Large-scale insertional mutagenesis using the Tnt1 retrotransposon in the model legume Medicago truncatula

Medicago truncatula is a fast-emerging model for the study of legume functional biology. We used the tobacco retrotransposon Tnt1 to tag the Medicago genome and generated over 7600 independent lines representing an estimated 190 000 insertion events. Tnt1 inserted on average at 25 different locations per genome during tissue culture, and insertions were stable during subsequent generations in soil. Analysis of 2461 Tnt1 flanking sequence tags (FSTs) revealed that Tnt1 appears to prefer gene-rich regions. The proportion of Tnt1 insertion in coding sequences was 34.1%, compared to the expected 15.9% if random insertions were to occur. However, Tnt1 showed neither unique target site specificity nor strong insertion hot spots, although some genes were more frequently tagged than others. Forward-genetic screening of 3237 R₁ lines resulted in identification of visible mutant phenotypes in approximately 30% of the regenerated lines. Tagging efficiency appears to be high, as all of the 20 mutants examined so far were found to be tagged. Taking the properties of Tnt1 into account and assuming 1.7 kb for the average M. truncatula gene size, we estimate that approximately 14 000–16 000 lines would be sufficient for 90% gene tagging coverage in M. truncatula . This is in contrast to more than 500 000 lines required to achieve the same saturation level using T-DNA tagging. Our data demonstrate that Tnt1 is an efficient insertional mutagen in M. truncatula , and could be a primary choice for other plant species with large genomes.     

Efficient transposition of the Tnt1 tobacco retrotransposon in the model legume Medicago truncatula

The tobacco element, Tnt1, is one of the few active retrotransposons in plants. Its transposition is activated during protoplast culture in tobacco and tissue culture in the heterologous host Arabidopsis thaliana. Here, we report its transposition in the R108 line of Medicago truncatula during the early steps of the in vitro transformation-regeneration process. Two hundred and twenty-five primary transformants containing Tnt1 were obtained. Among them, 11.2% contained only transposed copies of the element, indicating that Tnt1 transposed very early and efficiently during the in vitro transformation process, possibly even before the T-DNA integration. The average number of insertions per transgenic line was estimated to be about 15. These insertions were stable in the progeny and could be separated by segregation. Inspection of the sequences flanking the insertion sites revealed that Tnt1 had no insertion site specificity and often inserted in genes (one out of three insertions). Thus, our work demonstrates the functioning of an efficient transposable element in leguminous plants. These results indicate that Tnt1 can be used as a powerful tool for insertion mutagenesis in M. truncatula.     

Distribution dynamics of the Tnt1 retrotransposon in tobacco

Retrotransposons contribute significantly to the size, organization and genetic diversity of plant genomes. Although many retrotransposon families have been reported in plants, to this day, the tobacco Tnt1 retrotransposon remains one of the few elements for which active transposition has been shown. Demonstration that Tnt1 activation can be induced by stress has lent support to the hypothesis that, under adverse conditions, transposition can be an important source of genetic variability. Here, we compared the insertion site preference of a collection of newly transposed and pre-existing Tnt1 copies identified in plants regenerated from protoplasts or tissue culture. We find that newly transposed Tnt1 copies are targeted within or close to host gene coding sequences and that the distribution of pre-existing insertions does not vary significantly from this trend. Therefore, in spite of their potential to disrupt neighboring genes, insertions within or near CDS are not preferentially removed with age. Elimination of Tnt1 insertions within or near coding sequences may be relaxed due to the polyploid nature of the tobacco genome. Tnt1 insertions within or near CDS are thus better tolerated and can putatively contribute to the diversification of tobacco gene function. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The Tnt1 family member Retrosol copy number and structure disclose retrotransposon diversification in different <Emphasis Type="Italic">Solanum</Emphasis> species

Eukaryotic genome expansion/retraction caused by LTR-retrotransposon activity is dependent on the expression of full length copies to trigger efficient transposition and recombination-driven events. The Tnt1 family of retrotransposons has served as a model to evaluate the diversity among closely related elements within Solanaceae species and found that members of the family vary mainly in their U3 region of the long terminal repeats (LTRs). Recovery of a full length genomic copy of Retrosol was performed through a PCR-based approach from wild potato, Solanum oplocense. Further characterization focusing on both LTR sequences of the amplified copy allowed estimating an approximate insertion time at 2 million years ago thus supporting the occurrence of transposition cycles after genus divergence. Copy number of Tnt1-like elements in Solanum species were determined through genomic quantitative PCR whereby results sustain that Retrosol in Solanum species is a low copy number retrotransposon (1–4 copies) while Retrolyc1 has an intermediate copy number (38 copies) in S. peruvianum. Comparative analysis of retrotransposon content revealed no correlation between genome size or ploidy level and Retrosol copy number. The tetraploid cultivated potato with a cellular genome size of 1,715 Mbp harbours similar copy number per monoploid genome than other diploid Solanum species (613–884 Mbp). Conversely, S. peruvianum genome (1,125 Mbp) has a higher copy number. These results point towards a lineage specific dynamic flux regarding the history of amplification/activity of Tnt1-like elements in the genome of Solanum species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Distribution of the Tnt1 retrotransposon family in the amphidiploid tobacco (Nicotiana tabacum) and its wild Nicotiana relatives

Transposable elements can generate considerable genetic diversity. Here we examine the distribution of the Tnt1 retrotransposon family in representative species of the genus Nicotiana . We show that multiple Tnt1 insertions are found in all Nicotiana species. However, Tnt1 insertions are too polymorphic to reveal species relationships. This indicates that Tnt1 has amplified rapidly and independently after Nicotiana speciation. We compare patterns of Tnt1 insertion in allotetraploid tobacco ( N. tabacum ) with those in the diploid species that are most closely related to the progenitors of tobacco, N. sylvestris (S-genome donor) and N. tomentosiformis (T-genome donor). We found no evidence for Tnt1 insertion sites of N. otophora origin in tobacco. Nicotiana sylvestris has a higher Tnt1 content than N. tomentosiformis and the elements are distributed more uniformly across the genome. This is reflected in tobacco where there is a higher Tnt1 content in S-genome chromosomes. However, the total Tnt1 content of tobacco is not the sum of the two modern-day parental species. We also observed tobacco-specific Tnt1 insertions and an absence of tobacco Tnt1 insertion sites in the diploid relatives. These data indicate Tnt1 evolution subsequent to allopolyploidy. We explore the possibility that fast evolution of Tnt1 is associated with 'genomic-shock' arising out of interspecific hybridization and allopolyploidy.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 82 , 639–649.     

Constitutive activation of TORC1 signalling attenuates virulence in the cross-kingdom fungal pathogen Fusarium oxysporum

The filamentous fungus Fusarium oxysporum causes vascular wilt disease in a wide range of plant species and opportunistic infections in humans. Previous work suggested that invasive growth in this pathogen is controlled by environmental cues such as pH and nutrient status. Here we investigated the role of Target Of Rapamycin Complex 1 (TORC1), a global regulator of eukaryotic cell growth and development. Inactivation of the negative regulator Tuberous Sclerosis Complex 2 (Tsc2), but not constitutive activation of the positive regulator Gtr1, in F. oxysporum resulted in inappropriate activation of TORC1 signalling under nutrient-limiting conditions. The tsc2Δ mutants showed reduced colony growth on minimal medium with different nitrogen sources and increased sensitivity to cell wall or high temperature stress. Furthermore, these mutants were impaired in invasive hyphal growth across cellophane membranes and exhibited a marked decrease in virulence, both on tomato plants and on the invertebrate animal host Galleria mellonella. Importantly, invasive hyphal growth in tsc2Δ strains was rescued by rapamycin-mediated inhibition of TORC1. Collectively, these results reveal a key role of TORC1 signalling in the development and pathogenicity of F. oxysporum and suggest new potential targets for controlling fungal infections.     

人博卡病毒HBoV1非结构蛋白NP1对细胞转录因子的调控

【目的】研究人博卡病毒非结构蛋白NP1对细胞转录因子活性和炎症细胞因子TNF-α和IL-6表达的调控作用。【方法】通过双荧光素酶报告基因系统分析NP1对转录因子的调控作用,通过ELISA和荧光定量PCR分别从蛋白质和RNA水平检测NP1是否影响TNF-α、IL-6的表达,最后利用哺乳动物双杂交系统分析NP1是否通过寡聚化发挥功能。【结果】在293T细胞中,NP1可分别提高AP-1、STAT3和GAS转录因子报告载体荧光素酶活性3.69倍、2.45倍和3.03倍,但对NF-κB转录因子报告载体荧光素酶活性无明显影响(P>0.05)。转染24 h和48 h后,NP1表达细胞和对照细胞培养基中IL-6和TNF-α蛋白浓度没有显著性差异(P>0.05),但TNF-αmRNA的表达水平上调。哺乳动物双杂交实验表明NP1蛋白自身没有相互作用。【结论】结果首次表明HBoV1非结构蛋白NP1对细胞转录因子和炎症细胞因子具有调节作用,揭示NP1可能与HBoV1致病性有关,为进一步探究HBoV1病毒致病的分子机制提供参考。     

The evolutionary analysis of the Tnt1 retrotransposon in Nicotiana species reveals the high variability of its regulatory sequences

We studied the evolution of the tobacco Tnt1 retrotransposon by analyzing Tnt1 partial sequences containing both coding domains and U3 regulatory sequences obtained from a number of Nicotiana species. We detected three different subfamilies of Tnt1 elements, Tnt1A, Tnt1B, and Tnt1C, that differ completely in their U3 regions but share conserved flanking coding and LTR regions. U3 divergence between the three subfamilies is found in the region that contains the regulatory sequences that control the expression of the well-characterized Tnt1-94 element. This suggests that expression of the three Tnt1 subfamilies might be differently regulated. The three Tnt1 subfamilies were present in the Nicotiana genome at the time of species divergence, but have evolved independently since then in the different genomes. Each Tnt1 subfamily seems to have conserved its ability to transpose in a limited and different number of Nicotiana species. Our results illustrate the high variability of Tnt1 regulatory sequences. We propose that this high sequence variability could allow these elements to evolve regulatory mechanisms in order to optimize their coexistence with their host genome.