advantages and limitations of transposon tagging for isolating plant genes will be discussed.     

Reverse Genetic Approaches for Functional Genomics of Rice

T-DNA and transposable elements e.g., Ds and Tos17, are used to generate a large number of insertional mutant lines in rice. Some carry the GUS or GFP reporter for gene trap or enhancer trap. These reporter systems are valuable for identifying tissue- or organ-preferential genes. Activation tagging lines have also been generated for screening mutants and isolating mutagenized genes. To utilize these resources more efficiently, tagged lines have been produced for reverse genetic approaches. DNA pools of the T-DNA tagged lines and Tos17 lines have been prepared for PCR screening of insertional mutants in a given gene. Tag end sequences (TES) of the inserts have also been produced. TES databases are beneficial for analyzing the function of a large number of rice genes.     

A highly conserved, small LTR retrotransposon that preferentially targets genes in grass genomes

LTR retrotransposons are often the most abundant components of plant genomes and can impact gene and genome evolution. Most reported LTR retrotransposons are large elements (>4 kb) and are most often found in heterochromatic (gene poor) regions. We report the smallest LTR retrotransposon found to date, only 292 bp. The element is found in rice, maize, sorghum and other grass genomes, which indicates that it was present in the ancestor of grass species, at least 50-80 MYA. Estimated insertion times, comparisons between sequenced rice lines, and mRNA data indicate that this element may still be active in some genomes. Unlike other LTR retrotransposons, the small LTR retrotransposons (SMARTs) are distributed throughout the genomes and are often located within or near genes with insertion patterns similar to MITEs (miniature inverted repeat transposable elements). Our data suggests that insertions of SMARTs into or near genes can, in a few instances, alter both gene structures and gene expression. Further evidence for a role in regulating gene expression, SMART-specific small RNAs (sRNAs) were identified that may be involved in gene regulation. Thus, SMARTs may have played an important role in genome evolution and genic innovation and may provide a valuable tool for gene tagging systems in grass.     

Transposable element contributions to plant gene and genome evolution

Transposable elements were first discovered in plants because they can have tremendous effects on genome structure and gene function. Although only a few or no elements may be active within a genome at any time in any individual, the genomic alterations they cause can have major outcomes for a species. All major element types appear to be present in all plant species, but their quantitative and qualitative contributions are enormously variable even between closely related lineages. In some large-genome plants, mobile DNAs make up the majority of the nuclear genome. They can rearrange genomes and alter individual gene structure and regulation through any of the activities they promote: transposition, insertion, excision, chromosome breakage, and ectopic recombination. Many genes may have been assembled or amplified through the action of transposable elements, and it is likely that most plant genes contain legacies of multiple transposable element insertions into promoters. Because chromosomal rearrangements can lead to speciating infertility in heterozygous progeny, transposable elements may be responsible for the rate at which such incompatibility is generated in separated populations. For these reasons, understanding plant gene and genome evolution is only possible if we comprehend the contributions of transposable elements.     

Genetic resources offer efficient tools for rice functional genomics research

Rice is an important crop and major model plant for monocot functional genomics studies. With the establishment of various genetic resources for rice genomics, the next challenge is to systematically assign functions to predicted genes in the rice genome. Compared with the robustness of genome sequencing and bioinformatics techniques, progress in understanding the function of rice genes has lagged, hampering the utilization of rice genes for cereal crop improvement. The use of transfer DNA (T‐DNA) insertional mutagenesis offers the advantage of uniform distribution throughout the rice genome, but preferentially in gene‐rich regions, resulting in direct gene knockout or activation of genes within 20–30 kb up‐ and downstream of the T‐DNA insertion site and high gene tagging efficiency. Here, we summarize the recent progress in functional genomics using the T‐DNA‐tagged rice mutant population. We also discuss important features of T‐DNA activation‐ and knockout‐tagging and promoter‐trapping of the rice genome in relation to mutant and candidate gene characterizations and how to more efficiently utilize rice mutant populations and datasets for high‐throughput functional genomics and phenomics studies by forward and reverse genetics approaches. These studies may facilitate the translation of rice functional genomics research to improvements of rice and other cereal crops.     

Mass spectrometry analysis of the variants of histone H3 and H4 of soybean and their post-translational modifications

Background

Functional genomics tools provide researchers with the ability to apply high-throughput techniques to determine the function and interaction of a diverse range of genes. Mutagenised plant populations are one such resource that facilitate gene characterisation. They allow complex physiological responses to be correlated with the expression of single genes in planta, through either reverse genetics where target genes are mutagenised to assay the affect, or through forward genetics where populations of mutant lines are screened to identify those whose phenotype diverges from wild type for a particular trait. One limitation of these types of populations is the prevalence of gene redundancy within plant genomes, which can mask the affect of individual genes. Activation or enhancer populations, which not only provide knock-out but also dominant activation mutations, can facilitate the study of such genes.

Results

We have developed a population of almost 50,000 activation tagged A. thaliana lines that have been archived as individual lines to the T₃ generation. The population is an excellent tool for both reverse and forward genetic screens and has been used successfully to identify a number of novel mutants. Insertion site sequences have been generated and mapped for 15,507 lines to enable further application of the population, while providing a clear distribution of T-DNA insertions across the genome. The population is being screened for a number of biochemical and developmental phenotypes, provisional data identifying novel alleles and genes controlling steps in proanthocyanidin biosynthesis and trichome development is presented.

Conclusion

This publicly available population provides an additional tool for plant researcher's to assist with determining gene function for the many as yet uncharacterised genes annotated within the Arabidopsis genome sequence http://aafc-aac.usask.ca/FST. The presence of enhancer elements on the inserted T-DNA molecule allows both knock-out and dominant activation phenotypes to be identified for traits of interest.     

Moss (Physcomitrella patens) functional genomics--Gene discovery and tool development, with implications for crop plants and human health.

Recently, the moss Physcomitrella patens was established as a versatile tool in plant functional genomics. Mosses represent the oldest living clade of land plants, separated by approximately 450 million years of evolution from crop plants. Consequently, mosses contain metabolites and genes not known from these seed plants. In Physcomitrella, nuclear genes can be targeted by homologous recombination as efficiently as in yeast, allowing reverse genetics approaches in plants at high-throughput levels for the first time. Comprehensive expressed sequence tag databases gave new insights into the levels of diversity in land plants which are now ready to be exploited in plant biotechnology. In forward genetics screens, saturated tagged mutant collections help to unravel novel gene - function relationships. Additionally, proteomics tools are at hand to analyse subcellular proteomes, as well as the phosphoproteome, as the core of eukaryotic signal transduction. Moreover, specifically designed Physcomitrella strains can produce human therapeutic proteins safely and cost-effectively in bioreactors.     

Transposable elements in the fungal plant pathogenFusarium oxysporum

The genome of the fungal plant pathogenFusarium oxysporum contains at least six different families of transposable elements. Representatives of both DNA transposons and retrotransposons have been identified, either by cloning of dispersed repetitive sequences (Foret andpalm) or by trapping in the nitrate reductase gene (Fot1, Fot2 Impala andHop).Fot1 andImpala elements are related to theTc1 andmariner class of transposons. These transposable elements can affect gene structure and function in several ways: inactivation of the target gene through insertion, diversification of the nucleotide sequence by imprecise excisions, and probably chromosomal rearrangements as suggested by the extensive karyotype variation observed among field isolates. Comparisons of the distribution of these elements inFusarium populations have improved our understanding of population structure and epidemiology and provided support for horizontal genetic transfer. Also they could be developed as genetic tools for tagging genes, a cloning strategy that is particularly promising in imperfect fungi.     

A transgenic perspective on plant functional genomics

Transgenic crops are very much in the news due to the increasing public debate on their acceptance. In the scientific community though, transgenic plants are proving to be powerful tools to study various aspects of plant sciences. The emerging scientific revolution sparked by genomics based technologies is producing enormous amounts of DNA sequence information that, together with plant transformation methodology, is opening up new experimental opportunities for functional genomics analysis. An overview is provided here on the use of transgenic technology for the functional analysis of plant genes in model plants and a link made to their utilization in transgenic crops. In transgenic plants, insertional mutagenesis using heterologous maize transposons or Agrobacterium mediated T-DNA insertions, have been valuable tools for the identification and isolation of genes that display a mutant phenotype. To discover functions of genes that do not display phenotypes when mutated, insertion sequences have been engineered to monitor or change the expression pattern of adjacent genes. These gene detector insertions can detect adjacent promoters, enhancers or gene exons and precisely reflect the expression pattern of the tagged gene. Activation tag insertions can mis-express the adjacent gene and confer dominant phenotypes that help bridge the phenotype gap. Employment of various forms of gene silencing technology broadens the scope of recovering knockout phenotypes for genes with redundant function. All these transgenic strategies describing gene-phenotype relationships can be addressed by high throughput reverse genetics methods that will help provide functions to the genes discovered by genome sequencing. The gene functions discovered by insertional mutagenesis and silencing strategies along with expression pattern analysis will provide an integrated functional genomics perspective and offer unique applications in transgenic crops. This revised version was published online in August 2006 with corrections to the Cover Date.     

Distribution and characterization of more than 1000 T‐DNA tags in the genome of Brachypodium distachyon community standard line Bd21

A collection of 4117 fertile T‐DNA lines has been generated by Agrobacterium‐mediated transformation of the diploid community standard line Bd21 of Brachypodium distachyon. The regions flanking the T‐DNA left and right borders of the first 741 transformed plants were isolated by adapter‐ligation PCR and sequenced. A total of 1005 genomic sequences (representing 44.1% of all flanking sequences retrieved) characterized 660 independent T‐DNA loci assigned to a unique location in the Brachypodium genome sequence. Seventy‐six percent of the fertile plant lines contained at least one anchored T‐DNA locus (1.17 loci per tagged line on average). Analysis of the regions flanking both borders of the T‐DNA increased the number of T‐DNA loci tagged and the number of tagged lines by approximately 50% when compared to a single border analysis. T‐DNA integration (2.4 insertions per Mb on average) was proportional to chromosome size, however, varied greatly along each chromosome with often low insertion level around centromeres. The frequency of insertion within transposable elements (5.3%) was fivefold lower than expected if random insertion would have occurred. More than half of the T‐DNAs inserted in genic regions. On average, one gene could be tagged for every second fertile plant line produced and more than one plant line out of three contained a T‐DNA insertion directly within or 500 bp around the coding sequence. Approximately, 60% of the genes tagged corresponded to expressed genes. The T‐DNA lines generated by the BrachyTAG programme are available as a community resource and have been distributed internationally since 2008 via the BrachyTAG.org web site.     

Improved T-DNA vector for tagging plant promoters via high-throughput luciferase screening

Transferred DNA (T-DNA) tagging is a powerful tool for tagging and in planta characterization of plant genes on a genome-wide scale. An improved promoter tagging vector is described here, which contains the codon-optimized luciferase (luc+) reporter gene 31 bp from the right border of the T-DNA. Compared to the wild-type luciferase gene, this construct provides significantly increased reporter gene expression and a 40 times higher tagging frequency. The utility of the construct is demonstrated in banana, a tropical monocot species, by screening embryogenic cell colonies and regenerated plants with an ultrasensitive charged-coupled device (CCD) camera. The improved vector resulted in a luciferase activation frequency of 2.5% in 19,000 cell colonies screened. Detailed molecular analysis of flanking DNA sequences in a tagged line revealed insertion of the luciferase tag in a novel gene with near-constitutive expression.     

Mapped Ds/T-DNA launch pads for functional genomics in barley

A system for targeted gene tagging and local saturation mutagenesis based on maize transposable elements (Ac/Ds) was developed in barley (Hordeum vulgare L.). We generated large numbers of transgenic barley lines carrying a single copy of the non-autonomous maize Ds element at defined positions in the genome. Independent Ds lines were either generated by activating Ds elements in existing single-copy lines after crossing with AcTPase-expressing plants or by Agrobacterium-mediated transformation. Genomic DNA flanking Ds and T-DNA insertion sites from over 200 independent lines was isolated and sequenced, and was used for a sequence based mapping strategy in a barley reference population. More than 100 independent Ds insertion sites were mapped and can be used as launch pads for future targeted tagging of genes in the vicinity of the insertion sites. Sequence analysis of Ds and T-DNA flanking regions revealed a sevenfold preference of both mutagens for insertion into non-redundant, gene-containing regions of the barley genome. However, whilst transposed Ds elements preferentially inserted adjacent to regions with a high number of predicted and experimentally validated matrix attachment regions (nuclear MARs), this was not the case for T-DNA integration sites. These findings and an observed high transposition frequency from mapped launch pads demonstrate the future potential of gene tagging for functional genomics and gene discovery in barley.     

Insertional mutagenesis and promoter trapping in plants for the isolation of genes and the study of development

Insertional mutagenesis, whether by transposable elements or T-DNAs fromAgrobacterium tumefaciens, provides a powerful experimental strategy to investigate the genetic basis of plant growth, metabolism and development. The linkage of an insertion element with a mutant phenotype of interest greatly facilitates the isolation of the wild-type gene. A further refinement of this strategy is the incorporation of promoter traps or enhancer traps into the insertion elements. These can act as functional tags of regulatory elements associated with genes in the host genome, potentially can improve further the efficiency of screening for target mutant phenotypes, and may provide valuable markers of specific cell types for developmental analysis. We discuss the use of these techniques to study the molecular genetics of plant development.     

UTILLdb, a Pisum sativum in silico forward and reverse genetics tool

The systematic characterization of gene functions in species recalcitrant to Agrobacterium-based transformation, like Pisum sativum, remains a challenge. To develop a high throughput forward and reverse genetics tool in pea, we have constructed a reference ethylmethane sulfonate mutant population and developed a database, UTILLdb, that contains phenotypic as well as sequence information on mutant genes. UTILLdb can be searched online for TILLING alleles, through the BLAST tool, or for phenotypic information about mutants by keywords.     

Molecular genetics using T-DNA in rice

Now that sequencing of the rice genome is nearly completed, functional analysis of its large number of genes is the next challenge. Because rice is easy to transform, T-DNA has been used successfully to generate insertional mutant lines. Collectively, several laboratories throughout the world have established at least 200,000 T-DNA insertional lines. Some of those carry the GUS or GFP reporters for either gene or enhancer traps. Others are activation tagging lines for gain-of-function mutagenesis when T-DNA is inserted in the intergenic region. A forward genetic approach showed limited success because of somaclonal variations induced during tissue culture. To utilize these resources more efficiently, tagged lines have been produced for reverse genetics approaches. DNA pools of the T-DNA-tagged lines have been prepared for polymerase chain reaction (PCR) screening of insertional mutants in a given gene. Appropriate T-DNA insertion sites are determined by sequencing the region flanking the T-DNA. This information is then used to make databases that are shared with the scientific community. International efforts on seed amplification and maintenance are needed to exploit these valuable materials efficiently.