Excision of a transposable element from a viral vector introduced into maize plants by agroinfection

The geminivirus maize streak virus (MSV) was used as a vector to introduce the maize transposable element Dissociation (Ds) and to study its excision in maize plants. MSV carrying Ds1 in its genome was introduced into maize plants by agroinfection. Excision of the Ds1 element from the MSV genome was detected only when functions from the transposable element Activator (Ac) were supplied in trans, either endogenously by the recipient maize plant or by co-transformation with Agrobacterium carrying a genomic Ac clone. The excision of Ds1 could easily be visualized by the appearance of viral symptoms induced by the revertant virus. The junction sequences left on the MSV genome after excision revealed 'footprints' typical of transposition as described for maize. From these results, we conclude that transposition functions in our system and that the use of the MSV replicon provides a rapid and simple tool for the investigation of the excision of transposable elements in maize plants.     

The binding motifs for Ac transposase are absolutely required for excision of Ds1 in maize

A reverse genetic system for studying excision of the transposable elementDs1 in maize plants has been established previously. In this system, theDs1 element, as part of the genome of maize streak virus (MSV), is introduced into maize plants via agroinfection. In the presence of theAc element, excision ofDs1 from the MSV genome results in the appearance of viral symptoms on the maize plants. Here, we used this system to study DNA sequences requiredin cis for excision ofDs1. TheDs1 element contains theAc transposase binding motif AAACGG in only one of its subterminal regions (defined here as the 5′ subterminal region). We showed that mutation of these motifs abolished completely the excision capacity ofDs1. This is the first direct demonstration that the transposase binding motifs are essential for excision. Mutagenesis with oligonucleotide insertions in the other (3′) subterminal region resulted in elements with either a reduced or an increased excision efficiency, indicating that this subterminal region also has an important function.     

Mechanism of Ds1 excision from the genome of maize streak virus

Summary We have previously shown that the maize transposable element Ds1 introduced into maize plants by agroinfection can be excised from the genome of geminivirus maize streak virus (MSV). Excision depended strictly on the presence of an active Ac element in the plants. In this study, the excision products or footprints left in the MSV genome after Ds1 excision were extensively characterized and the effects of flanking sequences on Ds1 excision were analysed. Most types of footprints obtained were comparable to those described for Ds1 excision in the maize genome, and could be explained by the models proposed for excision of plant transposable elements. In two revertants, however, some terminal sequences of the Ds1 element were found to have been left behind at the excision site. The finding of this novel type of Ds1 footprint indicated that gene conversion events occurred during and/or after Ds1 excision from the MSV genome. A partial deletion of one copy of the 8 by duplications flanking the Ds1 element had no effect on the frequency or on the types of footprints of Ds1 excision from the MSV genome. Thus, the duplicated 8 by sequences flanking the transposable element are not involved in Ds1 excision. These results, as well as a statistical analysis of the modifications of the bases flanking the Ds1 element after excision, are discussed in terms of excision models.     

Gametophyte-specific transposition of the maize Ds element in transgenic tobacco

To assess the potential advantages of a transposon-tagging system based on gametophyte-specific transposition a fusion between the anther-specific Arabidopsis thaliana apg promoter and the maize Ac transposase gene was constructed and introduced into tobacco. The ability of this transposase source to activate Ds transposition in a developmentally controlled manner was monitored by crossing to plants harbouring the cell autonomous excision marker gene construct, Ds —SPT. A number of fully green, streptomycin-resistant seedlings resulting from germinal transposition events were observed in the progeny of apg -TPase x Ds —SPT F1 plants. Streptomycin-resistant sectors were not observed in either F₁ seedlings or F₂ progeny, indicating a complete lack of somatic excision. Further crosses of apg —TPase sources to plants containing Ds—bar herbicide selection excision marker constructs gave reproducible gametophytic excision frequencies of up to 0.3%. Sequencing of Ds excision sites from F₂ seedlings derived from single F₁ plants revealed various sequence alterations in the original Ds insertion 'footprint' indicative of independent Ds excision events. Independent re-insertion was confirmed by Southern analysis of F₂ siblings. It is concluded that apg -controlled Ac transposase expression activates male gametophyte-specific Ds transposition.     

Functional analysis of cis-elements, auxin response and early developmental profiles of the mannopine synthase bidirectional promoter

Summary The dual MAS1-2 promoter regulating two divergently transcribed mannopine synthase genes has been widely employed in plant expression vectors. As part of an effort towards its rational design as a genetic engineering tool, we have undertaken a functional analysis of the promoter by deletion mutagenesis and by the use of hybrid promoter constructs. Our results indicate that the central region of the intergenic promoter is composed of at least four domains. Three of these contain complementary sequences, which can potentially hybridize to form alternative palindromic structures. These three domains can function cooperatively, and in an orientation-independent manner, in imparting a sevenfold higher expression level at the 2 end relative to the corresponding 1. The remaining domain is characterized by tracts of repeated A/T-rich elements, and appears to confer the weak activity at the MAS1 promoter end. However, even though this A/T-rich DNA segment is functional, our deletion analysis provided strong evidence that it is completely dispensable for wild-type promoter activity. In addition, the relative distances between these enhancer domains and the 1–2 TATA-proximal regions can have a pronounced influence on the level of expression in both directions. In young tobacco seedlings, the two promoter ends are expressed in similar, if not identical, tissues in the aerial parts of the plants, but major differences can be observed in roots. Transient expression assays using hybrid promoter constructs showed that cis-elements that can respond to auxin induction signals are redundant in nature, in that they are dispersed throughout the promoter and showed no obvious consensus sequence.     

Somatic and germinal activities of maize Activator (Ac) transposase mutants in transgenic tobacco

The properties have been investigated of two deletion derivatives of the transposase protein (TPase) of maize transposable element Ac in transgenic tobacco. The wild-type and mutant TPases were expressed as fusions to the cauliflower mosaic virus 35S promoter. A deletion of 102 amino acids from the N-terminus, TPase(103–807), induces Ds excisions from a SPT::Ds reporter locus with a higher frequency than the wild-type TPase. The increased transpositional activity of TPase(103–807) is a dominant trait, as seedlings coexpressing truncated and wild-type TPase show the characteristic TPase(103–807) variegation pheno-type. A transpositionally inactive TPase deletion derivative lacking 188 amino acids from the N-terminus inhibits the transpositional activity of the wild-type TPase.     

Excision Patterns of Activator (Ac) and Dissociation (Ds) Elements in Zea Mays L.: Implications for the Regulation of Transposition

The pattern of aleurone variegation of maize kernels carrying Ac and bz-m2(DI) as reporter allele for Ac activity depends on the dosage of both Ac and Ds. Alterations of Ac dosage can abolish Ds excision at certain times and allow it to occur at other times. wx-m7 and wx-m9 are different Ac insertions in the Waxy gene which have different dosage effects on Ds excision. Kernels, heterozygous for the two Ac alleles and being either wx-m7/wx-m7/wx-m9 or wx-m9/wx-m9/wx-m7 exhibit characteristic patterns of predominantly late excisions; this is in strong contrast to the pattern of early excisions present on wx-m7/wx-m7/wx-m7 homozygotes. This observation supports the hypothesis that the Ac alleles express different amounts of transposase (TPase) during development and that above a certain level of TPase transposition is inhibited. Furthermore, experimental results suggest that the frequency of Ac-induced events is influenced by the dosage and composition of the transactivated Ds or Ac allele. Thus, transposition frequency seems not to be exclusively determined in trans by the amount of active TPase, but also by specific cis-acting properties of the TPase substrate.     

The binding motifs forAc transposase are absolutely required for excision ofDs1 in maize

A reverse genetic system for studying excision of the transposable elementDs1 in maize plants has been established previously. In this system, theDs1 element, as part of the genome of maize streak virus (MSV), is introduced into maize plants via agroinfection. In the presence of theAc element, excision ofDs1 from the MSV genome results in the appearance of viral symptoms on the maize plants. Here, we used this system to study DNA sequences requiredin cis for excision ofDs1. TheDs1 element contains theAc transposase binding motif AAACGG in only one of its subterminal regions (defined here as the 5′ subterminal region). We showed that mutation of these motifs abolished completely the excision capacity ofDs1. This is the first direct demonstration that the transposase binding motifs are essential for excision. Mutagenesis with oligonucleotide insertions in the other (3′) subterminal region resulted in elements with either a reduced or an increased excision efficiency, indicating that this subterminal region also has an important function.     

Differential expression of the Sesbania rostrata leghemoglobin glb3 gene promoter in transgenic legume and non-legume plants

The involvement of the Sesbania rostrata glb3 gene promoter NICE (nodule-infected cell expression) element in root-enhanced expression of 5-Srglb3-uidA-3nos chimeric gene was investigated in transgenic Nicotiana tabacum plants. The full-length wild-type Srglb3 promoter directed root meristem-enhanced expression in transgenic tobacco plants. The expression pattern of nine selected Srglb3 promoter mutations in the NICE element was examined in transgenic tobacco plants and compared with the pattern observed in nodules of transgenic Lotus corniculatus plants. The results suggest that the highly conserved motifs in the NICE element play an important role in expression in roots of non-legume plants.     

In vivo aggregation of maize Activator (Ac) transposase in nuclei of maize endosperm and Petunia protoplasts

The transposase (TPase) of the maize transposon Activator (Ac) accumulates in the nuclei of maize endosperm and transfected Petunia protoplasts, where it aggregates into rod-like structures about 2 μm in length. In petunia protoplasts the amount of TPase aggregates increases with the strength of the promoter fused to the Ac-coding region. The excision frequency of a Ds element, however, does not increase proportionally. The data suggest that the aggregated TPase is not responsible for the mobilization of the Ds element, but rather is a transpositionally inactive form of the protein. In contrast to the full-length TPase, a functional, N-terminally truncated TPase derivative is inefficiently transported into the nucleus at high expression levels and aggregates predominantly in the cytoplasm. Accordingly, the N-terminus of the TPase is involved in nuclear localization and/or aggregation.     

A –308 deletion of the tomato LAP promoters is able to direct flower-specific and MeJA-induced expression in transgenic plants

Tomato and potato leucine aminopeptidase (LAP) mRNAs are induced in response to mechanical wounding and the wound signal molecules, ABA and jasmonic acid. Here, we report the isolation of two LAP genes, LAP17.1A and LAP17.2, from tomato. Functional analysis in transgenic tomato and potato plants show that fusions of the corresponding 5 non-coding regions to the gusA gene are constitutively expressed in flowers and induced in leaves upon wounding or by treatment with methyl jasmonate (MeJA). Comparison of the 5 non-coding regions of the two genes revealed a region from –317 to –3 relative to the ATG, which is strongly conserved in both promoters. This 0.3 kb proximal promoter fragment is sufficient to direct flower-specific and MeJA-inducible GUS activity in transgenic potato plants, and thus contains a MeJA-responsive element that mediates induction by MeJA. Dimeric TGACG motifs or G-box elements similar to those found in other MeJA-inducible genes are not observed in this region, which suggests that a different DNA sequence is involved in MeJA induction of the LAP genes.     

Co-regulation with genes of phospholipid biosynthesis of the CTR/HNM1-encoded choline/nitrogen mustard permease in Saccbaromyces cerevisiae

An 815 by region of the promoter of the Saccharomyces cerevisiae gene CTR/HNM1, encoding choline permease was sequenced and its regulatory function analysed by deletion studies in an in-frame promoter-lacZ construct. In addition to the TATA box, a 10 by motif (consensus 5-CATGTGAAAT-3) was found to be mandatory for CTR/HNM1 expression. This decamer motif is located between nucleotides –262 and –271 and is identical in 9 of 10 by with the regulatory motif found in the S. cerevisiae INO1 and CHO1 genes. Constructs with the 10 by sequence show high constitutive expression, while elimination or alterations at three nucleotide positions, of the decamer motif in the context of an otherwise unchanged promoter leads to total loss of -galactosidase production. Expression of the CTR/HNM1 gene in wild-type cells is regulated by the phospholipid precursors inositol and choline; no such influence is seen in cells bearing mutations in the phospholipid regulatory genes INO2, INO4, and OPI1. There is no regulation by INO2 and OPI1 in the absence of the decamer motif. However constructs not containing this sequence (promoter intact to positions –213 or –152) are still controlled by INO4. Other substrates of the choline permease, i.e. ethanolamine, nitrogen mustard and nitrogen half mustard do not regulate expression of CTR/HNM1.     

Characterization of the U3 and U6 snRNA genes from wheat: U3 snRNA genes in monocot plants are transcribed by RNa polymerase III

We have demonstrated recently that the genes encoding the U3 small nuclear RNA (snRNA) in dicot plants are transcribed by RNA polymerase III (pol III), and not RNA polymerase II (pol II) as in all other organisms studied to date. The U3 gene was the first example of a gene transcribed by different polymerases in different organisms. Based on phylogenetic arguments we proposed that a polymerase specificity change of the U3 snRNA gene promoter occurred during plant evolution. To map such an event we are examining the U3 gene polymerase specificity in other plant species. We report here the characterization of a U3 gene from wheat, a monocot plant. This gene contains the conserved promoter elements, USE and TATA, in a pol III-specific spacing seen also in a wheat U6 snRNA gene characterized in this report. Both the U3 and the U6 genes possess typical pol III termination signals but lack the cis element, responsible for 3-end formation, found in all plant pol II-specific snRNA genes. In addition, expression of the U3 gene in transfected maize protoplasts is less sensitive to -amanitin than a pol II-transcribed U2 gene. Based on these data we conclude that the wheat U3 gene is transcribed by pol III. This observation suggests that the postulated RNA polymerase specificity switch of the U3 gene took place prior to the divergence of angiosperm plants into monocots and dicots.     

A 42 bp fragment of the pmas1′ promoter containing an ocs-like element confers a developmental,wound- and chemically inducible expression pattern

Synthesis of mannopine in plant tissues infected with Agrobacterium tumefaciens is controlled by a divergent promoter (pmas2 and pmas1) that in 479 bp contains all the cis-acting elements necessary to direct tissue-specific and wound-inducible expression. In this report, using transgenic tobacco plants harboring a pmas1--glucuronidase (GUS) gene fusion, we investigated the developmental expression pattern directed by pmas1 in the early stages of development and the responses of pmas1 to different chemical inducers. It was found that this promoter can respond to auxins, cytokinins, methyl jasmonate (MJ), salicylic acid (SA) and its analogue 2,6-dichloroisonicotinic acid (iNA). Treatment with chemical inducers also showed that the effects of iNA are organ-dependent, that wound-induction is a complex response mediated by at least two different chemical signals, and that MJ stimulates changes in the tissue-specific and developmental expression pattern directed by the pmas1 promoter. Using chimeric promoters we demonstrate that an ocs-like element (ocs+1) directs MJ responses in an orientation-dependent manner and that sequences around the ocs+1 are important to maintain the inducible and developmental properties of this cis-regulatory element.     

Infrequent transposition of Ac in lettuce,Lactuca sativa

The maize transposable element Activator (Ac) is being used to develop a transposon mutagenesis system in lettuce, Lactuca sativa. Two constructs containing the complete Ac from the waxy-m7 locus of maize were introduced into lettuce and monitored for activity using Southern analysis and PCR amplification of the excision site. No transposition of Ac was detected in over 32 transgenic R₁ plants, although these constructs were known to provide frequent transposition in other species. Also, no transposition was observed in later generations. In subsequent experiments, transposition was detected in lettuce calli using constructs that allowed selection for excision events. In these constructs, the neomycin phosphotransferase II gene was interrupted by either Ac or Ds. Excision was detected as the ability of callus to grow on kanamycin. Synthesis of the transposase from the cDNA of Ac expressed from the T-DNA 2 promoter resulted in more frequent excision of Ds than was observed with the wild-type Ac. No excision was observed with Ds in the absence of the transposase. The excision events were confirmed by amplification of the excision site by PCR followed by DNA sequencing. Excision and reintegration were also confirmed by Southern analysis. Ac/Ds is therefore capable of transposition in at least calli of lettuce.     

Stable transfer and expression of chimeric genes in licorice (Glycyrrhiza uralensis) using an Ri plasmid binary vector

The pharmaceutically important plant, licorice (Glycyrrhiza uralenesis Fisher), was transformed with a binary vector system of an Ri plasmid, pRi15834, and a mini Ti vector, pGSGluc1, containing chimeric neo and gus genes. The transgenic state of transformed roots was confirmed by detection of agropine and mannopine and by Southern blot hybridization with T-DNA of pGSGluc1. One to three copies of T-DNA of pGSGluc1 was integrated into the genomic DNA of G. uralensis. The expression of chimeric neo and gus genes driven by TR 1 and 2 promoters, respectively, was demonstrated by enzymatic assays. Histochemical analysis showed that the chimeric TR2-gus gene was expressed specifically in phloem and pericycle tissues of the transformed licorice roots.Abbreviations NPT-II neomycin phosphotransferase II - neo NPT-II gene from Tn5 - GUS ß-glucuronidase - gus GUS gene from Escherichia coli - TR 1–2 genes 1 and 2 of TR-DNA of pTiAch5 - Rif rifampicin