Translocation of cell-penetrating peptides and delivery of their cargoes in triticale microspores

Microspore culture is contributing significantly in the field of plant breeding for crop improvement in general and cereals, in particular. In the present study, we investigated the uptake of fluorescently labeled cell-penetrating peptides (CPP; Tat, Tat₂, M-Tat, peptide vascular endothelial-cadherin, transportan) in the freshly isolated triticale microspores (mid-late uninucleate stage). We demonstrated that Tat (RKKRRQRRR) and Tat₂ (RKKRRQRRRRKKRRQRRR) are able to efficiently transduce GUS enzyme (272 kDa) in its functional form in 5 and 14% of the microspores, respectively, in a noncovalent manner. Pep-1, a synthetic CPP, was able to transduce GUS enzyme in its active form in 31% of the microspores. The effect of various endocytic and macropinocytic inhibitors on Tat₂-mediated GUS enzyme delivery was studied and revealed a preferred micropinocytosis entry. DNase I protection assay and confocal laser microscopy was carried out to recommend a ratio of 4:1 Tat₂-linear plasmid DNA (pActGUS) in complex preparation for microspore transfection. We further show that Tat₂ can successfully deliver GUS gene in near to 2% triticale microspores. The negative control mutated Tat (M-Tat: AKKRRQRRR) failed to transducer the GUS protein and transfect the GUS gene in microspore nucleus. The ability of CPPs to deliver macromolecules (protein as well as linear plasmid DNA) noncovalently has been demonstrated in triticale isolated microspores. It further confirms potential applications of CPPs in developing simple, time saving, cost effective plant genetic engineering technologies.     

Plant transformation by particle bombardment of embryogenic pollen

Summary Direct delivery of DNA into embryogenic pollen was used to produce transgenic plants in tobacco. A plasmid bearing the ß-glucuronidase (GUS) marker gene in fusion with the 35S-promoter was introduced by microprojectile bombardment into mid-binucleate pollen of Nicotiana tabacum that had been induced to form embryos by a starvation treatment. In cytochemical expression assays, 5 out of 10⁴ pollen grains were GUS⁺. Visual selection by staining with a non-lethal substrate for GUS was used to manually isolate transformed embryos. From the initial population of embryogenic GUS⁺ pollen, 1–5% developed into multicellular structures and 0.02% formed regenerable embryos. Two haploid transformants were regenerated. GUS expression was detected in different parts of the plants, and Southern analysis confirmed stable integration of the foreign DNA. Diploidisation was induced by injection of colchicine into the stem near adventitious buds. Offspring from selfings and backcrosses of one transformant were tested for GUS expression and by Southern blots. All F1-plants were transgenic, in accordance with Mendelian inheritance.Abbreviations GUS ß-glucuronidase - CaMV Cauliflower Mosaic Virus - MCS multicellular structure - NPTII neomycin phosphotransferase - PEG polyethylene glycol - X-gluc 5-bromo-4-chloro-3-indolyl glucuronide - DAPI 4,6-diamidino-2-phenylindole - Tris Tris(hydroxymethyl)aminomethane hydrochloride - EDTA ethylenedinitrilo tetraacetic acid, disodium salt dihydrate     

DNA addition or deletion is associated with a major karyotype polymorphism in the fungal phytopathogen Colletotrichum gloeosporioides

The bacterial GUS (β-glucuronidase) gene has been used as a reporter gene in plants and bacteria and was recently expressed in filamentous fungi. Here, we report the application of GUS for the establishment of transient and stable gene expression systems in the phytopathogenic fungus Cochliobolus heterostrophus. The utility of the transient expression system is demonstrated in applications involving promoter analysis and in tests of various parameters of a transformation system, for comparing the rates of stable and transient transformation events using GUS as sole screening marker and for comparing different transformation systems using either GUS or a dominant selection marker. For these purposes two plasmids were constructed harbouring the GUS gene and the hph gene of Escherichia coli which confers resistance to the antibiotic hygromycin B (HygB), ligated either to the P1 or GPD1 (glyceraldehyde 3 phosphate dehydrogenase) promoter of C. heterostrophus. In transient expression studies the first appearance of GUS activity was observed within 2 h after transformation and maximal values were obtained after 7 or 10 h, depending on the promoter fused to the GUS gene. At peak activity, the GPD1 promoter was revealed to be five fold stronger than the P1 promoter. The same difference in promoter strenght was observed when the vectors were stably integrated in the fungal genome. Using the GUS gene as a colour selection marker in plate assays, it was possible to detect transformants and monitor the process of transient gene expression visually. Blue transformants obtained by screening for the GUS phenotype were mitotically unstable. Transformants obtained by selecting for HygB resistance were mitotically stable and expressed the β-glucuronidase gene constitutively. GUS activity in fungal colonies was detected fluorometrically in a nondestructive plate assay. The pathogenicity of these strains was unaltered compared with wild type. The GUS phenotype allowed selective blue staining of the colonizing mycelia on maize leaves.     

Study of uptake of cell penetrating peptides and their cargoes in permeabilized wheat immature embryos

The uptake of five fluorescein labeled cell-penetrating peptides (Tat, Tat(2), mutated-Tat, peptide vascular endothelial-cadherin and transportan) was studied in wheat immature embryos. Interestingly, permeabilization treatment of the embryos with toluene/ethanol (1 : 20, v/v with permeabilization buffer) resulted in a remarkably higher uptake of cell-penetrating peptides, whereas nonpermeabilized embryos failed to show significant cell-penetrating peptide uptake, as observed under fluorescence microscope and by fluorimetric analysis. Among the cell-penetrating peptides investigated, Tat monomer (Tat) showed highest fluorescence uptake (4.2-fold greater) in permeabilized embryos than the nonpermeabilized embryos. On the other hand, mutated-Tat serving as negative control did not show comparable fluorescence levels even in permeabilized embryos. A glucuronidase histochemical assay revealed that Tat peptides can efficiently deliver functionally active beta-glucuronidase (GUS) enzyme in permeabilized immature embryos. Tat(2)-mediated GUS enzyme delivery showed the highest number of embryos with GUS uptake (92.2%) upon permeabilization treatment with toluene/ethanol (1 : 40, v/v with permeabilization buffer) whereas only 51.8% of nonpermeabilized embryos showed Tat(2)-mediated GUS uptake. Low temperature, endocytosis and macropinocytosis inhibitors reduced delivery of the Tat(2)-GUS enzyme cargo complex. The results suggest that more than one mechanism of cell entry is involved simultaneously in cell-penetrating peptide-cargo uptake in wheat immature embryos. We also studied Tat(2)-plasmid DNA (carrying Act-1GUS) complex formation by gel retardation assay, DNaseI protection assay and confocal laser microscopy. Permeabilized embryos transfected with Tat(2)-plasmid DNA complex showed 3.3-fold higher transient GUS gene expression than the nonpermeabilized embryos. Furthermore, addition of cationic transfecting agent Lipofectamine 2000 to the Tat(2)-plasmid DNA complex resulted in 1.5-fold higher transient GUS gene expression in the embryos. This is the first report demonstrating translocation of various cell-penetrating peptides and their potential to deliver macromolecules in wheat immature embryos in the presence of a cell membrane permeabilizing agent.     

GUS expression driven by constitutive and phloem-specific promoters in citrus hybrid US-802

Transgenic solutions are being widely explored to develop huanglongbing (HLB) resistance in citrus. A critical component of a transgenic construct is the promoter, which determines tissue specificity and level of target gene expression. This study compares the characteristics of five promoters regulating the beta-glucuronidase (GUS) reporter gene in the trifoliate hybrid rootstock US-802. Two of the selected promoters direct high levels of constitutive transgene expression in other dicotyledonous plants: 2X35S, the tandem-repeat promoter of the cauliflower mosaic virus 35S gene and bul409S, a truncation of the potato polyubiquitin promoter. Because Candidatus Liberibacter, the Gram-negative bacterium associated with HLB, infects only the phloem tissue, it may be advantageous to limit transgene expression to the vascular tissue and reduce expression in the fruit. Thus, we also tested three promoters that demonstrate phloem specificity when transformed and expressed in other plants: WDV, from wheat dwarf geminivirus; AtSUC2, the sucrose-H+ symporter gene promoter from Arabidopsis; and CsSUS, the sucrose synthase promoter from citrus. Histochemical staining for GUS activity was observed throughout leaf and stem tissues for the constitutive promoters, while the three phloem-specific promoters largely showed the expected tissue-specific staining. Expression of GUS in some individual transformants with promoters CsSUS and WDV appeared leaky, with some laminar tissue staining. Relative quantification of qRT-PCR data revealed a wide range of mRNA abundance from transgenics with each of the five promoters. Fluorometry also revealed that GUS activity differed depending on the promoter used, but mRNA levels and enzyme activity were not highly correlated.     

High frequency regeneration via direct somatic embryogenesis and efficient Agrobacterium- mediated genetic transformation of tobacco

A direct somatic embryogenesis protocol was developed for four cultivars of Nicotiana species, by using leaf disc as an explant. Direct somatic embryogenesis of Nicotiana by using BAP and IAA has not been investigated so far. This method does not require formation of callus tissues which leads to somaclonal variations. The frequency of somatic embryogenesis was strongly influenced by the plant growth hormones. The somatic embryos developing directly from explant tissue were noticed after 6 d of culture. Somatic embryogenesis of a high frequency (87–96%) was observed in cultures of the all four genotypes (Nicotiana tabacum, N. benthamiyana, N. xanthi, N. t cv petihavana). The results showed that the best medium for direct somatic embryogenesis was MS supplemented with 2.5 mg/l, 0.2 mg/l IAA and 2% sucrose. Subculture of somatic embryos onto hormone free MS medium resulted in their conversion into plants for all genotypes. About 95% of the regenerated somatic embryos germinated into complete plantlets. The plants showed morphological and growth characteristics similar to those of seed-derived plants. Explants were transformed using Agrobacterium tumifacious LBA4404 plasmid pCAMBIA1301 harboring the GUS gene. The regenerated transgenic plants were confirmed by PCR analysis and histochemical GUS assay. The transformation efficiency obtained by using the Agrobacterium- mediated transformation was more than 95%. This method takes 6 wk to accomplish complete transgenic plants through direct somatic embryogenesis. The transgenic plantlets were acclimatized successfully with 98% survival in greenhouse and they showed normal morphological characteristics and were fertile. The regeneration and transformation method described herein is very simple, highly efficient and fast for the introduction of any foreign gene directly in tobacco through direct somatic embryogenesis.     

A 286 bp upstream regulatory region of a rice anther-specific gene, OSIPP3, confers pollen-specific expression in Arabidopsis

OSIPP3 gene (coding for pectin methylesterase inhibitor protein) was isolated from a pre-pollinated inflorescence-specific cDNA library by differential screening of stage-specific libraries from Oryza sativa. OSIPP3 is present in the genome of rice as a single copy gene. OSIPP3 gene was expressed exclusively in the pre-pollinated spikelets of rice. Upstream regulatory region (URR) of OSIPP3 was isolated and a series of 5′-deletions were cloned upstream of GUS reporter gene and were used to transform Arabidopsis. OSIPP3_del1 and del2 transgenic plants showed GUS expression in root, anther and silique, while OSIPP3_del3 showed GUS activity only in anthers and siliques. Pollen-specific expression was observed in case of plants harboring OSIPP3_del4 construct. It can, therefore, be concluded that the OSIPP3 URR between ?178 and +108 bp is necessary for conferring pollen-specific expression in Arabidopsis.     

Transformation of microspore-derived embryos of winter oilseed rape (Brassica napus L.) by usingAgrobacterium tumefaciens

Haploid microspore-derived embryos (MDEs) constitute a unique material for the introduction of new traits into winter oilseed rape (Brassica napus). MDEs have been transformed by usingAgrobacterium tumefaciens strains EHA105 and LBA4404, both carrying the binary vector pKGIB containing theuidA gene encoding β-glucuronidase (GUS) and thebar gene as a marker of resistance to phosphinotricin. Transformed embryos expressed GUS and regenerated plants that were resistant to herbicide Basta, as confirmed by a leaf-painting test. Progeny plants of the transformant T-39 were all transgenic, as they inherited T-DNA from their doubled haploid parental plant. Southern-blot analysis confirmed the integration and transmission of T-DNA into T₁ plants. Transformation of MDEs facilitates the obtaining of winter oilseed rape homozygous for the introduced genes.     

GUS activity for miR165a/166b, REV, and WUS/CLV3 in in vitro direct Arabidopsis thaliana shoot regeneration

In Arabidopsis thaliana, the process of shoot regeneration in vitro requires the presence of specific miRNAs. We describe here the β-glucuronidase (GUS) expression domains for miR165a/166b, REV, and WUS/CLV3 during direct shoot regeneration. Increased GUS activity of miR166b and REV were first detected within the shoot apical meristem of explants, while no pmiR165a::GUS activity appeared there. The zone of pWUS::GUS activity covered the inner sides of developing protuberances, while that of pCLV3::GUS was more restricted. Once the primary shoot had emerged from the protuberance, pREV::GUS activity was turned on throughout the protuberance. pmiR165a::GUS activity was detected in a small number of protuberance surface cells, while pWUS::GUS activity was restricted to within a few cells beneath the protuberance surface. After the differentiation of leaf-like structures, GUS activity for miR165a and miR166b appeared largely on their abaxial surface, while pWUS::GUS activity was concentrated at the apex of the primary shoot, and no pCLV3::GUS activity was detectable. Following the formation of secondary shoots, pmiR165a::GUS activity was detected on their abaxial surface. GUS activity for miR166b, REV, and WUS/CLV3 were concentrated in the stem apical meristem. The observations suggested that each member of this set of genes might play a distinct role in both primary and secondary shoot regeneration.     

Pineapple translation factor SUI1 and ribosomal protein L36 promoters drive constitutive transgene expression patterns in Arabidopsis thaliana

The availability of a variety of promoter sequences is necessary for the genetic engineering of plants, in basic research studies and for the development of transgenic crops. In this study, the promoter and 5′ untranslated regions of the evolutionally conserved protein translation factor SUI1 gene and ribosomal protein L36 gene were isolated from pineapple and sequenced. Each promoter was translationally fused to the GUS reporter gene and transformed into the heterologous plant system Arabidopsis thaliana. Both the pineapple SUI1 and L36 promoters drove GUS expression in all tissues of Arabidopsis at levels comparable to the CaMV35S promoter. Transient assays determined that the pineapple SUI1 promoter also drove GUS expression in a variety of climacteric and non-climacteric fruit species. Thus the pineapple SUI1 and L36 promoters demonstrate the potential for using translation factor and ribosomal protein genes as a source of promoter sequences that can drive constitutive transgene expression patterns.