A highly conserved domain of the maize activator transposase is involved in dimerization

Previous studies have presented indirect evidence that the transposase of the maize transposable element Activator (TPase) is active as an oligomer and forms inactive macromolecular complexes expressed in large amounts. Here, we have identified and characterized a dimerization domain at the C terminus of the protein. This domain is the most highly conserved region in the transposases of elements belonging to the Activator superfamily (hAT element superfamily) and contains a characteristic signature motif. The isolated dimerization domain forms extremely stable dimers in vitro. Interestingly, mutations in five of the six conserved residues of the signature motif do not affect in vitro dimerization, whereas mutations in other, less strictly conserved residues of the signature motif do. Loss of dimerization in vitro correlates with loss of TPase activity in vivo. As revealed by in situ immunofluorescence staining of mutant TPase proteins, the dimerization domain also is involved in forming inactive macromolecular aggregates when overexpressed, and the TPase contains one or more additional interaction functions.     

Excision of Ds1 from the genome of maize streak virus in response to different transposase-encoding genes

We have previously established a reverse genetic system for studying excision of the transposable element Ds1 in maize plants. Ds1 carried by the genome of maize streak virus (MSV) is introduced into maize plants by agroinfection. Excision of Ds1 from the MSV genome depends on the presence of an active Ac element in the recipient maize plants. With the purpose of exploiting MSV-Ds1 as vector for maize transformation, we studied different genes encoding the transposase (TPase) for their efficiency of activating Ds1 excision. These genes were inserted in the same T-DNA carrying MSV-Ds1 and introduced into maize plants by Agrobacterium-mediated transformation. We showed that the wild-type TPase transcribed by the 2 promoter produced much higher efficiency of Ds1 excision than that transcribed by the Ac promoter. In contrast to what had been observed in tobacco and petunia, the truncated TPase (103–807) lacking the amino-terminal 102 amino acids gave a much more reduced Ds1 excision efficiency than the wild-type TPase when both genes were transcribed by the 2 promoter.     

Transposition of the maize Ds element from a viral vector to the rice genome

The geminivirus miscanthus streak virus (MiSV) was used as a gene vector to study the transposition of the maize Ds element in rice protoplasts. Efficient excision of the Ds from the MISV vector was observed only when the MiSV vector was allowed to replicate and the plasmid expressing the transposase gene encoded by Ac was co-transfected. Under the same condition, the Ds carrying a hygromycin phosphotransferase gene (Ds::HPT) was also efficiently excised. Hygromycin-resistant calli were obtained by culturing these transfected protoplasts in order to examine the transposition of the excised Ds::HPT into the rice genome. In five out of 16 calli examined, the Ds::HPT, but not the vector sequence, was integrated into the rice genome and 8 bp target site duplication typical of Ac/Ds transposition was generated. These results show that the Ds::HPT inserted in the MISV vector transposed directly into the rice genome. This demonstrates the direct transposition of a cloned plant transposable element into the plant genome. Implications of these finding are discussed.     

One of three nuclear localization signals of maize Activator (Ac) transposase overlaps the DNA-binding domain

The nuclear localization sequences (NLSs) of the Ac transposase (TPase) protein have been characterized by indirect immunofluorescence detection of TPase deletion derivatives and TPase/β-glucuronidase (GUS) fusion proteins in transiently transfected Petunia cells. The TPase contains three NLSs near its amino-terminal end, NLS(44–62), NLS(159–178) and NLS(174–206), each of which is sufficient to redirect GUS to the nucleus. Deletion of the N-terminal 102 TPase residues including NLS(44–62) results in strongly reduced nuclear import of the truncated TPase. NLS(44–62) and NLS(159–178) are bipartite NLSs, whereas the structure of NLS(174–206) does not allow a classification into one of the three major NLS categories. NLS(174–206) overlaps with the basic DNA-binding domain of TPase. A substitution of two amino acids in this segment (HiS₁₉₁→Arg and Arg₁₉₃→His) results in a total loss of DNA-binding activity, but retains reduced NLS activity. Accordingly, the two functions can be separated. In addition, we show that a NLS-deficient 71 kDa TPase derivative is co-imported into the nucleus in the presence of wildtype TPase.     

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.     

A candidate autonomous version of the wheat MITE <Emphasis Type="Italic">Hikkoshi</Emphasis> is present in the rice genome

A miniature inverted-repeat transposable element (MITE), designated as Hikkoshi, was previously identified in the null Wx-A1 allele of Turkish bread wheat lines. This MITE is 165 bp in size and has 12-bp terminal inverted repeats (TIRs) flanked by 8-bp target site duplications (TSDs). Southern and PCR analyses demonstrated the presence of multiple copies of Hikkoshi in the wheat genome. Database searches indicated that Hikkoshi MITEs are also present in barley, rice and maize. A 3.4-kb element that has Hikkoshi-like TIRs flanked by 8-bp TSDs has now been identified in the rice genome. This element shows high similarity to the 5 subterminal region of the wheat Hikkoshi MITE and contains a transposase (TPase) coding region. The TPase has two conserved domains, ZnF_TTF and hATC, and its amino acid sequence shows a high degree of homology to TPases encoded by Tip100 transposable elements belonging to the hAT superfamily. We designated the 3.4-kb element as OsHikkoshi. Several wheat clones deposited in EST databases showed sequence similarity to the TPase ORF of OsHikkoshi. The sequence information from the TPase of OsHikkoshi will thus be useful in isolating the autonomous element of the Hikkoshi system from wheat.     

Mechanical release and lectin labeling of maize root protoplasts

Summary An improved method for the mechanical release of protoplasts from plant tissues is described. The historically-low yield of mechanically-released protoplasts is greatly increased by use of a simple electrically-driven tissue sheer and by optimization of various other steps in the procedure. As counted by light microscopy of a purified preparation, the number of mechanically-released protoplasts obtained is about 6×10⁴ per gram fresh weight of cortical tissue from the primary root of maize (Zea mays L. WF9×Mo 17) seedlings. Nuclear staining of the preparation, however, shows that about half of these protoplasts lack a nucleus and thus are actually subprotoplasts. Comparison of lectin binding to the plasma membranes of mechanically-and enzymatically-released protoplasts shows that both types contain binding sites forRicinus communis agglutinin. Binding sites for peanut (Arachis hypogaea) agglutinin are not naturally present on mechanically-released protoplasts but are generated by exposure to a mixture of Cellulysin and Pectolyase Y-23, the cell wall-degrading enzymes used to prepare enzymatically-released protoplasts.Abbreviations BSA bovine serum albumin - DDT dithiothreitol - gfw gram fresh weight - Mes 2-(N-morpholino) ethanesulfonic acid - PNA peanut (Arachis hypogaea) agglutinin - RCA Ricinus communis agglutinin - Tris tris(hydroxymethyl)aminomethane     

Maize Activator transposase has a bipartite DNA binding domain that recognizes subterminal sequences and the terminal inverted repeats

 The mobility of maize transposable element Activator (Ac) is dependent on the 11-bp terminal inverted repeats (IRs) and approximately 250 subterminal nucleotides at each end. These sequences flank the coding region for the transposase (TPase) protein, which is required for the transposition reaction. Here we show that Ac TPase has a bipartite DNA binding domain, and recognizes the IRs and subterminal sequences in the Ac ends. TPase binds cooperatively to repetitive ACG and TCG sequences, of which 25 copies are found in the 5′ and 20 copies in the 3′ subterminal regions. TPase affinity is highest when these sites are flanked on the 3′ side by an additional G residue (A/TCGG), which is found at 75% of binding sites. Moreover, TPase binds specifically to the Ac IRs, albeit with much lower affinity. Two mutations within the IRs that immobilize Ac abolish TPase binding completely. The basic DNA binding domain of TPase is split into two subdomains. Binding to the subterminal motifs is accomplished by the C-terminal subdomain alone, whereas recognition of the IRs requires the N-terminal subdomain in addition. Furthermore, TPase is extremely flexible in DNA binding. Two direct or inverted binding sites are bound equally well, and sites that are five to twelve bases apart are similarly well bound. The consequences of these findings for the Ac transposition reaction are discussed. Received: 3 June 1996 / Accepted: 29 July 1996     

PEG-mediated transient gene expression and silencing system in maize mesophyll protoplasts: a valuable tool for signal transduction study in maize

Polyethylene glycol (PEG)-mediated transient gene expression and silencing in protoplasts is widely applied in model plants such as Arabidopsis thaliana and rice. Here, we developed an efficient transient gene expression system based on the PEG-mediated method both in etiolated and green maize mesophyll protoplasts. The results showed that both yellow fluorescent protein encoding gene and glucuronidase encoding gene were efficiently expressed in maize protoplasts. More importantly, double-stranded RNAs (dsRNAs) can also be transfected into maize protoplasts by the PEG-mediated method to specifically silence exogenous and endogenous genes. Our results showed that dsRNA can be used to knockdown both exogenous and endogenous gene expression. Furthermore, bimolecular fluorescence complementation system for the detection of protein–protein interactions in maize protoplasts was developed. We also overexpressed and knockdowned the mitogen-activated protein kinase encoding gene ZmMPK5 to investigate the role of ZmMPK5 in abscisic acid (ABA)-induced antioxidant defense in maize protoplasts. This method here we reported will be valuable for signal transduction study in maize.     

Introduction and transposition of the maize transposable element Ac in rice (Oryza sativa L.).

Summary To develop a transposon tagging system in an important cereal plant, rice (Oryza sativa L.), the maize transposable element Ac (Activator) was introduced into rice protoplasts by electroporation. We employed a phenotypic assay for excision of Ac from the selectable hph gene encoding resistance to hygromycin B. Southern blot analysis of hygromycin B-resistant calli showed that the Ac element can transpose from the introduced hph gene into the rice chromosomes. Sequence analysis of several Ac excision sites in the hph gene revealed sequence alterations characteristic of the excision sites of this plant transposable element. The Ac element appears to be active during development of transgenic rice plants from calli. Moreover, hybridization patterns of different leaves from the same plant indicated that some Ac elements are stable whereas others are able to transpose further during development of leaves. The results indicate that the introduced Ac element can transpose efficiently in transgenic rice plants.     

Analysis of the frequency of inheritance of transposed Ds elements in Arabidopsis after activation by a CaMV 35S promoter fusion to the Ac transposase gene

The Ac/Ds transposon system of maize shows low activity in Arabidopsis. However, fusion of the CaMV 35S promoter to the transposase gene (35S::TPase) increases the abundance of the single Ac mRNA encoded by Ac and increases the frequency of Ds excision. In the experiments reported here it is examined whether this high excision frequency is associated with efficient re-insertion of the transposon. This was measured by using a Ds that carried a hygromycin resistance gene (HPT) and was inserted within a streptomycin resistance gene (SPT). Excision of Ds therefore gives rise to streptomycin resistance, while hygromycin resistance is associated with the presence of a transposed Ds or with retention of the element at its original location. Self-fertilisation of most individuals heterozygous for Ds and 35S::TPase produced many streptomycin-resistant (strep^r) progeny, but in many of these families a small proportion of strep^r seedlings were also resistant to hygromycin (hyg^r). Nevertheless, 70% of families tested did give rise to at least one strep^r, hyg^r seedling, and over 90% of these individuals carried a transposed Ds. In contrast, the Ac promoter fusion to the transposase gene (Ac::TPase) produced fewer strep^rhyg^r progeny, and only 53% of these carried a transposed Ds. However, a higher proportion of the strep^r seedlings were also hyg^r than after activation by 35S::TPase. We also examined the genotype of strep^r, hyg^r seedlings and demonstrated that after activation by 35S::TPase many of these were homozygous for the transposed Ds, while this did not occur after activation by Ac::TPase. From these and other data we conclude that excisions driven by 35S::TPase usually occur prior to floral development, and that although a low proportion of strep^r progeny plants inherit a transposed Ds, those that do can be efficiently selected with an antibiotic resistance gene contained within the element. Our data have important implications for transposon tagging strategies in transgenic plants and these are discussed.     

Microcallus formation from maize protoplasts prepared from embryogenic callus

Conditions have been developed that induce maize (Zea mays L.) protoplasts to re-synthesize cell walls and to initiate cell divisions. Two types of embryogenic maize callus were used as a source of protoplasts: a heterogeneous callus (Type I) derived from immature embryos after three weeks in culture, and a friable, rapidly growing callus (Type II) selected from portions of the Type I callus. Many variables in the growth conditions of the donor tissue (type of medium, transfer schedule, age of callus), protoplast isolation solutions (pH, osmolarity, type and concentration of cell wall hydrolyzing enzymes, addition of polyamines) and conditions (amount of time in enzyme, amount of tissue per volume of enzyme incubation medium, agitation, preplasmolysis of source tissue, type of callus), and purification procedures (filtration and-or flotation), were found to affect both yield and viability of protoplasts (based upon fluorescein-diacetate staining). Our isolation procedure yielded high numbers of viable, uninucleated maize callus protoplasts which were densely cytoplasmic and varied in size from 20 to 50 m in diameter. Protoplasts plated in solid medium formed walls and divided several times. Of several gelling agents tested for protoplast propagation, only agarose resulted in protoplasts capable of sustained divisions leading to the formation of microcalli. Plating efficiency was established over a wide range of protoplast densities (10³–10⁷ protoplasts/ml). Highest plating efficiency (25%) was obtained at 1·10⁶ protoplasts/ml). The resulting microcalli grew to be dense clusters of about 0.1–0.5 mm in diameter and then stopped growing. Nurse cultures of maize and carrot (Daucus carota L.), were used to establish that individual protoplasts (not contaminating cells or cell clusters) formed walls and divided. Nurse cultures also increased the efficiency of microcallus formation from protoplasts.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) salts - MS 1D Murashige and Skoog salts with 1 mg/l 2,4-D - MS 2D Murashige and Skoog salts with 2 mg/l 2,4-D - N₆ medium of Chu et al. (1975) - NN67-mod medium of Nitsch and Nitsch (1967) as modified in the present paper - FDA fluorescein diacetate - LMP low melting point     

Homologous recombination between plasmid DNA molecules in maize protoplasts

Summary The requirements for homologous recombination between plasmid DNA molecules have been studied using the PEG (polyethylene glycol)-mediated transformation system of maize (Zea mays L.) protoplasts coupled with the transient expression assay for -glucuronidase (GUS). Two plasmids were introduced into maize protoplasts; one plasmid (pB×26) contained a genomic clone of the Adh1 maize gene; the other plasmid (piGUS) was a promoterless construction containing part of intron A of the Adhl gene fused to the gusA coding sequence. Thus, the two vectors shared an effective homologous region consisting of a 459 by (Hindlll—PvuII) fragment of the yAdh1 intron A sequence. An active gusA fusion gene would result upon homologous recombination between the plasmids within the intron A sequence, and indeed GUS activity was observed in extracts following co-transformation of maize protoplasts with the two plasmids. The presence of recombinant DNA molecules in protoplast DNA isolated 1 day after co-transformation was verified using polymerase chain reactions (PCR) and Southern blots. For efficient homologous recombination, both plasmids had to be linearized. The recombination reaction was induced by restriction of the plasmid molecules either inside the effective homologous region or at the borders of the intron sequence. However, the presence of even small, terminal, nonhomologous sequences at the 3 end of the pB×26 fragment inhibited the recombination reaction. Also, both ends of the linearized piGUS DNA molecules were involved in the recombination reaction. The results revealed some features of homologous recombination reactions occurring in plant cells which cannot be accommodated by mechanisms postulated for similar reactions in animal system and in lower eukaryotes.     

Establishment of somatic hybrid cell lines between Zea mays L. (maize) and Triticum sect,trititrigia MacKey (trititrigia)

Summary Somatic hybrid cell lines were constructed by the fusion of protoplasts isolated from cell suspensions of Zea mays L. (maize, 2n = 20) and Triticum sect, trititrigia MacKey (trititrigia, 2n = 35), a perennial hybrid of T. durum Desf. and Elytrigia intermedium (Host) Nevski. Iodoacetamide-inactivated protoplasts of maize were fused with trititrigia protoplasts, which were sensitive to the PEG/DMSO fusion treatment at high pH and high calcium. Based on physiological complementation, approximately 0.002% of the total protoplasts cultured following fusion treatment developed into cell colonies, and 79 lines of them, almost a half, were singled out and subcultured. Among the subcultured lines three were, in comparison with the parents, identified as somatic hybrids by their coupled XbaI restriction patterns of total DNAs probed with the ribosomal DNA of rice. Southern analysis of the digested total DNAs with a mitochondrial gene, atpA., from pea, or a chloroplast gene, trnK, from rice, revealed that all the hybrids carried only the organellar DNAs of trititrigia, which excluded the possibilities of a chimeric callus or any DNA contamination. Cytogenetically, one hybrid was mixoploid with a 2n of 46–67 in which chromosomal endoreduplication, characterized by the appearance of diplochromosomes, was occasionally observed. Its hybridity was reconfirmed by the fact that it bore the satellite chromosomes of both maize and trititrigia, which were distinguishable from each other by size. In contrast, the other two hybrids were aneuploids. The potential of gene transfer between Zea and Triticum species was thus conclusively established.     

Effect of cellulases on spontaneous fusion of maize protoplasts

Summary The effect of protoplast-isolating enzymes on spontaneous fusion of maize protoplasts (Zea mays L. cv. Black Mexican Sweet) was investigated using a convenient ethidium bromide nuclear staining procedure. After 2–2.5 hour digestion in an enzyme solution containing 1% Cellulysin, 0.5% Rhozyme, and 0.02% Pectolyase Y-23, 50–75% of the protoplasts contained multiple nuclei. The cellulase Cellulysin was identified as the factor causing the spontaneous protoplast fusion; when Cellulysin was replaced by CELF cellulase, most protoplasts were uninucleate. Calcium and other components in the enzyme solution did not affect spontaneous fusion. Cellulysin also increased the percentage of multinucleate protoplasts from rice and asparagus suspensions. Presence of multiple nuclei might affect genetic manipulations involving protoplasts.     

Mixing of maize and wheat genomic DNA by somatic hybridization in regenerated sterile maize plants

Intergeneric somatic hybridization was performed between albino maize (Zea mays L.) protoplasts and mesophyll protoplasts of wheat (Triticum aestivum L.) by polyethylene glycol (PEG) treatments. None of the parental protoplasts were able to produce green plants without fusion. The maize cells regenerated only rudimentary albino plantlets of limited viability, and the wheat mesophyll protoplasts were unable to divide. PEG-mediated fusion treatments resulted in hybrid cells with mixed cytoplasm. Six months after fusion green embryogenic calli were selected as putative hybrids. The first-regenerates were discovered as aborted embryos. Regeneration of intact, green, maize-like plants needed 6 months of further subcultures on hormone-free medium. These plants were sterile, although had both male and female flowers. The cytological analysis of cells from callus tissues and root tips revealed 56 chromosomes, but intact wheat chromosomes were not observed. Using total DNA from hybrid plants, three RAPD primer combinations produced bands resembling the wheat profile. Genomic in situ hybridization (GISH) using total wheat DNA as a probe revealed the presence of wheat DNA islands in the maize chromosomal background. The increased viability and the restored green color were the most-significant new traits as compared to the original maize parent. Other intermediate morphological traits of plants with hybrid origin were not found.     

High frequency callus formation from maize protoplasts

Summary A solid feeder layer technique was developed to improve callus formation of Black Mexican Sweet maize (Zea mays L.) suspension culture protoplasts. Protoplasts were plated in 0.2 ml liquid media onto a cellulose nitrate filter on top of agarose-solidified media in which Black Mexican Sweet suspension feeder cells were embedded. Callus colony formation frequencies exceeding 10% of the plated protoplasts were obtained for densities of 10³–10⁵ protoplasts/ 0.2 ml, which was 100- to 1,000-fold higher than colony formation frequencies obtained for conventional protoplast plating methods such as liquid culture or embedding in agarose media. Compared with conventional methods, the feeder layer method gave higher colony formation frequencies for three independently maintained Black Mexican Sweet suspension lines. Differences among the three lines indicated that colony formation frequencies might also be influenced by the suspension culture maintenance regime and length of time on different 2,4-dichlorophenoxyacetic acid concentrations. The callus colony formation frequency reported is an essential prerequesite for recovering rare mutants or genetically transformed maize protoplasts.     

Maize Activator transposase has a bipartite DNA binding domain that recognizes subterminal sequences and the terminal inverted repeats

The mobility of maize transposable element Activator (Ac) is dependent on the 11-bp terminal inverted repeats (IRs) and approximately 250 subterminal nucleotides at each end. These sequences flank the coding region for the transposase (TPase) protein, which is required for the transposition reaction. Here we show that Ac TPase has a bipartite DNA binding domain, and recognizes the IRs and subterminal sequences in the Ac ends. TPase binds cooperatively to repetitive ACG and TCG sequences, of which 25 copies are found in the 5′ and 20 copies in the 3′ subterminal regions. TPase affinity is highest when these sites are flanked on the 3′ side by an additional G residue (A/TCGG), which is found at 75% of binding sites. Moreover, TPase binds specifically to the Ac IRs, albeit with much lower affinity. Two mutations within the IRs that immobilize Ac abolish TPase binding completely. The basic DNA binding domain of TPase is split into two subdomains. Binding to the subterminal motifs is accomplished by the C-terminal subdomain alone, whereas recognition of the IRs requires the N-terminal subdomain in addition. Furthermore, TPase is extremely flexible in DNA binding. Two direct or inverted binding sites are bound equally well, and sites that are five to twelve bases apart are similarly well bound. The consequences of these findings for the Ac transposition reaction are discussed.     

In vitro fertilization of single,isolated gametes of maize mediated by electrofusion

Summary Electrofusion-mediated in vitro fertilization of maize using single sperm and egg cells was performed. Sperm cells were released from pollen grains after rupture of the latter by osmotic shock in the fusion medium (0.55 M mannitol). Egg cells were isolated by enzyme treatment (pectinase, pectolyase, hemicellulase, and cellulase) followed by mechanical isolation. The conditions generally used for the electrical fusion of protoplasts of somatic cells were also applied to the protoplasts of gametic cells of maize. Electrofusion was performed with single pairs of gametes under microscopic observation. The mean fusion frequency was 79%. Isolated egg cells of maize showed protoplasmic streaming during 22 days of culture, but they did not divide. However, after fusion of the sperm with the egg cells, these fused cells did develop, with a mean division frequency of 83%, and grew to multicellular structures. Egg cells and fusion products were cultivated with a maize feeder-cell system.