Agrobacterium-mediated Transient Transformation Research Articles

Identification and functional characterization of the MdHB-1 gene promoter sequence from Malus×domestica

AbstractHomeobox 1 in Malus×domestica (MdHB-1) is a transcription factor that belongs to homeodomain-leucine zipper I (HD-Zip I) protein subfamily. According to previous reports, MdHB-1 could regulate ethylene synthesis by binding with the MdACO1 promoter, but other functions of MdHB-1 are still unknown. To reveal more clues concerning the characters of the MdHB-1 gene promoter and the functions of MdHB-1, the promoter region of MdHB-1 was cloned from the Royal Gala apple genome and recombined with the β-glucuronidase (GUS) gene in this study. This research was conducted in Nicotiana tabacum and supported by Agrobacterium-mediated transient transformation and bioinformatics analysis. Deletion analysis of the MdHB-1 promoter showed that the GUS gene could be activated by serially deleted promoters, and the activity promoted by 680 nucleotides (nt) was the lowest. The region, which is 266 nt upstream of the initiation code (ATG), was effective for GUS expression. Meanwhile, the activity of the MdHB-1 promoter (−1 057 nt), which was stronger than MdHB-1 promoter (−1 057 to −266 nt) and lack the 5′-untranslated region (5′-UTR), showed that 5′-UTR may have a positive effect on gene transcription. After the sequence analysis, the cis-acting elements that respond to hormones and environmental stresses were identified in the promoter region. The MdHB-1 promoter (1 057 nt) activity in Nicotiana tabacum was positively induced by ethrel and darkness, and it was suppressed by gibberellic acid (GA), whereas abscisic acid (ABA), salicylic acid (SA), wounding, and Pseudomonas syringae pv. tomato (DC3000) treatments revealed a slight auxo-action. These results reveal that the MdHB-1 promoter receive internal or external signals, and MdHB-1 may refer to many biological activities in apple, such as its stress response, development, and ripening.

Agrobacterium-mediated transient expression via root absorption in flowering Chinese cabbage.

BackgroundBecause most transient transformation techniques are inadequate for functional genomics research in roots, we aimed to develop a simple and efficient Agrobacterium-mediated transient transformation system that utilized root absorption for research in flowering Chinese cabbage.ResultsBoth semi-quantitative and fluorescent quantitative RT-PCR confirmed that the target gene BcAMT1;3 was more highly expressed in plants that were infected with the transformed Agrobacterium strain (EHA105-p35S-BcAMT1;3) than in control plants that were infected with the control strain (EHA105-p35S). Furthermore, GUS staining analysis conformed the availability of this transient transformation system. In addition, we found that the highest transformation efficiency was achieved using an Agrobacterium cell density of OD600 = 0.3 for 3–6 h, without hyperosmotic pretreatment, and under these conditions, the peak transformation efficiency was observed at 2 and 4 d after infection.ConclusionsThe transformation method developed by the present study is simple and convenient, since no special equipment is required, and since the method causes no damage, the plants can be used for subsequent experiments.

In vitro shoot multiplication and ex vitro rooting of Eustoma grandiflorum by shoot tips using 2,4-D and BAP

An Agrobacterium-mediated transient transformation system was established for gene functional analysis in persimmon (Diospyros kaki Thunb.) utilizing sonication followed by vacuum infiltration of seedlings in vitro. The optimal vacuum conditions for agroinfiltration of persimmon were evaluated by monitoring expression of GUS reporter gene. The technique was further applied to evaluate five different genotypes. Among the genotypes examined in vitro, ‘Xiaoguo-tianshi’ (Chinese PCNA type) produced the highest expression levels of both GUS and GFP, and was the most ideal genotype. Furthermore, the developed system was successfully utilized for DkLAR (leucoanthocyanidin reductase) gene transient silencing. DMACA (4- dimethylaminocinnamaldehyde) staining and the Folin-Ciocalteau assay revealed that the proanthocyanidins (PAs) levels was markedly decrease in ‘Xiaoguo-tianshi’ and ‘Mopanshi’ (Chinese PCA type) leaves. These results confirmed that DkLAR plays an important role in the accumulation of PAs in persimmon. In conclusion, based on the Agrobacterium-mediated vacuum infiltration, we developed a reliable and highly efficient transient transformation system that can be used for gene functional analysis through the seedlings in vitro.

Protocol: transient expression system for functional genomics in the tropical tree Theobroma cacao L.

BackgroundTheobroma cacao L., the source of cocoa, is a crop of significant economic value around the world. To facilitate the study of gene function in cacao we have developed a rapid Agrobacterium-mediated transient genetic transformation protocol. Here we present a detailed methodology for our transformation assay, as well as an assay for inoculation of cacao leaves with pathogens.ResultsAgrobacteriumtumefaciens cultures are induced then vacuum-infiltrated into cacao leaves. Transformation success can be gauged 48 h after infiltration by observation of green fluorescent protein and by qRT-PCR. We clarify the characteristics of cacao leaf stages and demonstrate that our strategy efficiently transforms leaves of developmental stage C. The transformation protocol has high efficacy in stage C leaves of four of eight tested genotypes. We also present the functional analysis of cacao chitinase overexpression using the transient transformation system, which resulted in decreased pathogen biomass and lesion size after infection with Phytophthora tropicalis.ConclusionsLeaves expressing transgenes of interest can be used in subsequent functional genetic assays such as pathogen bioassay, metabolic analysis, gene expression analysis etc. This transformation protocol can be carried out in 1 day, and the transgenes expressing leaf tissue can be maintained in petri dishes for 5–7 days, allowing sufficient time for performance of additional downstream gene functional analysis. Application of these methods greatly increases the rapidity with which candidate genes with roles in defense can be tested.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-016-0119-5) contains supplementary material, which is available to authorized users.

Agrobacterium-mediated transient MaFT expression in mulberry (Morus alba L.) leaves.

To optimize Agrobacterium-mediated transient transformation assay in mulberry (Morus alba L.), various infiltration methods, Agrobacterium tumefaciens (A. tumefaciens) strains, and bacterial concentrations were tested in mulberry seedlings. Compared with LBA4404, GV3101 harboring pBE2133 plasmids presented stronger GUS signals at 3 days post infiltration using syringe. Recombinant plasmids pBE2133:GFP and pBE2133:GFP:MaFT were successfully constructed. Transient expression of MaFT:GFP protein was found in leaves, petiole (cross section), and shoot apical meristem (SAM) of mulberry according to the GFP signal. Moreover, MaFT:GFP mRNA was also detected in leaves and SAM via RT-PCR and qRT-PCR. An efficient transient transformation system could be achieved in mulberry seedlings by syringe using A. tumefaciens GV3101 at the OD600 of 0.5. The movement of MaFT expression from leaves to SAM might trigger the precocious flowering of mulberry.

Characterization of FeDREB1 promoter involved in cold- and drought-inducible expression from common buckwheat (Fagopyrum esculentum).

C-repeat-binding factor (CBF)/dehydration-responsive element (DREB) transcription factors play key roles in plant stress responses. However, little information is available on the regulation of CBF/DREB expression. In this study, we isolated and characterized the FeDREB1 promoter sequence from the common buckwheat accession Xinong 9976. To identify the upstream region of the FeDREB1 gene required for promoter activity, we constructed a series of FeDREB1 promoter deletion derivatives. Each deletion construct was analyzed through Agrobacterium-mediated transient transformation in tobacco leaves treated with 4°C cold or drought stress. Promoter-beta-glucuronidase fusion assays revealed that the pCD1 (-270 bp) deletion in the upstream region of FeDREB1 could activate expression of the GUS gene at 4°C. The pCD1 (-270 bp), pCD2 (-530 bp), and pCD3 (-904 bp) deletion induced low-level GUS expression under drought stress. However, the pCD4 (-1278 bp) deletion clearly activated GUS gene expression. Our results suggest that sections pCD1 (-270 bp) and pCD4 (-1278 bp) in the FeDREB1 gene promoter are new sources of induced promoters for adversity-resistance breeding in plant genetic engineering.

The mannose‐binding lectin gene FaMBL1 is involved in the resistance of unripe strawberry fruits to Colletotrichum acutatum

The fungal pathogen Colletotrichum acutatum is the causal agent of strawberry (Fragaria × ananassa) anthracnose. Although the fungus can infect strawberry fruits at both unripe and ripe stages, the symptoms appear only on red ripe fruits. On white unripe fruits, the pathogen becomes quiescent as melanized appressoria after 24 h of interaction. Previous transcriptome analysis has indicated that a mannose-binding lectin (MBL) gene is the most up-regulated gene in 24-h-infected white strawberries, suggesting a role for this gene in the low susceptibility of unripe stages. A time course analysis of the expression of this MBL gene, named FaMBL1 (Fragaria × ananassa MBL 1a), was undertaken to monitor its expression profile in white and red fruits at early interaction times: FaMBL1 was expressed exclusively in white fruit after 24 h, when the pathogen was quiescent. Agrobacterium-mediated transient transformation was used to silence and overexpress the FaMBL1 gene in 24-h-infected white and red strawberries, respectively. FaMBL1-silenced unripe fruits showed an increase in susceptibility to C. acutatum. These 24-h-infected tissues contained subcuticular hyphae, indicating pathogen penetration and active growth. In contrast, overexpression of FaMBL1 in ripe fruits decreased susceptibility; here, 24-h-infected tissues showed a high percentage of ungerminated appressoria, suggesting that the growth of the pathogen had slowed. These data suggest that FaMBL1 plays a crucial role in the resistance of unripe strawberry fruits to C. acutatum.

A rapid, highly efficient and economical method of Agrobacterium-mediated in planta transient transformation in living onion epidermis.

Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale.

A mutation in negative regulator of basal resistance WRKY17 of Arabidopsis increases susceptibility to Agrobacterium-mediated transient genetic transformation

Agrobacterium is a phytopathogenic bacterium that induces crown gall disease in many plant species by transferring and integrating a segment of its own DNA (T-DNA) into its host genome. Whereas Agrobacterium usually does not trigger an extensive defense response in its host plants, it induces the expression of several defense-related genes and activates plant stress reactions. In the complex interplay between Agrobacterium and its host plant, Agrobacterium has evolved to take advantage of these plant defense pathways for its own purpose of advancement of the infection process. For example, Agrobacterium utilizes the host stress response transcriptional regulator VIP1 to facilitate nuclear import and proteasomal uncoating of its T-DNA during genetic transformation of the host cell. In Arabidopsis, the VIP1 gene expression is repressed by WRKY17, a negative regulator of basal resistance to Pseudomonas. Thus, we examined whether WRKY17 is also involved in plant susceptibility to genetic transformation by Agrobacterium. Using reverse genetics, we showed that a wrky17 mutant displays higher expression of the VIP1 gene in roots, but not in shoots. In a root infection assay, the wrky17 mutant plants were hyper-susceptible to Agrobacterium compared to wild type plants. WRKY17, therefore, may act as a positive regulator of Arabidopsis resistance to Agrobacterium. This notion is important for understanding the complex regulation of Agrobacterium-mediated transient genetic transformation; thus, although this paper reports a relatively small set of data that we do not plan to pursue further in our lab, we believe it might be useful for the broad community of plant pathologists and plant biotechnologists.

A mutation in negative regulator of basal resistance WRKY17 of Arabidopsis increases susceptibility to Agrobacterium-mediated transient genetic transformation

A mutation in negative regulator of basal resistance WRKY17 of Arabidopsis increases susceptibility to Agrobacterium-mediated transient genetic transformation

Expression patterns and promoter characteristics of the gene encoding Actinidia deliciosa l-galactose-1-phosphate phosphatase involved in the response to light and abiotic stresses

The gene encoding L-galactose-1-phosphate phosphatase (GPP) plays a central role in ascorbic acid (AsA) biosynthesis in plants. Here, we report AsA contents, GPP expression, and functioning of its promoter in response to light, exogenous stress-signalling hormones, or abiotic stresses in kiwifruit (Actinidia deliciosa). To identify the upstream region of GPP required for promoter activity, we constructed a series of promoter deletion derivatives. Each construct was analyzed by Agrobacterium-mediated transient transformation in tobacco leaves after various treatments. Some correlation was observed between the relative levels of GPP mRNA and AsA contents when kiwi leaves were exposed to varying light conditions, treatment with ABA or SA, wounding, or a hypoxic environment. Analysis of a series of 5' deletions in tobacco leaves indicated that the proximal area 390bp from the transcription initiation site was needed for establishing both the constitutive and the induced patterns of expression. This promoter was induced by light or one of our abiotic treatments. These results suggest that GPP is regulated by light or abiotic stress and that it plays an important role in controlling AsA contents in kiwifruit.

An in planta, Agrobacterium-mediated transient gene expression method for inducing gene silencing in rice (Oryza sativa L.) leaves

BackgroundLocalized introduction and transient expression of T-DNA constructs mediated by agro-infiltration of leaf tissues has been largely used in dicot plants for analyzing the transitivity and the cell-to cell movement of the RNAi signal. In cereals, however, the morphology of the leaf and particularly the structure of the leaf epidermis, prevent infiltration of a bacterial suspension in cells by simple pressure, a method otherwise successful in dicots leaves. This study aimed at establishing a rapid method for the functional analysis of rice genes based on the triggering of RNA interference (RNAi) following Agrobacterium-mediated transient transformation of leaves.ResultsUsing an agro-infection protocol combining a wound treatment and a surfactant, we were able to obtain in a reliable manner transient expression of a T-DNA-borne uidA gene in leaf cells of japonica and indica rice cultivars. Using this protocol to transiently inhibit gene expression in leaf cells, we introduced hairpin RNA (hpRNA) T-DNA constructs containing gene specific tags of the phytoene desaturase (OsPDS) and of the SLENDER 1 (OsSLR1) genes previously proven to trigger RNAi of target genes in stable transformants. SiRNA accumulation was observed in the agro-infected leaf area for both constructs indicating successful triggering of the silencing signal. Accumulation of secondary siRNA was observed in both stably and transiently transformed leaf tissues expressing the HpRNA OsSLR1 construct. Gene silencing signalling was investigated in monitoring the parallel time course of OsPDS-derived mRNA and siRNA accumulation in the agro-infiltrated leaf area and adjacent systemic sectors. The sensitive RT-Q-PCR method evidenced a consistent, parallel decrease of OsPDS transcripts in both the agroinfiltred and adjacent tissues, with a time lag for the latter.ConclusionsThese results indicate that the method is efficient at inducing gene silencing in the agro-infected leaf area. The transfer of low amounts of siRNA, probably occurring passively through the symplastic pathway from the agro-infected area, seemed sufficient to trigger degradation of target transcripts in the adjacent tissues. This method is therefore well suited to study the cell-to-cell movement of the silencing signal in a monocot plant and further test the functionality of natural and artificial miRNA expression constructs.Electronic supplementary materialThe online version of this article (doi:10.1186/1939-8433-5-23) contains supplementary material, which is available to authorized users.

Genomic Structure, Sub-Cellular Localization, and Promoter Analysis of the Gene Encoding Sorbitol-6-Phosphate Dehydrogenase from Apple

Sorbitol is the primary photosynthetic product and the translocatable and storage carbohydrate in apple (Malus domestica) and other fruit tree species within the Rosaceae family. Sorbitol-6-phosphate dehydrogenase (S6PDH, EC 1.1.1.200) is the key enzyme in the biosynthesis of sorbitol. In this study, we isolated two full-length genomic sequences for S6PDH from “Gala” apple. The two sequences have same six exons and first two introns, but the sizes of their last three introns are different. The two sequences were mapped to the same loci on chromosome 10. Immunogold electron microscopy analysis demonstrates that the S6PDH is localized mainly in leaf cytosol and chloroplasts. We also isolated and analyzed the promoter region of S6PDH and constructed a series of promoter deletion derivatives with β-glucuronidase (GUS) gene to identify the upstream region of the S6PDH gene required for promoter activity. The GUS activity in Agrobacterium-mediated transient transformation of tobacco leaves reveals that the -1719 region is more important for gene expression contrasting with other regions in the S6PDH promoter. The promoter region can be induced by cold, dark, and abscisic acid treatment.

An efficient Agrobacterium‐mediated transient transformation of Arabidopsis

Agrobacterium tumefaciens-mediated transient transformation has been a useful procedure for characterization of proteins and their functions in plants, including analysis of protein-protein interactions. Agrobacterium-mediated transient transformation of Nicotiana benthamiana by leaf infiltration has been widely used due to its ease and high efficiency. However, in Arabidopsis this procedure has been challenging. Previous studies suggested that this difficulty was caused by plant immune responses triggered by perception of Agrobacterium. Here, we report a simple and robust method for Agrobacterium-mediated transient transformation in Arabidopsis. AvrPto is an effector protein from the bacterial plant pathogen Pseudomonas syringae that suppresses plant immunity by interfering with plant immune receptors. We used transgenic Arabidopsis plants that conditionally express AvrPto under the control of a dexamethasone (DEX)-inducible promoter. When the transgenic plants were pretreated with DEX prior to infection with Agrobacterium carrying a β-glucuronidase (GUS, uidA) gene with an artificial intron and driven by the CaMV 35S promoter, transient GUS expression was dramatically enhanced compared to that in mock-pretreated plants. This transient expression system was successfully applied to analysis of the subcellular localization of a cyan fluorescent protein (CFP) fusion and a protein-protein interaction in Arabidopsis. Our findings enable efficient use of Agrobacterium-mediated transient transformation in Arabidopsis thaliana.

Functional characterization of sugarcane MYB transcription factor gene promoter (PScMYBAS1) in response to abiotic stresses and hormones

The sugarcane (Saccharum officinarum) stress-related MYB transcription factor gene, ScMYBAS1, demonstrated induced response to water deficit and salt stress in our previous study. To elucidate its stress tolerance mechanism at the transcriptional level, we isolated and characterized the promoter (PScMYBAS1, 1,033 bp) flanking the 5' ScMYBAS1 coding region from the sugarcane genome. A series of PScMYBAS1 deletion derivatives from the transcription start site (-56, -152, -303, -442, -613, -777, -843, -1,033) was fused to the uidA reporter gene (GUS) and each deletion construct was analyzed by Agrobacterium-mediated transient transformation in tobacco leaves subjected to dehydration, salinity, cold, wounding, gibberellic acid (GA), salicylic acid (SA), and methyl jasmonic acid (MeJA). Deletion analysis of the promoter, PScMYBAS1, suggested that the 303-bp promoter region was required for basal expression. Promoter fragments, 777 bp or longer showed ~twofold to ~fourfold increased induction of GUS in response to abiotic stress (dehydration, salt, cold, wounding) and hormone (SA, MeJA) treatments. These findings further our understanding of the regulation of ScMYBAS1 expression and provide a new stress-inducible promoter system in transgenic plants.

Mitochondrial and chloroplastic targeting signals of NADP+-dependent isocitrate dehydrogenase

Many mitochondrial and chloroplast proteins are encoded in the nucleus and subsequently imported into the organelles via active protein transport systems. While usually highly specific, some proteins are dual-targeted to both organelles. In tobacco (Nicotiana tabacum L.), the cDNA encoding the mitochondrial isoform of NADP+-dependent isocitrate dehydrogenase (NADP+-ICDH) contains two translational ATG start sites, suggesting the possibility of tandem targeting signals. In this work, the putative mitochondrial and chloroplastic targeting signals from NADP+-ICDH were fused to a yellow fluorescent protein (YFP) reporter to generate a series of constructs and introduced into tobacco leaves by Agrobacterium-mediated transient transformation. The subsequent sub-cellular locations of the ICDH:YFP fusion proteins were then examined using confocal microscopy. Constructs predicted to be targeted to the chloroplast all localized to the chloroplast. However, this was not the case for all of the constructs that were predicted to be mitochondrial targeted. Although some constructs localized to mitochondria as expected, others appeared to be chloroplast localized. This was attributed to an additional 50 amino acid residues of the mature NADP+-ICDH protein that were present in those constructs, generated from either 'Xanthi' or 'Petit Havana' cultivars of tobacco. The results of this study raise interesting questions regarding the targeting and processing of organellar isoforms of NADP+-ICDH.

Characterization of a novel stilbene synthase promoter involved in pathogen- and stress-inducible expression from Chinese wild Vitis pseudoreticulata

Stilbene synthase is a plant-specific polyketide synthase, and plays important roles in diverse metabolic processes. The genomic stilbene synthase gene was cloned from accession "Baihe-35-1" of Chinese wild Vitis pseudoreticulata, and a stilbene synthase of V. pseudoreticulata (VpSTS) transcripts expressed in the grape-powdery mildew interaction were determined by semi-quantitative RT-PCR. To monitor VpSTS expression in plant, the promoter region flanking the 5' VpSTS coding region was isolated from the genomic DNA of Chinese wild V. pseudoreticulata accession Baihe-35-1. Alignment of the VpSTS promoter sequence showed a 56.4% identity to Vitis vinifera. To identify the upstream region of the VpSTS gene required for promoter activity, a series of VpSTS promoter deletion derivatives was constructed. Each deletion construct was analyzed by Agrobacterium-mediated transient transformation in grapevine and tobacco leaves after infection by Uncinula necator and Alternaria alternata. In transiently transformed grapevine leaves, GUS activity was also determined after treatment with salicylic acid (SA) and 4 degrees C cold. Analysis of a series of 5' deletions of the VpSTS promoter in grapevine leaves indicated that the proximal 162 bp from the transcription initiation site was proved to be necessary for establishing both the constitutive and induced pattern of expression.

Transient transformation of sunflower leaf discs via an Agrobacterium-mediated method: applications for gene expression and silencing studies

The sunflower belongs to the Compositae family and is an economically important crop because of the quality of its oil. Unfortunately, molecular analyses are limited due to the lack of genomic information, mutant libraries and efficient and rapid transformation protocols. In a wide variety of species, Agrobacterium-mediated transient transformation is a useful tool that can provide valuable insight into many biological processes. However, this technology has not been routinely applied to the sunflower because of difficulties with infiltration. Here, we present an optimized protocol for Agrobacterium-mediated transient transformation of leaf discs. Using this procedure, we were able to quickly overexpress or silence a given gene, enabling us to study several biochemical processes and characterize sunflower regulatory sequences. One of the major advantages of this approach is that in only 1 work-week it is possible to acquire considerable molecular information while avoiding the use of controversial heterologous systems. Transforming heterologous species is frequently unacceptable, as the conservation of molecular events in many cases is not well documented.

Diversity and Evolution of Effector Loci in Natural Populations of the Plant Pathogen Melampsora lini

Genetic variation for pathogen infectivity is an important driver of disease incidence and prevalence in both natural and managed systems. Here, we use the interaction between the rust pathogen, Melampsora lini, and two host plants, Linum marginale and Linum usitatissimum, to examine how host-pathogen interactions influence the maintenance of polymorphism in genes underlying pathogen virulence. Extensive sequence variation at two effector loci (AvrP123, AvrP4) was found in M. lini isolates collected from across the native range of L. marginale in Australia, as well as in isolates collected from a second host, the cultivated species L. usitatissimum. A highly significant excess of nonsynonymous compared with synonymous polymorphism was found at both loci, suggesting that diversifying selection is important for the maintenance of the observed sequence diversity. Agrobacterium-mediated transient transformation assays were used to demonstrate that variants of both the AvrP123 and AvrP4 genes are differentially recognized by resistance genes in L. marginale. We further characterized patterns of nucleotide variation at AvrP123 and AvrP4 in 10 local populations of M. lini infecting the wild host L. marginale. Populations were significantly differentiated with respect to allelic representation at the Avr loci, suggesting the possibility of local selection maintaining distinct genetic structures between pathogen populations, whereas limited diversity may be explained via selective sweeps and demographic bottlenecks. Together, these results imply that interacting selective and nonselective factors, acting across a broad range of scales, are important for the generation and maintenance of adaptively significant variation in populations of M. lini.

The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species

BackgroundPlant genome sequencing has resulted in the identification of a large number of uncharacterized genes. To investigate these unknown gene functions, several transient transformation systems have been developed as quick and convenient alternatives to the lengthy transgenic assay. These transient assays include biolistic bombardment, protoplast transfection and Agrobacterium-mediated transient transformation, each having advantages and disadvantages depending on the research purposes.ResultsWe present a novel transient assay based on cocultivation of young Arabidopsis (Arabidopsis thaliana) seedlings with Agrobacterium tumefaciens in the presence of a surfactant which does not require any dedicated equipment and can be carried out within one week from sowing seeds to protein analysis. This Fast Agro-mediated Seedling Transformation (FAST) was used successfully to express a wide variety of constructs driven by different promoters in Arabidopsis seedling cotyledons (but not roots) in diverse genetic backgrounds. Localizations of three previously uncharacterized proteins were identified by cotransformation with fluorescent organelle markers. The FAST procedure requires minimal handling of seedlings and was also adaptable for use in 96-well plates. The high transformation efficiency of the FAST procedure enabled protein detection from eight transformed seedlings by immunoblotting. Protein-protein interaction, in this case HY5 homodimerization, was readily detected in FAST-treated seedlings with Förster resonance energy transfer and bimolecular fluorescence complementation techniques. Initial tests demonstrated that the FAST procedure can also be applied to other dicot and monocot species, including tobacco, tomato, rice and switchgrass.ConclusionThe FAST system provides a rapid, efficient and economical assay of gene function in intact plants with minimal manual handling and without dedicated device. This method is potentially ideal for future automated high-throughput analysis.