Identification and Genome Characterization of a Dahlia Common Mosaic Virus Isolate from China.

Dahlia (Dahlia variabilis) is a widely cultivated ornamental and medicinal plant in China. Recently, dahlia plants with symptoms of leaf mottling and distortion were collected in Hohhot, Inner Mongolia, China. The presence of dahlia common mosaic virus (DCMV), an unassigned species in the genus Caulimovirus, was confirmed by high-throughput sequencing. Three fragments of DCMV Inner Mongolia isolate (DCMV-IN) were PCR-amplified with specific primers, sequenced and assembled into the complete genome sequence with a GenBank accession number of OR494328. The double-stranded circular DNA genome of DCMV-IN consists of 7949 bp and contains six open reading frames (ORFs). Sequence analysis showed that DCMV-IN shared high sequence identities with other DCMV isolates available in the GenBank database. Phylogenetic analysis of DCMV isolates and other representative caulimoviruses based on genome sequence clustered four DCMV isolates to a single branch which was closest to dahlia mosaic virus (DMV). No recombination event was detected among the four DCMV isolates.

Complete genome sequence of a putative new caulimovirus which exists as endogenous pararetroviral sequences in Angelica dahurica.

A virus isolate designated Angelica bushy stunt virus (AnBSV), provisionally representing a new species in the genus Caulimovirus, was discovered in the medicinal plant Angelica dahurica. The complete 8,300-nt genomic DNA of AnBSV had seven putative open reading frames containing conserved domains/motifs, which are typical features of caulimoviruses, and showed the greatest nucleotide sequence identity (74% identity and 27% query coverage) to a lamium leaf distortion virus isolate. Interestingly, the new caulimovirus exists as endogenous pararetroviral sequences in the host plant and is considered to have multiple defective plant genome-integrated copies that may lead to the generation of subgenomic DNA species.

Complete genome sequence of a tentative new caulimovirus from the medicinal plant Atractylodes macrocephala.

A total of nine contigs related to caulimovirus-like sequences were detected using high-throughput paired-end RNA sequencing. An attempt to find the plant sample infected with this type of virus identified the medicinal plant Atractylodes macrocephala Koidzumi showing mild mottle symptoms. Subsequently, the complete DNA genome sequence of the Atractylodes virus was determined. The 8,105-nt genome of the virus was composed of six open reading frames and displayed the highest nucleotide sequence identity (70%) with soybean Putnam virus. Based upon the symptoms observed on the source plant, we propose to refer to this new member of the genus Caulimovirus as atractylodes mild mottle virus.

A comparison study of Agrobacterium-mediated transformation methods for root-specific promoter analysis in soybean.

KEY MESSAGE: Both in vitro and in vivo hairy root transformation systems could not replace whole plant transformation for promoter analysis of root-specific and low-P induced genes in soybean. An efficient genetic transformation system is crucial for promoter analysis in plants. Agrobacterium-mediated transformation is the most popular method to produce transgenic hairy roots or plants. In the present study, first, we compared the two different Agrobacterium rhizogenes-mediated hairy root transformation methods using either constitutive CaMV35S or the promoters of root-preferential genes, GmEXPB2 and GmPAP21, in soybean, and found the efficiency of in vitro hairy root transformation was significantly higher than that of in vivo transformation. We compared Agrobacterium rhizogenes-mediated hairy root and Agrobacterium tumefaciens-mediated whole plant transformation systems. The results showed that low-phosphorous (P) inducible GmEXPB2 and GmPAP21 promoters could not induce the increased expression of the GUS reporter gene under low P stress in both in vivo and in vitro transgenic hairy roots. Conversely, GUS activity of GmPAP21 promoter was significantly higher at low P than high P in whole plant transformation. Therefore, both in vitro and in vivo hairy root transformation systems could not replace whole plant transformation for promoter analysis of root-specific and low-P induced genes in soybean.

Development of an efficient bi-directional promoter with tripartite enhancer employing three viral promoters.

We have developed a novel bi-directional promoter (FsFfCBD) by placing two heterogeneous core-promoters from the Figwort mosaic virus sub-genomic transcript promoter (FsCP, -69 to +31) and Cauliflower mosaic virus 35S promoter (CCP, -89 to +1) respectively on upstream (5') and downstream (3') ends of a tri-hybrid enhancer (FsEFfECE), in reverse orientation. The FsEFfECE domain encompasses three heterologous enhancer fragments from Figwort mosaic virus sub-genomic transcript promoter (FsE, 101 bp, -70 to -170), Figwort mosaic virus full-length transcript promoter (FfE, 196 bp, -249 to -54) and Cauliflower mosaic virus 35S promoter (CE, 254 bp, -343 to -90). The bi-directional nature of the FsFfCBD promoter (coupled to GFP and GUS) was established both in transient systems (onion epidermal cells and tobacco protoplasts) and transgenic plant (Nicotiana tabacum samsun NN) by monitoring the simultaneous expression of GFP and GUS employing fluorescence (for GFP) and biochemical (for GUS) based assays. In transgenic plants, the FsFfCBD promoter was found to be 6.8 and 2.5 times stronger than two parent promoters; Fs and FfC respectively. The bi-directional compound promoter FsFfCBD, composed of three heterologous enhancers with enhanced activity could become a valuable additional tool for efficient plant metabolic engineering and molecular pharming.

Characterization of the fungal gibberellin desaturase as a 2-oxoglutarate-dependent dioxygenase and its utilization for enhancing plant growth.

The biosynthesis of gibberellic acid (GA(3)) by the fungus Fusarium fujikuroi is catalyzed by seven enzymes encoded in a gene cluster. While four of these enzymes are characterized as cytochrome P450 monooxygenases, the nature of a fifth oxidase, GA(4) desaturase (DES), is unknown. DES converts GA(4) to GA(7) by the formation of a carbon-1,2 double bond in the penultimate step of the pathway. Here, we show by expression of the des complementary DNA in Escherichia coli that DES has the characteristics of a 2-oxoglutarate-dependent dioxygenase. Although it has low amino acid sequence homology with known 2-oxoglutarate-dependent dioxygenases, putative iron- and 2-oxoglutarate-binding residues, typical of such enzymes, are apparent in its primary sequence. A survey of sequence databases revealed that homologs of DES are widespread in the ascomycetes, although in most cases the homologs must participate in non-gibberellin (GA) pathways. Expression of des from the cauliflower mosaic virus 35S promoter in the plant species Solanum nigrum, Solanum dulcamara, and Nicotiana sylvestris resulted in substantial growth stimulation, with a 3-fold increase in height in S. dulcamara compared with controls. In S. nigrum, the height increase was accompanied by a 20-fold higher concentration of GA(3) in the growing shoots than in controls, although GA(1) content was reduced. Expression of des was also shown to partially restore growth in plants dwarfed by ectopic expression of a GA 2-oxidase (GA-deactivating) gene, consistent with GA(3) being protected from 2-oxidation. Thus, des has the potential to enable substantial growth increases, with practical implications, for example, in biomass production.

Expression of recombinant staphylokinase, a fibrin-specific plasminogen activator of bacterial origin, in potato (Solanum tuberosum L.) plants.

One of the most dynamically developing sectors of green biotechnology is molecular farming using transgenic plants as natural bioreactors for the large scale production of recombinant proteins with biopharmaceutical and therapeutic values. Such properties are characteristic of certain proteins of bacterial origin, including staphylokinase. For many years, work has been carried out on the use of this protein in thrombolytic therapy. In this study, transgenic Solanum tuberosum plants expressing a CaMV::sak-mgpf-gusA gene fusion, were obtained. AGL1 A. tumefaciens strain was used in the process of transformation. The presence of the staphylokinase gene was confirmed by PCR in 22.5% of the investigated plants. The expression of the fusion transgene was detected using the ß-glucuronidase activity assay in 32 putative transgenic plants. Furthermore, on the basis of the GUS histochemical reaction, the transgene expression pattern had a strong, constitutive character in seven of the transformants. The polyacrylamide gel electrophoresis of a protein extract from the SAK/PCR-positive plants, revealed the presence of a119 kDa protein that corresponds to that of the fusion protein SAK-mGFP-GUSA. Western blot analysis, using an antibody against staphylokinase, showed the presence of the staphylokinase domain in the 119 kDa protein in six analyzed transformants. However, the enzymatic test revealed amidolytic activity characteristic of staphylokinase in the protein extract of only one plant. This is the first report on a Solanum tuberosum plant producing a recombinant staphylokinase protein, a plasminogen activator of bacterial origin.

Overexpression of cinnamate 4-hydroxylase gene enhances biosynthesis of decursinol angelate in Angelica gigas hairy roots.

Angelica gigas is a medicinal plant that produces pyranocoumarins, including decursin (D) and decursinol angelate (DA), which have neuroprotective, anticancer, and antiandrogenic effects. In this study, the coumarin biosynthetic pathway was engineered to increase the production of DA. Specifically, a vector was constructed which contained the A. gigas phenylalanine ammonia-lyase (AgPAL) and cinnamate 4-hydroxylase (AgC4H) genes that were driven by the cauliflower mosaic virus (CaMV) 35S promoter. Transgenic hairy roots that overexpressed AgPAL or AgC4H genes were obtained by using an Agrobacterium rhizogenes-mediated transformation system. Among them, only AgC4H-transgenic hairy root lines produced more DA than control transgenic hairy root lines. The enhanced gene expression corresponded to elevated C4H activities. This study showed the importance of C4H in the production of DA in A. gigas hairy root culture.

Isolation and characterization of the organ-specific and light-inducible promoter of the gene encoding rubisco activase in potato (Solanum tuberosum).

Constitutive promoters have been widely used in crop biotechnology applications. Tissue-specific or inducible promoters, however, have advantages in some cases. We isolated the 731-bp 5' flanking sequence of a potato (Solanum tuberosum) gene, encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase (RCA), which was isolated by genome walking. By using GUS as a reporter and with Northern blot analysis, the 702-bp fragment (referred to as StRCAp), ranging from nt -731 to -30 relative to the initiation code of the RCA gene, was analyzed in transgenic tobacco plants. The activity of StRCAp in leaves was 0.4-fold less than that of cauliflower mosaic virus 35S promoter, and was expressed throughout the green part of the light-grown transgenic T(1) seedlings, including cytoledons, leaves and young stems, but not roots. Further deletion analysis revealed that a shorter fragment (nt -249 to -30, StRCAp2) retained light-inducible features in cytoledons and leaves, but showed no detectable activity in young stems and roots. Although the activity of StRCAp2 in leaves was reduced significantly compared with that of StRCAp, the overall data indicated that cis-elements sufficient to regulate organ-specific and light-inducible transcription are within the 220-bp fragment. There is potential for application of StRCAp in plant genetic engineering.

Increasing seed mass and oil content in transgenic Arabidopsis by the overexpression of wri1-like gene from Brassica napus.

Rapeseed (Brassica napus) is one of the most important edible oilseed crops in the world and is increasingly used globally to produce bio-diesel. Therefore, increasing oil content of oilseed corps is of importance economically in both food and oil industries. The wri1 genes are differentially expressed in B. napus lines with different oil content. To investigate the effects of B. napus WRI1 (BnWRI1) on oil content, two Bnwri1 genes with different lengths, Bnwri1-1 and Bnwri1-2, were identified and sequenced. Homology analysis shows 80% amino acids of Bnwri1s are homologous to Arabidopsis thaliana WRI1 (AtWRI1). Overexpression of Bnwri1 cDNAs driven by cauliflower mosaic virus 35S-promoter in 51 transgenic A. thaliana lines resulted in 10-40% increased seed oil content and enlarged seed size and mass. Detailed analysis on transgenic embryos indicates an increased cell size other than cell number. In addition, Bnwri1 sequence polymorphism is highly related to oil content (p < 0.001). Taking together, Bnwri1 has potential applications in food and oil industries and in rapeseed breeding.

[Transgenic Belarussian-bred potato plants expressing genes for antimicrobial peptides of the cecropin-melittin type].

Binary vectors for Agrobacterium-mediated transformation were constructed to express the genes for antimicrobial peptides (APs) of the cectropin-melittin type under the control of the cauliflower mosaic virus 35S RNA promoter in plants. It was shown with Escherichia coli and Agrobacterium tumefaciens cells that the cassettes could be cloned in pB1121-based vectors with deletion of the 3-D-glycuronidase gene only in the orientation opposite to that of the original vector. Transgenic potato plants were obtained using the Belarussian varieties Odyssey, Vetraz, and Scarb. Their cells expressed the MsrA1 or CEMA peptides of the cecropin-melittin type. The expression was shown to confer higher resistance to bacterial (Erwinia carotovora) infection and extremely high resistance to fungal (Phytophtora infestans and Alternarla solani) infections.

Enhancement of heavy metal accumulation by tissue specific co-expression of iaaM and ACC deaminase genes in plants.

1-Aminocyclopropane deaminase (ACC) and tryptophan monooxygenase are two enzymes involved in plant senescence-inhibiting and growth-promoting regulation, respectively. In this study, two binary vectors were constructed in which the Agrobacterium iaaM gene was under the transcriptional control of a xylem-specific glycine-rich protein promoter alone, or co-expressed with the bacterial ACC deaminase gene, which was driven by the constitutive CaMV 35S promoter. Transgenic petunia shoots co-expressing both genes were able to root on medium supplemented with 7.5 mg l(-1) CoCl2. When T1 transgenic tobacco plants were grown in sand supplemented with Cu2+ and Co2+, tissue specific co-expression of both iaaM and ACC deaminase genes showed faster growth with larger biomass with a more extensive root system, and accumulated a greater amount of heavy metals than the empty vector control plants. When T1 transgenic tobacco plants were grown in soil watered with different concentrations of CuSO4, xylem specific expression of the iaaM gene caused the accumulation of more Cu2+ than the empty vector control at lower CuSO4 concentrations, but showed severe toxic symptoms at concentration of 100 mg l(-1) CuSO4. T1 transgenic plants co-expressing both genes accumulated more heavy metals into the plant shoots and can tolerate CuSO4 at 150 mg l(-1). In addition, plants co-expressing these two genes can grow well in a complex contaminated soil containing both inorganic and organic pollutants, while the growth of the control plants was greatly inhibited.

Polyubiquitin promoter-based binary vectors for overexpression and gene silencing in Lotus japonicus.

In this study, we compared the transcriptional activities between Cauliflower mosaic virus (CaMV)35S promoter and polyubiquitin (Ljubq1) promoter from Lotus japonicus using beta-glucuronidase (gus) reporter gene in transgenic plants of L. japonicus. The promoter analysis demonstrated that the Ljubq1 promoter possessed higher activity than the CaMV35S promoter in leaves, stems, roots, nodules, and pollen. Finally, we created GATEWAY conversion technology-compatible binary vectors for over-expression and RNA interference under the Ljubq1 promoter. These materials could provide alternative choice for studies in L. japonicus.

The transcription factor IDEF1 regulates the response to and tolerance of iron deficiency in plants.

Iron is essential for most living organisms and is often the major limiting nutrient for normal growth. Plants induce iron utilization systems under conditions of low iron availability, but the molecular mechanisms of gene regulation under iron deficiency remain largely unknown. We identified the rice transcription factor IDEF1, which specifically binds the iron deficiency-responsive cis-acting element IDE1. IDEF1 belongs to an uncharacterized branch of the plant-specific transcription factor family ABI3/VP1 and exhibits the sequence recognition property of efficiently binding to the CATGC sequence within IDE1. IDEF1 transcripts are constitutively present in rice roots and leaves. Transgenic tobacco plants expressing IDEF1 under the control of the constitutive cauliflower mosaic virus 35S promoter transactivate IDE1-mediated expression only in iron-deficient roots. Transgenic rice plants expressing an introduced IDEF1 exhibit substantial tolerance to iron deficiency in both hydroponic culture and calcareous soil. IDEF1 overexpression leads to the enhanced expression of the iron deficiency-induced transcription factor gene OsIRO2, suggesting the presence of a sequential gene regulatory network. These findings reveal cis element/trans factor interactions that are functionally linked to the iron deficiency response. Manipulation of IDEF1 also provides another approach for producing crops tolerant of iron deficiency to enhance food and biomass production in calcareous soils.

Chloroplast-like organelles were found in enucleate sieve elements of transgenic plants overexpressing a proteinase inhibitor.

SaPIN2a, a plant proteinase inhibitor from nightshade (Solanum americanum), was located to the enucleate sieve elements (SEs) of phloem. The expressed SaPIN2a in transgenic lettuce showed inhibition of plant endogenous trypsin- and chymotrypsin-like activities, suggesting that SaPIN2a can regulate proteolysis in plant cells. To further investigate the physiological role of SaPIN2a, we produced transgenic nightshade and lettuce plants overexpressing SaPIN2a from the cauliflower mosaic virus (CaMV) 35S promoter using Agrobacterium-mediated transformation. Overexpression of SaPIN2a in transgenic plants was demonstrated by northern blot and western blot analysis. SaPIN2a-overexpressing transgenic nightshade plants showed significantly lower height than wild-type plants. Transmission electron microscopy analysis showed that chloroplast-like organelles with thylakoids, which are not present in enucleate SEs of wild-type plants, were present in the enucleate SEs of SaPIN2a-overexpressing transgenic plants. This finding is discussed in terms of the possible role played by SaPIN2a in the regulation of proteolysis in SEs.

Glyphosate-induced anther indehiscence in cotton is partially temperature dependent and involves cytoskeleton and secondary wall modifications and auxin accumulation.

Yield reduction caused by late application of glyphosate to glyphosate-resistant cotton (Gossypium hirsutum; GRC) expressing CP4 5-enol-pyruvylshikmate-3-P synthase under the cauliflower mosaic virus-35S promoter has been attributed to male sterility. This study was aimed to elucidate the factors and mechanisms involved in this phenomenon. Western and tissue-print blots demonstrated a reduced expression of the transgene in anthers of GRC compared to ovules of the same plants. Glyphosate application to GRC grown at a high temperature regime after the initiation of flower buds caused a complete loss of pollen viability and inhibition of anther dehiscence, while at a moderate temperature regime only 50% of the pollen grains were disrupted and anther dehiscence was normal. Glyphosate-damaged anthers exhibited a change in the deposition of the secondary cell wall thickenings (SWT) in the endothecium cells, from the normal longitudinal orientation to a transverse orientation, and hindered septum disintegration. These changes occurred only at the high temperature regime. The reorientation of SWT in GRC was accompanied by a similar change in microtubule orientation. A similar reorientation of microtubules was also observed in Arabidopsis (Arabidopsis thaliana) seedlings expressing green fluorescent protein tubulin (tubulin alpha 6) following glyphosate treatment. Glyphosate treatment induced the accumulation of high levels of indole-3-acetic acid in GRC anthers. Cotton plants treated with 2,4-dichlorophenoxyacetic acid had male sterile flowers, with SWT abnormalities in the endothecium layer similar to those observed in glyphosate-treated plants. Our data demonstrate that glyphosate inhibits anther dehiscence by inducing changes in the microtubule and cell wall organization in the endothecium cells, which are mediated by auxin.

A microarray-based detection system for genetically modified (GM) food ingredients.

A multiplex DNA microarray chip was developed for simultaneous identification of nine genetically modified organisms (GMOs), five plant species and three GMO screening elements, i.e. the 35S promoter, the nos terminator and the nptII gene. The chips also include several controls, such as that for the possible presence of CaMV. The on-chip detection was performed directly with PCR amplified products. Particular emphasis was placed on the reduction of the number of PCR reactions required and on the number of primers present per amplification tube. The targets were biotin labelled and the arrays were detected using a colorimetric methodology. Specificity was provided by specific capture probes designed for each GMO and for the common screening elements. The sensitivity of the assay was tested by experiments carried out in five different laboratories. The limit of detection was lower than 0.3% GMO for all tests and in general around 0.1% for most GMOs. The chip detection system complies with the requirements of current EU regulations and other countries where thresholds are established for the labelling of GMO.

Mechanism of ribosome shunting in Rice tungro bacilliform pararetrovirus.

In plant pararetroviruses, pregenomic RNA serves both as a template for replication through reverse transcription and a polysictronic mRNA. This RNA has a complex leader sequence preceding the first large ORF. The leader contains multiple short ORFs and strong secondary structure, both inhibiting ribosome scanning. Translation on this RNA is initiated by shunting, in which scanning ribosomes bypass a large portion of the leader with the inhibitory secondary structure and short ORFs. In Cauliflower mosaic virus (CaMV), the ribosome shunting mechanism involves translation of the 5'-proximal short ORF terminating in front of the secondary structure that appears to force ribosomes to take off and resume scanning at a landing site downstream of the structure. Using two plant protoplast systems and shunt-competent wheat-germ extracts, we demonstrate that in Rice tungro bacilliform virus (RTBV) shunting also depends on the first short ORF followed by strong secondary structure. Swapping of the conserved shunt elements between CaMV and RTBV revealed the importance of nucleotide composition of the landing sequence for efficient shunting. The results suggest that the mechanism of ribosome shunting is evolutionary conserved in plant pararetroviruses.

A novel microsurgery method for intact plant tissue at the single cell level using ArF excimer laser microprojection.

A novel microsurgery technique for the partial removal of rigid cell-walls in intact plant tissue is established. Using a size-variable slit, an ArF excimer laser was microprojected on the surface of the targeted cell, and this method enabled the area- and depth-controllable processing of the cortical structure of plant cells including the cuticle and cell wall layer. In epidermal cells of all tested plants, viabilities of more than 90% were retained 24 h after irradiation. Scanning electron microscope (SEM) observation revealed that the cuticle layer of the irradiated region was completely ablated, and the cellulose microfibrils of the secondary cell wall were partially removed; furthermore, 4 days after laser treatment, the regeneration of cell wall fibrils was observed. As a model experiment, the transient expression of synthetic green fluorescent protein (sGFP) was performed by the microinjection of cauliflower mosaic virus (CMV) 35S promoter-derived sGFP gene through an "aperture" in the treated cell surface. Moreover, micron-sized fluorescent beads were successfully introduced by the same method into the onion cells indicating that this method can be used to introduce foreign materials as large as organelles.

Plants with increased expression of ent-kaurene oxidase are resistant to chemical inhibitors of this gibberellin biosynthesis enzyme.

The gibberellin (GA) biosynthetic pathway includes the three-step oxidation of ent-kaurene to ent-kaurenoic acid, catalyzed by the enzyme ent-kaurene oxidase (KO). Arabidopsis plants overexpressing the KO cDNA under the control of the cauliflower mosaic virus 35S promoter, with or without a translational fusion to a modified green fluorescent protein (GFP), are very similar to wild-type (WT) plants under normal growth conditions. In contrast, when WT and 35S:KO (or 35S:KO-GFP) seeds, seedlings or pollen tubes are grown in the presence of chemical inhibitors of KO, such as paclobutrazol and uniconazole, plants with increased KO expression are partially resistant to the effects of these inhibitors. In combination with the observation that decreased KO levels increase the sensitivity to KO inhibitors, the 35S:KO phenotypes demonstrate that the modification of KO enzyme levels could be used to create transgenic crop plants with altered KO inhibitor response. These results also suggest that the KO gene could be used as a selectable marker for plant regeneration based on resistance to KO inhibitors. Finally, the observation that pollen tubes expressing 35S:KO or 35S:KO-GFP have decreased sensitivity to KO inhibitors provides further evidence for a physiological role for GAs in pollen tube elongation.