Tnt1 Insertion Research Articles

Genome-wide methylation landscape during somatic embryogenesis in Medicago truncatula reveals correlation between Tnt1 retrotransposition and hyperactive methylation regions.

Medicago truncatula is a model legume for fundamental research on legume biology and symbiotic nitrogen fixation. Tnt1, a retrotransposon from tobacco, was used to generate insertion mutants in M. truncatula R108. Approximately 21 000 insertion lines have been generated and publicly available. Tnt1 retro-transposition event occurs during somatic embryogenesis (SE), a pivotal process that triggers massive methylation changes. We studied the SE of M. truncatula R108 using leaf explants and explored the dynamic shifts in the methylation landscape from leaf explants to callus formation and finally embryogenesis. Higher cytosine methylation in all three contexts of CG, CHG, and CHH patterns was observed during SE compared to the controls. Higher methylation patterns were observed in assumed promoter regions (~2-kb upstream regions of transcription start site) of the genes, while lowest was recorded in the untranslated regions. Differentially methylated promoter region analysis showed a higher CHH methylation in embryogenesis tissue samples when compared to CG and CHG methylation. Strong correlation (89.71%) was identified between the differentially methylated regions (DMRs) and the site of Tnt1 insertions in M.truncatula R108 and stronger hypermethylation of genes correlated with higher number of Tnt1 insertions in all contexts of CG, CHG, and CHH methylation. Gene ontology enrichment and KEGG pathway enrichment analysis identified genes and pathways enriched in the signal peptide processing, ATP hydrolysis, RNA polymerase activity, transport, secondary metabolites, and nitrogen metabolism pathways. Combined gene expression analysis and methylation profiling showed an inverse relationship between methylation in the DMRs (regions spanning genes) and the expression of genes. Our results show that a dynamic shift in methylation happens during the SE process in the context of CG, CHH and CHG methylation, and the Tnt1 retrotransposition correlates with the hyperactive methylation regions.

MtTCP18 Regulates Plant Structure in Medicago truncatula.

Plant structure has a large influence on crop yield formation, with branching and plant height being the important factors that make it up. We identified a gene, MtTCP18, encoding a TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factor highly conserved with Arabidopsis gene BRC1 (BRANCHED1) in Medicago truncatula. Sequence analysis revealed that MtTCP18 included a conserved basic helix-loop-helix (BHLH) motif and R domain. Expression analysis showed that MtTCP18 was expressed in all organs examined, with relatively higher expression in pods and axillary buds. Subcellular localization analysis showed that MtTCP18 was localized in the nucleus and exhibited transcriptional activation activity. These results supported its role as a transcription factor. Meanwhile, we identified a homozygous mutant line (NF14875) with a mutation caused by Tnt1 insertion into MtTCP18. Mutant analysis showed that the mutation of MtTCP18 altered plant structure, with increased plant height and branch number. Moreover, we found that the expression of auxin early response genes was modulated in the mutant. Therefore, MtTCP18 may be a promising candidate gene for breeders to optimize plant structure for crop improvement.

NODULE INCEPTION: a direct regulator of gibberellin biosynthesis during symbiotic nodulation.

This article is a Commentary on Gao et al. (2023), 239: 459–465.

Medicago truncatula resources to study legume biology and symbiotic nitrogen fixation.

Medicago truncatula is a chosen model for legumes towards deciphering fundamental legume biology, especially symbiotic nitrogen fixation. Current genomic resources for M. truncatula include a completed whole genome sequence information for R108 and Jemalong A17 accessions along with the sparse draft genome sequences for other 226 M. truncatula accessions. These genomic resources are complemented by the availability of mutant resources such as retrotransposon (Tnt1) insertion mutants in R108 and fast neutron bombardment (FNB) mutants in A17. In addition, several M. truncatula databases such as small secreted peptides (SSPs) database, transporter protein database, gene expression atlas, proteomic atlas, and metabolite atlas are available to the research community. This review describes these resources and provide information regarding how to access these resources.

MtING2 encodes an ING domain PHD finger protein which affects Medicago growth, flowering, global patterns of H3K4me3, and gene expression.

Flowering of the reference legume Medicago truncatula is promoted by winter cold (vernalization) followed by long-day photoperiods (VLD) similar to winter annual Arabidopsis. However, Medicago lacks FLC and CO, key regulators of Arabidopsis VLD flowering. Most plants have two INHIBITOR OF GROWTH (ING) genes (ING1 and ING2), encoding proteins with an ING domain with two anti-parallel alpha-helices and a plant homeodomain (PHD) finger, but their genetic role has not been previously described. In Medicago, Mting1 gene-edited mutants developed and flowered normally, but an Mting2-1 Tnt1 insertion mutant and gene-edited Mting2 mutants had developmental abnormalities including delayed flowering particularly in VLD, compact architecture, abnormal leaves with extra leaflets but no trichomes, and smaller seeds and barrels. Mting2 mutants had reduced expression of activators of flowering, including the FT-like gene MtFTa1, and increased expression of the candidate repressor MtTFL1c, consistent with the delayed flowering of the mutant. MtING2 overexpression complemented Mting2-1, but did not accelerate flowering in wild type. The MtING2 PHD finger bound H3K4me2/3 peptides weakly in vitro, but analysis of gene-edited mutants indicated that it was dispensable to MtING2 function in wild-type plants. RNA sequencing experiments indicated that >7000 genes are mis-expressed in the Mting2-1 mutant, consistent with its strong mutant phenotypes. Interestingly, ChIP-seq analysis identified >5000 novel H3K4me3 locations in the genome of Mting2-1 mutants compared to wild type R108. Overall, our mutant study has uncovered an important physiological role of a plant ING2 gene in development, flowering, and gene expression, which likely involves an epigenetic mechanism.

KIN3 impacts arbuscular mycorrhizal symbiosis and promotes fungal colonisation in Medicago truncatula.

Arbuscular mycorrhizal fungi help their host plant in the acquisition of nutrients, and this association is itself impacted by soil nutrient levels. High phosphorus levels inhibit the symbiosis, whereas high nitrogen levels enhance it. The genetic mechanisms regulating the symbiosis in response to soil nutrients are poorly understood. Here, we characterised the symbiotic phenotypes in four Medicago truncatula Tnt1-insertion mutants affected in arbuscular mycorrhizal colonisation. We located their Tnt1 insertions and identified alleles for two genes known to be involved in mycorrhization, RAM1 and KIN3. We compared the effects of the kin3-2 and ram1-4 mutations on gene expression, revealing that the two genes alter the expression of overlapping but not identical gene sets, suggesting that RAM1 acts upstream of KIN3. Additionally, KIN3 appears to be involved in the suppression of plant defences in response to the fungal symbiont. KIN3 is located on the endoplasmic reticulum of arbuscule-containing cortical cells, and kin3-2 mutants plants hosted significantly fewer arbuscules than the wild type. KIN3 plays an essential role in the symbiotic response to soil nitrogen levels, as, contrary to wild-type plants, the kin3-2 mutant did not exhibit increased root colonisation under high nitrogen.

Two linked resistance genes function divergently in defence against Verticillium Wilt in Alfalfa.

Two linked resistance genes function divergently in defence against Verticillium Wilt in Alfalfa.

Delineating the Tnt1 Insertion Landscape of the Model Legume Medicago truncatula cv. R108 at the Hi-C Resolution Using a Chromosome-Length Genome Assembly.

Legumes are of great interest for sustainable agricultural production as they fix atmospheric nitrogen to improve the soil. Medicago truncatula is a well-established model legume, and extensive studies in fundamental molecular, physiological, and developmental biology have been undertaken to translate into trait improvements in economically important legume crops worldwide. However, M. truncatula reference genome was generated in the accession Jemalong A17, which is highly recalcitrant to transformation. M. truncatula R108 is more attractive for genetic studies due to its high transformation efficiency and Tnt1-insertion population resource for functional genomics. The need to perform accurate synteny analysis and comprehensive genome-scale comparisons necessitates a chromosome-length genome assembly for M. truncatula cv. R108. Here, we performed in situ Hi-C (48×) to anchor, order, orient scaffolds, and correct misjoins of contigs in a previously published genome assembly (R108 v1.0), resulting in an improved genome assembly containing eight chromosome-length scaffolds that span 97.62% of the sequenced bases in the input assembly. The long-range physical information data generated using Hi-C allowed us to obtain a chromosome-length ordering of the genome assembly, better validate previous draft misjoins, and provide further insights accurately predicting synteny between A17 and R108 regions corresponding to the known chromosome 4/8 translocation. Furthermore, mapping the Tnt1 insertion landscape on this reference assembly presents an important resource for M. truncatula functional genomics by supporting efficient mutant gene identification in Tnt1 insertion lines. Our data provide a much-needed foundational resource that supports functional and molecular research into the Leguminosae for sustainable agriculture and feeding the future.

MtFULc controls inflorescence development by directly repressing MtTFL1 in Medicago truncatula

Flowering plants display a vast diversity of inflorescence architecture, which plays an important role in determining seed yield and fruit production. Unlike the model eudicot Arabidopsis thaliana that has simple inflorescences, most legume plants have compound types of inflorescences. Recent studies in the model legume species Pisum sativum and Medicago truncatula showed that the MADS-box transcription factors VEGETATIVE1/PsFRUITFULc/MtFRUITFULc (VEG1/PsFULc and MtFULc) are essential for the development of compound inflorescences by specifying the secondary inflorescence meristem identity. In this study, we report the isolation and characterization of two new mtfulc alleles by screening the M. truncatula Tnt1 insertion mutant collection. We found that MtFULc specifies M. truncatula secondary inflorescence meristem identity in a dose-dependent manner. Biochemical analysis and chromatin immunoprecipitation (ChIP) assays revealed that MtFULc acts as a transcriptional repressor to directly repress the expression of MtTFL1 through its promoter and 3′ intergenic region. Comprehensive genetic analysis suggest MtFULc coordinates with the primary inflorescence meristem maintainer MtTFL1 and floral meristem regulator MtPIM to control M. truncatula inflorescence development. Our findings help to elucidate the mechanism of MtFULc-mediated regulation of secondary inflorescence meristem identity and provide insights into understanding the genetic regulatory network underlying compound inflorescence development in legumes.

Genome-wide identification and characterization of cytokinin oxidase/dehydrogenase family genes in Medicago truncatula

Cytokinin oxidase/dehydrogenases (CKXs) play a key role in the irreversible degradation of phytohormone cytokinin that is necessary for various plant growth and development processes. However, thus far, detailed investigations of the CKX gene family in the model legume Medicago truncatula are limited. In this study, we identified 9 putative CKX homologues with conserved FAD- and cytokinin-binding domains in the M. truncatula genome. We analyzed their phylogenetic relationship, gene structure, conserved domain, expression pattern, protein subcellular locations and other properties. The tissue-specific expression profiles of the MtCKX genes are different among different members and these MtCKXs also displayed different patterns in response to synthetic cytokinin 6-benzylaminopurine (6-BA) and indole-3-acetic acid (IAA), suggesting their diverse roles in M. truncatula development. To further understand the biological function of MtCKXs, we identified and characterized mutants of each MtCKX by taking advantage of the Tnt1 mutant population in M. truncatula. Results indicated that M. truncatula plants harboring Tnt1 insertions in each single MtCKX genes showed no morphological changes in aerial parts, suggesting functional redundancy of MtCKXs in M. truncatula shoot development. However, disruption of Medtr4g126160, which is predominantly expressed in roots, leads to an obvious reduced primary root length and increased lateral root number, indicating the specific roles of cytokinin in regulating root architecture. We systematically analyzed the MtCKX gene family at the genome-wide level and revealed their possible roles in M. truncatula shoot and root development, which shed lights on understanding the biological function of CKX family genes in related legume plants.

The 3-ketoacyl-CoA synthase WFL is involved in lateral organ development and cuticular wax synthesis in Medicago truncatula.

A 3-ketoacyl-CoA synthase involved in biosynthesis of very long chain fatty acids and cuticular wax plays a vital role in aerial organ development in M. truncatula. Cuticular wax is composed of very long chain fatty acids and their derivatives. Defects in cuticular wax often result in organ fusion, but little is known about the role of cuticular wax in compound leaf and flower development in Medicago truncatula. In this study, through an extensive screen of a Tnt1 retrotransposon insertion population in M. truncatula, we identified four mutant lines, named wrinkled flower and leaf (wfl) for their phenotype. The phenotype of the wfl mutants is caused by a Tnt1 insertion in Medtr3g105550, encoding 3-ketoacyl-CoA synthase (KCS), which functions as a rate-limiting enzyme in very long chain fatty acid elongation. Reverse transcription-quantitative PCR showed that WFL was broadly expressed in aerial organs of the wild type, such as leaves, floral organs, and the shoot apical meristem, but was expressed at lower levels in roots. In situ hybridization showed a similar expression pattern, mainly detecting the WFL transcript in epidermal cells of the shoot apical meristem, leaf primordia, and floral organs. The wfl mutant leaves showed sparser epicuticular wax crystals on the surface and increased water permeability compared with wild type. Further analysis showed that in wfl leaves, the percentage of C20:0, C22:0, and C24:0 fatty acids was significantly increased, the amount of cuticular wax was markedly reduced, and wax constituents were altered compared to the wild type. The reduced formation of cuticular wax and wax composition changes on the leaf surface might lead to the developmental defects observed in the wfl mutants. These findings suggest that WFL plays a key role in cuticular wax formation and in the late stage of leaf and flower development in M. truncatula.

Dwarf and Increased Branching 1 controls plant height and axillary bud outgrowth in Medicago truncatula.

Optimizing plant architecture is an efficient approach for breeders to increase crop yields, and phytohormones such as gibberellins (GAs) play an important role in controlling growth. Medicago truncatula is a model legume species, but the molecular mechanisms underlying its architecture are largely unknown. In this study, we examined a tobacco retrotransposon Tnt1-tagged mutant collection of M. truncatula and identified dwarf and increased branching 1 (dib1), which exhibited extreme dwarfism and increased numbers of lateral branches. By analysis of the flanking sequences of Tnt1 insertions in different alleles of the tagged lines, we were able to clone DIB1. Linkage analysis and reverse screening of the flanking-sequence tags identified Medtr2g102570 as the gene corresponding to the DIB1 locus in the dib1 loss-of-function mutants. Phylogenetic analysis indicated that DIB1 was the ortholog of PsGA3ox1/Le in Pisum sativum. Expression analysis using a GUS-staining reporter line showed that DIB1 was expressed in the root apex, pods, and immature seeds. Endogenous GA4 concentrations were markedly decreased whilst some of representative GA biosynthetic enzymes were up-regulated in the dib1 mutant. In addition, exogenous application of GA3 rescued the dib1 mutant phenotypes. Overall, our results suggest that DIB1 controls plant height and axillary bud outgrowth via an influence on the biosynthesis of bioactive GAs. DIB1 could therefore be a good candidate gene for breeders to optimize plant architecture for crop improvement.

Blkn, a novel Medicago truncatula mutant achieving black nodule phenotype

blkn is a Medicago truncatula mutant that is achieving null function-black nodule phenotype. blkn is a Tnt1-retrotransposon mutant, Tnt1 is Nicotiana tabacum retro-transposon which is replicated via RNA copy and integrated in plant genome. Interestingly, blkn exhibited double contents of phenolic compounds comparing to R108 wild type. The mutated black nodule is displaying cells abnormality in both infection and nitrogen fixation zones. Transverse section of blkn nodule doesn’t display clearly characteristic shape like the control and the symbiotic cells don't totally filled with bacteroids along with high lignification at the cell wall periphery. Our goal was blkn mutant; phenotype, physiological, and molecular characterizations. AFLP-based PCR method was used to detect the mutated gene(s) in this mutant line. About 25 Tnt1-tagged fragments ranging from ~100 to ~500 bp were isolated, sequenced and submitted to Genbank. The Tnt1 insertion was precisely located next to the base number 303 post ATG start codon of M. truncatula L-type lectin-domain receptor kinase VII.2 gene encodes Lectin_LegB Receptor Like Kinase (MtLectinRLK). MtLectinRLK contains Lectin_legB domain, two transmembrane helix (TMhilex) and an extracellular Receptor Protein kinase (Pkinase). MtLectinRLK is an ancestry related to probable L-type lectin-domain containing receptor kinase Cicer arietinum, Trifolium pretense, Phaseolus vulgaris, Vigna radiate and Glycine soja.

The seed-specific heat shock factor A9 regulates the depth of dormancy in Medicago truncatula seeds via ABA signalling.

During the later stages of seed maturation, two key adaptive traits are acquired that contribute to seed lifespan and dispersal, longevity and dormancy. The seed-specific heat shock transcription factor A9 is an important hub gene in the transcriptional network of late seed maturation. Here, we demonstrate that HSFA9 plays a role in thermotolerance rather than in ex situ seed conservation. Storage of hsfa9 seeds of Medicago truncatula and Arabidopsis had comparable lifespan at moderate storage relative humidity (RH), whereas at high RH, hsfa9 seeds lost their viability much faster than wild type seeds. Furthermore, we show that in M. truncatula, Mthsfa9 seeds acquired more dormancy during late maturation than wild type. Transient expression of MtHSFA9 in hairy roots and transcriptome analysis of Mthsfa9 Tnt1 insertion mutants identified a deregulation of genes involved in ABA biosynthesis, catabolism and signalling. Consistent with these results, Mthsfa9 seeds exhibited increased ABA levels and higher sensitivity to ABA. These data suggest that in legumes, HSFA9 acts as a negative regulator of the depth of seed dormancy during seed development via the modulation of hormonal balance.

Medicago PHYA promotes flowering, primary stem elongation and expression of flowering time genes in long days

BackgroundFlowering time is an important trait for productivity in legumes, which include many food and fodder plants. Medicago truncatula (Medicago) is a model temperate legume used to study flowering time pathways. Like Arabidopsis thaliana (Arabidopsis), its flowering is promoted by extended periods of cold (vernalization, V), followed by warm long day (LD) photoperiods. However, Arabidopsis flowering-time genes such as the FLOWERING LOCUS C (FLC)/ MADS AFFECTING FLOWERING (MAF) clade are missing and CONSTANS-LIKE (CO-LIKE) genes do not appear to have a role in Medicago or Pisum sativum (pea). Another photoperiodic regulator, the red/far red photoreceptor PHYTOCHROME A (PHYA), promotes Arabidopsis flowering by stabilizing the CO protein in LD. Interestingly, despite the absence of CO-LIKE function in pea, PsPHYA plays a key role in promoting LD photoperiodic flowering and plant architecture. Medicago has one homolog of PHYA, MtPHYA, but its function is not known.ResultsGenetic analysis of two MtPHYA Tnt1 insertion mutant alleles indicates that MtPHYA has an important role in promoting Medicago flowering and primary stem elongation in VLD and LD and in perception of far-red wavelengths in seedlings. MtPHYA positively regulates the expression of MtE1-like (MtE1L), a homologue of an important legume-specific flowering time gene, E1 in soybean and other Medicago LD-regulated flowering-time gene homologues, including the three FLOWERING LOCUS T-LIKE (FT-LIKE) genes, MtFTa1, MtFTb1 and MtFTb2 and the two FRUITFULL-LIKE (FUL-LIKE) genes MtFULa and MtFULb. MtPHYA also modulates the expression of the circadian clock genes, GIGANTEA (GI) and TIMING OF CAB EXPRESSION 1a (TOC1a). Genetic analyses indicate that Mtphya-1 Mte1l double mutants flowered at the same time as the single mutants. However, Mtphya-1 Mtfta1 double mutants had a weak additive effect in delaying flowering and in reduction of primary axis lengths beyond what was conferred by either of the single mutants.ConclusionMtPHYA has an important role in LD photoperiodic control of flowering, plant architecture and seedling de-etiolation under far-red wavelengths in Medicago. It promotes the expression of LD-induced flowering time genes and modulates clock-related genes. In addition to MtFTa1, MtPHYA likely regulates other targets during LD floral induction in Medicago.

Lateral Leaflet Suppression 1 (LLS1), encoding the MtYUCCA1 protein, regulates lateral leaflet development in Medicago truncatula.

In species with compound leaves, the positions of leaflet primordiuminitiation are associated with local peaks of auxin accumulation. However, the role of auxin during the late developmental stages and outgrowth of compound leaves remains largely unknown. Using genome resequencing approaches, we identified insertion sites at four alleles of the LATERAL LEAFLET SUPPRESSION1 (LLS1) gene, encoding the auxin biosynthetic enzyme YUCCA1 in Medicago truncatula. Linkage analysis and complementation tests showed that the lls1 mutant phenotypes were caused by the Tnt1 insertions that disrupted the LLS1 gene. The transcripts of LLS1 can be detected in primordia at early stages of leaf initiation and later in the basal regions of leaflets, and finally in vein tissues at late leaf developmental stages. Vein numbers and auxin content are reduced in the lls1-1 mutant. Analysis of the lls1 sgl1 and lls1 palm1 double mutants revealed that SGL1 is epistatic to LLS1, and LLS1 works with PALM1 in an independent pathway to regulate the growth of lateral leaflets. Our work demonstrates that the YUCCA1/YUCCA4 subgroup plays very important roles in the outgrowth of lateral leaflets during compound leaf development of M.truncatula, in addition to leaf venation.

SMALL LEAF AND BUSHY1 controls organ size and lateral branching by modulating the stability of BIG SEEDS1 in Medicago truncatula.

Summary Organ size is a major agronomic trait that determines grain yield and biomass production in crops. However, the molecular mechanisms controlling organ size, especially in legumes, are poorly understood.Using forward genetic approaches in a Tnt1 insertion mutant population of the model legume Medicago truncatula, we identified SMALL LEAF AND BUSHY1 (SLB1), which is required for the control of organ size and lateral branching.Loss of function of SLB1 led to reduced leaf and flower size but increased lateral branch formation in M. truncatula. SLB1 encodes an F‐box protein, an orthologue of Arabidopsis thaliana STERILE APETALA (SAP), that forms part of an SKP1/Cullin/F‐box E3 ubiquitin ligase complex. Biochemical and genetic analyses revealed that SLB1 controls M. truncatula organ growth and lateral branching by modulating the stability of BIG SEEDS1 (BS1). Moreover, the overexpression of SLB1 increased seed and leaf size in both M. truncatula and soybean (Glycine max), indicating functional conservation.Our findings revealed a novel mechanism by which SLB1 targets BS1 for degradation to regulate M. truncatula organ size and shoot branching, providing a new genetic tool for increasing seed yield and biomass production in crop and forage legumes.

The MYB Activator WHITE PETAL1 Associates with MtTT8 and MtWD40-1 to Regulate Carotenoid-Derived Flower Pigmentation in Medicago truncatula.

Carotenoids are a group of natural tetraterpenoid pigments with indispensable roles in the plant life cycle and the human diet. Although the carotenoid biosynthetic pathway has been well characterized, the regulatory mechanisms that control carotenoid metabolism, especially in floral organs, remain poorly understood. In this study, we identified an anthocyanin-related R2R3-MYB protein, WHITE PETAL1 (WP1), that plays a critical role in regulating floral carotenoid pigmentation in Medicago truncatula Carotenoid analyses showed that the yellow petals of the wild-type M. truncatula contained high concentrations of carotenoids that largely consisted of esterified lutein and that disruption of WP1 function via Tnt1 insertion led to substantially reduced lutein accumulation. WP1 mainly functions as a transcriptional activator and directly regulates the expression of carotenoid biosynthetic genes including MtLYCe and MtLYCb through its C-terminal acidic activation motif. Further molecular and genetic analyses revealed that WP1 physically interacts with MtTT8 and MtWD40-1 proteins and that this interaction facilitates WP1's function in the transcriptional activation of both carotenoid and anthocyanin biosynthetic genes. Our findings demonstrate the molecular mechanism of WP1-mediated regulation of floral carotenoid pigmentation and suggest that the conserved MYB-basic-helix-loop-helix-WD40 regulatory module functions in carotenoid biosynthesis in M. truncatula, with specificity imposed by the MYB partner.

Medicago TERPENE SYNTHASE 10 Is Involved in Defense Against an Oomycete Root Pathogen.

In nature, plants interact with numerous beneficial or pathogenic soil-borne microorganisms. Plants have developed various defense strategies to expel pathogenic microbes, some of which function soon after pathogen infection. We used Medicago truncatula and its oomycete pathogen Aphanomyces euteiches to elucidate early responses of the infected root. A. euteiches causes root rot disease in legumes and is a limiting factor in legume production. Transcript profiling of seedlings and adult plant roots inoculated with A. euteiches zoospores for 2 h revealed specific upregulation of a gene encoding a putative sesquiterpene synthase (M. truncatula TERPENE SYNTHASE 10 [MtTPS10]) in both developmental stages. MtTPS10 was specifically expressed in roots upon oomycete infection. Heterologous expression of MtTPS10 in yeast led to production of a blend of sesquiterpenes and sesquiterpene alcohols, with NMR identifying a major peak corresponding to himalachol. Moreover, plants carrying a tobacco (Nicotiana tabacum) retrotransposon Tnt1 insertion in MtTPS10 lacked the emission of sesquiterpenes upon A. euteiches infection, supporting the assumption that the identified gene encodes a multiproduct sesquiterpene synthase. Mttps10 plants and plants with reduced MtTPS10 transcript levels created by expression of an MtTPS10-artificial microRNA in roots were more susceptible to A. euteiches infection than were the corresponding wild-type plants and roots transformed with the empty vector, respectively. Sesquiterpenes produced by expression of MtTPS10 in yeast also inhibited mycelial growth and A. euteiches zoospore germination. These data suggest that sesquiterpene production in roots by MtTPS10 plays a previously unrecognized role in the defense response of M. truncatula against A. euteiches.

Genome-wide analysis of flanking sequences reveals thatTnt1 insertion is positively correlated with gene methylation in Medicago truncatula.

From a single transgenic line harboring five Tnt1 transposon insertions, we generated a near-saturated insertion population in Medicago truncatula. Using thermal asymmetric interlaced-polymerase chain reaction followed by sequencing, we recovered 388888 flanking sequence tags (FSTs) from 21 741 insertion lines in this population. FST recovery from 14 Tnt1 lines using the whole-genome sequencing (WGS) and/or Tnt1-capture sequencing approaches suggests an average of 80 insertions per line, which is more than the previous estimation of 25 insertions. Analysis of the distribution pattern and preference of Tnt1 insertions showed that Tnt1 is overall randomly distributed throughout the M. truncatula genome. At the chromosomal level, Tnt1 insertions occurred on both arms of all chromosomes, with insertion frequency negatively correlated with the GC content. Based on 174 546 filtered FSTs that show exact insertion locations in the M. truncatula genome version 4.0 (Mt4.0), 0.44 Tnt1 insertions occurred per kb, and 19 583 genes contained Tnt1 with an average of 3.43 insertions per gene. Pathway and gene ontology analyses revealed that Tnt1-inserted genes are significantly enriched in processes associated with 'stress', 'transport', 'signaling' and 'stimulus response'. Surprisingly, gene groups with higher methylation frequency were more frequently targeted for insertion. Analysis of 19 583 Tnt1-inserted genes revealed that 59% (1265) of 2144 transcription factors, 63% (765) of 1216 receptor kinases and 56% (343) of 616 nucleotide-binding site-leucine-rich repeat genes harbored at least one Tnt1 insertion, compared with the overall 38% of Tnt1-inserted genes out of 50 894 annotated genes in the genome.