Cauliflower Mosaic Virus 35S Enhancer Research Articles

Constitutive overexpression of the pollen specific gene SKS13 in leaves reduces aphid performance on Arabidopsis thaliana.

BackgroundPlants have developed a variety of mechanisms to counteract aphid attacks. They activate their defences by changing the expression of specific genes. Previously we identified an activation tag mutant of Arabidopsis thaliana on which Myzus persicae population development was reduced. Activation tag mutants are gain-of-function in which the expression of a gene is increased by the insertion of the Cauliflower mosaic virus 35S enhancer that acts on the natural promoter. By further characterizing this previously identified mutant we identified a gene that reduces performance of M. persicae and also provided clues about the mechanism involved.ResultsWe show that SKU5 SIMILAR 13 (SKS13), a gene whose expression in wild type plants is restricted to pollen and non-responsive to M. persicae attack, is overexpressed in the A. thaliana mutant showing reduced performance of M. persicae. Monitoring M. persicae feeding behaviour on SKS13 overexpressing plants indicated that M. persicae have difficulties feeding from the phloem. The constitutive expression of SKS13 results in accumulation of reactive oxygen species, which is possibly regulated through the jasmonic acid pathway. The enhanced resistance is not aphid species specific as also the population development of Brevicoryne brassicae was affected.ConclusionsWe demonstrate that constitutive expression in leaves of the pollen-specific gene SKS13 can enhance plant defence, resulting in a reduction of M. persicae population development and also decreases the transmission of persistent viruses. Overexpression of SKS13 in A. thaliana also affects B. brassicae and possibly other phloem feeding insects as well. Identifying genes that can enhance plant defence against insects will be important to open up new avenues for the development of insect resistant crop plants.

The CaMV 35S enhancer has a function to change the histone modification state at insertion loci in Arabidopsis thaliana

Chromatin regions with different states usually harbor distinct epigenetic information, through which gene expression is regulated. Recent studies using mammalian cells showed that a chromatin state signature is associated with active developmental enhancers, defined by high levels of histone H3 lysine 27 acetylation (H3K27ac) and strong depletion of H3K27 trimethylation (H3K27me3). These findings also imply that active enhancers may play a role in creating a chromatin state by changing histone modification markers, which in turn affects gene expression. To explore whether an active enhancer in plants affect histone modifications, we investigated the cauliflower mosaic virus 35S enhancer (35Senh) for understanding its action model in Arabidopsis. We report that the 35Senh has a function to change the histone modification pattern at its presenting loci, by characterization of the 35Senh activated BREVIPEDICELLUS (BP) silencing lines and the randomly selected 35Senh activation tagging lines. By analyzing histone modification markers reflecting the plant chromatin state, we show that the 35Senh is generally correlated with the reduced level of H3K27me3 and the increased level of H3K4me3 at the insertion loci. Our data are consistent with those in mammals and suggest that the enhancer sequence correlating with the active chromatin state signature may be generally present in the eukaryotic kingdom.

A 1-kb Bacteriophage Lambda Fragment Functions as an Insulator to Effectively Block Enhancer–Promoter Interactions in Arabidopsis thaliana

Enhancers are known to be capable of overriding the specificity of nearby promoters in a distance-dependent manner, which is problematic when multiple promoters coexist in a single transgene unit. In an attempt to determine whether enhancer activation function is inversely related to its distance from the target promoter, we inserted 1-, 2-, and 4-kb bacteriophage λ fragments, respectively, between the cauliflower mosaic virus 35S enhancer and a flower-specific AGAMOUS second intron-derived promoter (AGIP) fused to the β-glucuronidase (GUS) coding region. In the absence of an insert sequence, the 35S enhancer activates AGIP-driven GUS expression in vegetative tissues of transgenic Arabidopsis thaliana lines. Moreover, neither the 2-kb nor the 4-kb λ fragment was able to block GUS expression in transgenic leaves, implying that the 35S enhancer can override a distance barrier of at least 4 kb in our system. Unexpectedly, insertion of the 1-kb λ insert into the same site resulted in diminished GUS expression in transgenic leaves. Our data indicate that this fragment functions as a true enhancer-blocking insulator that could potentially be utilized to minimize enhancer–promoter interference between multiple transcriptional units within a plasmid vector during plant transformation experiments.

A transformation booster sequence (TBS) from Petunia hybrida functions as an enhancer-blocking insulator in Arabidopsis thaliana

Several matrix-attachment regions (MARs) from animals have been shown to block interactions between an enhancer and promoter when situated between the two. Since a similar function for plant MARs has not been discerned, we tested the Zea mays ADH1 5' MAR, Nicotiana tabacum Rb7 3' MAR and a transformation booster sequence (TBS) MAR from Petunia hybrida for their ability to impede enhancer-promoter interactions in Arabidopsis thaliana. Stable transgenic lines containing vectors in which one of the three MAR elements or a 4 kb control sequence were interposed between the cauliflower mosaic virus 35S enhancer and a flower-specific AGAMOUS second intron-derived promoter (AGIP)::beta-glucuronidase (GUS) fusion were assayed for GUS expression in vegetative tissues. We demonstrate that the TBS MAR element, but not the ADH1 or Rb7 MARs, is able to block interactions between the 35S enhancer and AGIP without compromising the function of either with elements from which they are not insulated.

Activation of Plant Plasma Membrane H+-ATPase by 14-3-3 Proteins Is Negatively Controlled by Two Phosphorylation Sites within the H+-ATPase C-terminal Region

The proton pump ATPase (H(+)-ATPase) of the plant plasma membrane is regulated by an autoinhibitory C-terminal domain, which can be displaced by phosphorylation of the penultimate Thr residue and the subsequent binding of 14-3-3 proteins. We performed a mass spectrometric analysis of PMA2 (plasma membrane H(+)-ATPase isoform 2) isolated from Nicotiana tabacum suspension cells and identified two new phosphorylated residues in the enzyme 14-3-3 protein binding site: Thr(931) and Ser(938). When PMA2 was expressed in Saccharomyces cerevisiae, mutagenesis of each of these two residues into Asp prevented growth of a yeast strain devoid of its own H(+)-ATPases. When the Asp mutations were individually introduced in a constitutively activated mutant of PMA2 (E14D), they still allowed yeast growth but at a reduced rate. Purification of His-tagged PMA2 showed that the T931D or S938D mutation prevented 14-3-3 protein binding, although the penultimate Thr(955) was still phosphorylated, indicating that Thr(955) phosphorylation is not sufficient for full enzyme activation. Expression of PMA2 in an N. tabacum cell line also showed an absence of 14-3-3 protein binding resulting from the T931D or S938D mutation. Together, the data show that activation of H(+)-ATPase by the binding of 14-3-3 proteins is negatively controlled by phosphorylation of two residues in the H(+)-ATPase 14-3-3 protein binding site. The data also show that phosphorylation of the penultimate Thr and 14-3-3 binding each contribute in part to H(+)-ATPase activation.

The GIGANTEA-regulated microRNA172 mediates photoperiodic flowering independent of CONSTANS in Arabidopsis.

Regulated RNA metabolism appears to be a critical component of molecular mechanisms directing flowering initiation in plants. A group of RNA binding proteins exerts their roles through the autonomous flowering pathway. Posttranscriptional mechanisms regulated by microRNAs (miRNAs) also play a key role in flowering-time control. Here, we demonstrate that the GIGANTEA (GI)-regulated miR172 defines a unique genetic pathway that regulates photoperiodic flowering by inducing FLOWERING LOCUS T (FT) independent of CONSTANS (CO). A late-flowering mutant in which a miR172 target gene, TARGET OF EAT1, is constitutively activated by the nearby insertion of the cauliflower mosaic virus 35S enhancer normally responded to vernalization and gibberellic acid treatments. By contrast, its response to daylength changes was severely disrupted. In the mutant, FT was significantly repressed, but other flowering genes were unaffected. Notably, miR172 abundance is regulated by photoperiod via GI-mediated miRNA processing. Accordingly, miR172-overproducing plants exhibit early flowering under both long days and short days, even in the absence of functional CO, indicating that miR172 promotes photoperiodic flowering through a CO-independent genetic pathway. Therefore, it appears that GI-mediated photoperiodic flowering is governed by the coordinated interaction of two distinct genetic pathways: one mediated via CO and the other mediated via miR172 and its targets.

(285) Characterization of a Composite Promoter from Genomic Sequences of Grapevine

Genetic transformation of plants necessitates the use of promoters to control transgene expression. Numerous promoters have been isolated from a wide range of organisms for use in plants. However, many of these natural promoters exhibit relatively low activity and/or have limited use. To provide an alternative, we constructed a composite promoter (EP) using a genomic DNA sequence and a 35 bp TATA-containing fragment from the 2S albumin (VvAlb1) gene core promoter of grapevine. The 0.9-kb genomic sequence was identified after TAIL-PCR, based on the presence of several unique cis-acting elements. The sequence showed no homology to any known plant gene, enhancer, and promoter. Two binary vectors, pEP-EGFP/NPT and pEP-GUS, containing a bifunctional EGFP/NPTII fusion gene and a GUS gene, respectively, were constructed to test transcriptional activity of the composite promoter both qualitatively and quantitatively. Transient GFP expression was observed in somatic embryos (SE) of Vitis vinifera `Thompson Seedless' after Agrobacterium-mediated transformation using pEP-EGFP/NPT. Quantitative GUS assay of stably transformed SE containing pEP-GUS indicated that the EP composite promoter was capable of producing GUS activity as high as 12% of that from a doubly enhanced Cauliflower Mosaic Virus 35S promoter or eight times higher than that from a doubly enhanced Cassava Vein Mosaic Virus promoter. In addition, transformation of Arabidopsis with pEP-GUS yielded comparable GUS activity throughout the plant. These data indicate that the EP composite promoter can be used in transformation studies to provide sustained constitutive gene expression in plants.

Assessment of the Effectiveness of a Nuclear-Launched TMV-Based Replicon as a Tool for Foreign Gene Expression in Plants in Comparison to Direct Gene Expression from a Nuclear Promoter

An environmentally safe Tobacco Mosaic Virus (TMV)-based expression replicon was constructed that lacks movement protein (MP) and coat protein (CP), and which expresses the green fluorescent protein (GFP) gene from a full CP subgenomic promoter. The TMV replicon, whose cDNA was positioned between an enhanced Cauliflower Mosaic Virus 35S promoter (CaMV) and a self-cleaving hammerhead ribozyme with a downstream nopaline synthase gene polyadenylation signal [nos-poly(A)], was assessed for its effectiveness to accumulate GFP upon agroinfiltration into plant leaves compared to a control construct in which GFP was directly expressed from the enhanced CaMV 35S promoter. It was determined that individually expressing cells produced ca. 9-fold more GFP from the TMV-based replicon than from the enhanced 35S promoter. In contrast, GFP measurements from total leaf extracts determined that leaves infiltrated with the TMV-based replicon produced ca. 7-fold less GFP than the control construct. These apparently contradictory results can be explained by the low infectivity of the TMV-based replicon as it was found that the number of foci expressing GFP produced in leaves agroinfiltrated with the TMV-based replicon was ca. 66-fold lower than produced by the control.

Molecular characterization of the flavonoid biosynthesis of Verbena hybrida and the functional analysis of verbena and Clitoria ternatea F3'5'H genes in transgenic verbena

Homologues of the flavonoid 3′,5′-hydroxylase (F3′5′H) gene, a key gene determining flower color, were obtained from a Verbena hybrida (verbena) cultivar Temari Violet, verbena cultivar Tapien Pink, and Clitoria ternatea (butterfly pea). The expression of the Temari and butterfly pea homologues in yeast confirmed that they encoded F3′5′H. The two genes under the control of an enhanced cauliflower mosaic virus 35S promoter were introduced into verbena Temari Sakura. Some of the transgenic verbena plants had elevated delphinidin contents and flower color altered toward violet. Interestingly, the butterfly pea F3′5′H gene yielded more delphinidin and gave clearer flower color change than the verbena Temari gene in the transgenic verbena plants. The results indicate that the choice of the gene source should be considered to obtain strong phenotypic changes, even if the genes encode the same enzymatic activity. We also cloned some flavonoid biosynthetic genes from verbenas. The potential usefulness of verbena in the phytomonitoring of environmental pollutants is also discussed.

Development and Characterization of a CP4 EPSPS-Based, Glyphosate-Tolerant Corn Event

5-Enol-pyruvylshikimate-3-phosphate synthase from Agrobacterium sp. CP4 (CP4 EPSPS) confers tolerance to the nonselective herbicide glyphosate (marketed under the trade name Roundup11 Roundup Ready and Roundup herbicide are trademarks of the Monsanto Company.) when sufficiently expressed in transgenic plants. Dual CP4 EPSPS transgene cassettes were transformed into corn (Zea mays L.) under the transcriptional regulatory control of the rice (Oryza sativa L.) actin 1 (P-Ract1) and the enhanced Cauliflower mosaic virus 35S (P-e35S) promoters, respectively, to impart fully constitutive expression in corn. Resulting events were tested for lack of chlorosis and malformation injury after two sequential applications of 1.68 kg acid equivalents (a.e.) ha−1 glyphosate. Agronomic parameters, male fertility, appropriate Mendelian segregation of the trait, plus characteristics of the transgenic integration site were also evaluated. From this selection process, the NK603 event was chosen for commercialization as the event that embodied the most optimal profile of tolerance, agronomics, and molecular characteristics. The NK603 event exhibited high glyphosate tolerance from one transgenic locus bearing a single copy of the dual cassettes integrated into the corn genome with a minimum of target sequence disruption. Trait expression in the NK603 event has remained stable over more than eight generations as shown through tolerance testing, western blots of CP4 EPSPS accumulation, and Southern blot analysis of the transgene.

Low frequency of T-DNA based activation tagging in Arabidopsis is correlated with methylation of CaMV 35S enhancer sequences

A powerful system to create gain-of-function mutants in plants is activation tagging using T-DNA based vehicles to introduce transcriptional enhancer sequences. Large Arabidopsis populations of individual plants carrying a quadruple cauliflower mosaic virus (CaMV) 35S enhancer are frequently used for mutant screenings, however the frequency of morphological mutants remains very low. To clarify this low frequency we analyzed a subset of lines generated by this method. The correlation between the number of T-DNA insertion sites, the methylation status of the 35S enhancer sequence and 35S enhancer activity was determined. All plants containing more than a single T-DNA insertion showed methylation of the 35S enhancer and revealed a dramatic decrease in 35S enhancer activity. The results support the notion that in a large proportion of the T-DNA based activation tagged lines the 35S transcriptional enhancer is silenced due to methylation, which is induced by multiple T-DNA integrations.

Delayed abscission and shorter Internodes correlate with a reduction in the ethylene receptor LeETR1 transcript in transgenic tomato.

Stable transformation of tomato (Lycopersicon esculentum cv Ailsa Craig) plants with a construct containing the antisense sequence for the receiver domain and 3'-untranslated portion of the tomato ethylene receptor (LeETR1) under the control of an enhanced cauliflower mosaic virus 35S promoter resulted in some expected and unexpected phenotypes. In addition to reduced LeETR1 transcript levels, the two most consistently observed phenotypes in the transgenic lines were delayed abscission and reduced plant size. Fruit coloration and softening were essentially unaffected, and all the seedlings from first generation seed displayed a normal triple response to ethylene. Two independent lines with a single copy of the transgene and reduced LeETR1 transcript accumulation were selected for detailed phenotypic analysis of second generation (R1) plants. Delayed abscission, shorter internode length, and reduced auxin movement all correlated with the presence of the transgene and the degree of reduced LeETR1 transcript accumulation. No significant differences were noted for fruit coloration or fruit softening on R1 plants and all seedlings from R1 and R2 seed displayed a normal triple response. LeETR2 transcript accumulation was only slightly reduced in the R1 plants compared with azygous plants, and LeETR3 (NR) transcript levels appeared to be unaffected by the transgene. We propose that ethylene signal transduction occurs through parallel paths that partially intersect to regulate shared ethylene responses.

Biotransformation of an exogenously supplied isoflavonoid by transgenic tobacco cells expressing alfalfa isoflavone reductase

Isoflavone reductase (IFR) from alfalfa catalyzes the NADPH-dependent reduction of 2′-hydroxy isoflavones to 2′-hydroxyisoflavanones such as vestitone, thereby performing a key step in isoflavonoid phytoalexin biosynthesis. Tobacco (Nicotiana tabacum L. cv. Xanthi) was transformed with an alfalfa cDNA encoding IFR regulated by an enhanced cauliflower mosaic virus 35S promoter and used to generate cell suspension cultures. Transformed cells expressed high levels of IFR activity and could take up 2′-hydroxyformononetin (2′OHF; IFR substrate) and convert it to vestitone. Both 2′OHF and vestitone were each converted to two sugar conjugates. The structure of the most abundant vestitone conjugate was determined using NMR spectroscopic analysis to be a novel vestitone 2′-O-glucoside. Liquid chromatography-mass spectroscopy (LC-MS) indicated that the molecular weights of the three less abundant conjugates were consistent with hexose conjugates of vestitone and 2′OHF. The IFR reduction and conjugation reactions were complete within 4 h, with the net percentage conversion of 2′OHF to vestitone ranging as high as 60%.

Effect of an enhanced CaMV 35S promoter and a fruit-specific promoter on uida gene expression in transgenic tomato plants

Two different promoters, a cauliflower mosaic virus (CaMV) 35S promoter with a 5′-untranslated leader sequence from alfalfa mosaic virus RNA4 (designated as CaMV 35S/AMV) and an E-8 fruit-ripening-specific promoter, were compared to evaluate their effects on expression of the uidA reporter gene in transgenic tomato plants. In order to generate sufficient numbers of transgenic tomato plants, both a reliable regeneration system and an efficient Agrobacterium transformation protocol were developed using 8-d-old cotyledons of tomato (Lycopersicon ecsulentum Mill. cv. Swifty Belle). Two sets of constructs, both derivatives of the binary vector pBI121, were used in transformation of tomato whereby the uidA gene was driven either by the CaMV 35S/AMV or the E-8 fruit-ripening-specific promoter. Southern blot hybridization confirmed the stable integration of the chimeric uidA gene into the tomato genome. Fruit and leaf tissues were collected from T0 and T1 plants, and assayed for β-glucuronidase (GUS) enzyme activity. As expected, both vegetative and fruit tissues of transgenic plants carrying the uidA gene under the control of CaMV 35S/AMV showed varying levels of GUS activity, while no expression was observed in vegetative tissues of transgenic plants carrying the uidA gene driven by the E-8 promoter. All fruits from transgenic plants produced with both sets of constructs displayed expression of the uidA gene. However, when this reporter gene was driven by the CaMV 35S/AMV, GUS activity levels were significantly higher than when it was driven by the E-8 fruit-specific promoter. The presence/absence of the uidA gene in T1 plants segregated in a 3∶1 Mendelian ratio.

Plant selection principle based on xylose isomerase

The xylose isomerase genes (xylA) from Thermoanaerobacterium thermosulfurogenes and Streptomyces rubiginosus were introduced and expressed in three plant species (potato, tobacco and tomato) and transgenic plants were selected on xylose-containing medium. The xylose isomerase genes were transferred to explants of the target plant by Agrobacterium-mediated transformation. The xylose isomerase genes were expressed under the control of the enhanced cauliflower mosaic virus 35S promoter and the Ω′ translation enhancer sequence from tobacco mosaic virus. In potato and tomato, xylose isomerase selection was more efficient than the established kanamycin selection. The level of enzyme activity in the regenerated transgenic plants selected on xylose was 5–25-fold higher than the enzyme activity in control plants selected on kanamycin. The xylose isomerase system enables transgenic cells to utilize xylose as a carbohydrate source. In contrast to antibiotic or herbicide resistance-based system where transgenic cells survive on a selective medium but nontransgenic cells are killed, the xylose system is an example of a positive selection system where transgenic cells proliferate while non-transgenic cells are starved but still survive. The results show that a new selection method, is established. The xylose system is devoid of the disadvantages of antibiotic or herbicide selection, and depends on an enzyme which is already being widely utilized in specific food processes and that is generally recognized as safe for use in the starch industry.

Functional expression and subcellular localization of the Nectria haematococca Mak1 phytoalexin detoxification enzyme in transgenic tobacco.

Medicarpin and maackiain are antifungal pterocarpan phytoalexins produced by many legumes, and are thought to be important components of the defense response of these legumes to certain fungal pathogens. The Mak1 gene from the fungal pathogen Nectria haematococca encodes an FAD-dependent mono-oxygenase, known to specifically hydroxylate the phytoalexins medicarpin and maackiain, converting them to less fungitoxic derivatives. Two binary vector constructs were made containing the coding regions from two fungal clones, a Mak1 cDNA (intronless) and a genomic (including three fungal introns) clone, regulated by an enhanced cauliflower mosaic virus 35S promoter. The constructs were introduced into tobacco to check for expression of active fungal enzyme in plant cells and for splicing of fungal introns. Leaves of tobacco plants transformed with the Mak1 cDNA construct readily metabolized infiltrated medicarpin to 1a-hydroxymedicarpin, indicating high levels of active enzyme. RT-PCR analysis of tobacco plants transformed with the Mak1 genomic construct indicated no processing of Mak1 introns, and no Mak1 activity was detected in these plants. When using plants containing the Mak1 cDNA construct, immunolocalization with a Mak1-specific antibody together with cellular fractionation indicated that Mak1 protein accumulated in the plant cytoplasm, associated with endoplasmic reticulum membranes; medicarpin biosynthetic enzymes have been localized to the same subcellular region. The Mak1 cDNA construct is therefore suitable for use in studies to selectively eliminate medicarpin accumulation to assess the relative importance of medicarpin in the antifungal defense mechanisms of alfalfa and other legumes.

Stable Genetic Transformation of EschscholziaCalifornica Expressing Synthetic Green Fluorescent Proteins

An efficient protocol is described for the stable genetic transformation of Eschscholzia californica (California poppy) using Agrobacterium tumefaciens as a vector. We have employed the disarmed A. tumefaciens LBA4404 encoding a synthetic green fluorescent protein reporter gene that is further controlled by an enhanced cauliflower mosaic virus 35S promoter. Stably transformed E. californica cells appear 3 weeks after initial cocultivation of A. tumefaciens with poppy leaves, stems, or roots. Transformed poppy calli were visualized by exposure to long-wave UV or blue light and analyzed in detail by fluorescent microscopy and laser-scanning microscopy. Moreover, green fluorescent calli have been maintained through continual subculture and grow well either on Gamborg's B5 agarose or liquid medium.

Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis.

Plants produce a wide array of natural products, many of which are likely to be useful bioactive structures. Unfortunately, these complex natural products usually occur at very low abundance and with restricted tissue distribution, thereby hindering their evaluation. Here, we report a novel approach for enhancing the accumulation of natural products based on activation tagging by Agrobacterium-mediated transformation with a T-DNA that carries cauliflower mosaic virus 35S enhancer sequences at its right border. Among approximately 5000 Arabidopsis activation-tagged lines, we found a plant that exhibited intense purple pigmentation in many vegetative organs throughout development. This upregulation of pigmentation reflected a dominant mutation that resulted in massive activation of phenylpropanoid biosynthetic genes and enhanced accumulation of lignin, hydroxycinnamic acid esters, and flavonoids, including various anthocyanins that were responsible for the purple color. These phenotypes, caused by insertion of the viral enhancer sequences adjacent to an MYB transcription factor gene, indicate that activation tagging can overcome the stringent genetic controls regulating the accumulation of specific natural products during plant development. Our findings suggest a functional genomics approach to the biotechnological evaluation of phytochemical biodiversity through the generation of massively enriched tissue sources for drug screening and for isolating underlying regulatory and biosynthetic genes.

Overexpression of a gene encoding a cytochrome P450, CYP78A9, induces large and seedless fruit in arabidopsis.

An activation tagging screen in which the cauliflower mosaic virus 35S enhancer was inserted randomly into an Arabidopsis genome homozygous for the floral homeotic mutation apetala2-1 (ap2-1) resulted in a line (28-5) with extraordinarily wide, heart-shaped ovaries. The ovary of the 28-5 ap2-1 mutant shows an oval shape because of increased numbers of enlarged cells. When the ap2-1 mutation is crossed out of the genetic background, more elongated rather than wider fruits are obtained. Normally, Arabidopsis fruits will develop to a normal size only when the ovules are present and fertilized. In the 28-5 single mutant, the siliques keep growing despite failure of fertilization and can reach nearly normal size. When wild-type pollen was used to pollinate the mutant pistil, the pollinated 28-5 silique became >10% longer and 40% wider than a wild-type silique, although producing very few seeds. The enhancer insertion in line 28-5 acts by hyperactivating a cytochrome P450 gene, CYP78A9. The pistil of 28-5 ap2-1 mutant flowers shows a structure similar to that of Capsella bursa-pastoris, a distant mustard relative of Arabidopsis, suggesting that the processes regulated by the CYP78A9-encoded protein may be involved in evolutionary control of carpel shape.

Construction of a stable and highly infectious intron-containing cDNA clone of plum pox potyvirus and its use to infect plants by particle bombardment

An infectious plum pox potyvirus cDNA clone was constructed placing a copy of the full-length sequence of the virus genome between an enhanced cauliflower mosaic virus 35S promoter and a nopaline synthase termination signal. Stabilization of the clone and faster growth of bacteria, in addition to higher plasmid yield, followed a modification consisting of the insertion of an intron which interrupted the viral open reading frame at the P3 region. This intron-containing clone was infectious when inoculated into plants after undergoing in vivo transcription and splicing. Particle bombardment delivery of the cDNA greatly increased the efficiency of plant infection.