pMAA-Red: a new pPZP-derived vector for fast visual screening of transgenic Arabidopsis plants at the seed stage
© Ali et al.; licensee BioMed Central Ltd. 2012
Received: 16 March 2012
Accepted: 1 June 2012
Published: 2 July 2012
The production of transgenic plants, either for the overproduction of the protein of interest, for promoter: reporter lines, or for the downregulation of genes is an important prerequisite in modern plant research but is also very time-consuming.
We have produced additions to the pPZP family of vectors. Vector pPZP500 (derived from pPZP200) is devoid of NotI sites and vector pPZP600 (derived from pPZP500) contains a bacterial kanamycin resistance gene. Vector pMAA-Red contains a Pdf2.1: DsRed marker and a CaMV:: GUS cassette within the T-DNA and is useful for the production of promoter: GUS lines and overexpression lines. The Pdf2.1 promoter is expressed in seeds and syncytia induced by the beet cyst nematode Heterodera schachti in Arabidopsis roots. Transgenic seeds show red fluorescence which can be used for selection and the fluorescence level is indicative of the expression level of the transgene. The advantage is that plants can be grown on soil and that expression of the marker can be directly screened at the seed stage which saves time and resources. Due to the expression of the Pdf2.1: DsRed marker in syncytia, the vector is especially useful for the expression of a gene of interest in syncytia.
The vector pMAA-Red allows for fast and easy production of transgenic Arabidopsis plants with a strong expression level of the gene of interest.
Modern plant research relies heavily on the use of transient and stable transformation with the help of Agrobacterium tumefaciens[1, 2]. This bacterium is a natural genetic engineer and transfers a part of its large Ti plasmid into host plants to induce cell division and the synthesis of opines. For use in genetic engineering, the Ti plasmid has been divided into a helper plasmid which is devoid of the T-DNA and remains within the Agrobacteria and a binary vector carrying the T-DNA which can be manipulated in E. coli. The first widely used binary vector was pBIN19 . Many derivatives have been described and some are still in use today although other binary vectors which are smaller, have a higher copy number, and different selectable markers for use in bacteria (E. coli and Agrobacteria) and in plants have been introduced (for review see [4, 5]). One popular series are the pPZP vectors  which were also the basis for the pCAMBIA vectors . We have recently published an improved pPZP vector (pPZP3425) which was equipped with a kanamycin resistance gene for selection in Agrobacterium  . The strong 35 S CaMV promoter driving the plant resistance gene for kanamycin resistance was replaced by the weaker nos promoter because it had been shown that the 35 S promoter driving the plant resistance marker in the original pPZP vectors can lead to ectopic expression of the transgene [9, 10]. Furthermore, pPZP3425 contains an expression cassette which consists of an intron-containing GUS gene driven by a strong constitutive promoter (35 S promoter with doubled enhancer plus omega element as translational enhancer). This vector has successfully been used in our laboratory.
Plant selectable markers for the pPZP vectors include kanamycin and gentamycin. Both markers work well for a variety of plant species. Kanamycin is perhaps the most widely used selectable marker for plant transformation. Kanamycin and gentamycin as well as other antibiotic markers have the disadvantage that they are usually used under sterile conditions. In case of Arabidopsis this means that to isolate transgenic plants the seeds have to be sterilized and grown on a sterile agar medium containing the antibiotics. Recently it has been shown that the selection of transgenic plant lines containing a kanamycin marker gene can be done by culturing the seedlings on rockwool saturated with MS medium without sugar but containing the selective agent . Since the medium does not contain sugar, sterile conditions are not necessary, saving costs and labour. However, extreme care has to be taken that the seedlings do not run dry. Other markers that also circumvent the need to work under sterile conditions use resistance against herbicides, especially phosphinotricin (BASTA). The herbicide can be sprayed onto plants growing in soil to select for those containing the Bar gene which mediates resistance against phosphinotricin . Fluorescent proteins have also been reported as markers for plant transformation including Arabidopsis [13–17]. For Arabidopsis transformation, DsRed, GFP, and GFP variants have been used as markers driven by seed-specific promoters derived from other plant species [18, 19].
During cloning of a vector for transient expression we realized that the pPZP vectors contain 3 NotI sites in their backbone such that this eight-cutter could not be used in the polylinker. Starting with pPZP200, we have therefore removed all NotI sites from the vector backbone as well as other unnecessary parts to produce pPZP500. By replacing the spectinomycin resistance gene with the nptII gene we also produced the vector pPZP600.
The vector pPZP500 does not contain a plant selectable marker as this is not needed for transient expression. However, since pPZP500 was much smaller than the original pPZP vectors, it could be the basis of a new binary vector (pMAA-Red) for stable transformation of Arabidopsis. For that we included a DsRed gene driven by the Pdf2.1 promoter and the GUS cassette from pPZP3425. The Pdf2.1 promoter was chosen because it is strongly expressed in seeds and in syncytia, feeding sites induced by the beet cyst nematode H. schachtii in Arabidopsis roots [20, 21]. In addition, we replaced the spectinomycin resistance gene used for selection in Agrobacteria by a kanamycin resistance gene.
Construction of pPZP500 and pPZP600
Vector pPZP500 contains the streptomycin/spectinomycin resistance gene (aadA, encoding aminoglycoside-3"-adenyltransferase) which can be used in E. coli and in Agrobacteria. However, in our hands spectinomycin selection was not as tight as kanamycin selection. We, therefore, replaced the spectinomycin resistance gene with the nptII gene which yielded the vector pPZP600 (Additional file 2: Figure S2) as described in Methods.
Construction of pMAA-Red
List of oligonucleotides used in this work
Production of transgenic Arabidopsis lines using pMAA-Red
A large variety of binary vectors for plant transformation have been described. Among them the pPZP series of vectors  and the derived pCAMBIA vectors  are especially popular. A prominent feature of these vectors is their stability in bacteria, the high copy number, and the relatively small size. One disadvantage was the use of the CaMV promoter for the plant selectable marker and the use of spectinomycin and chloramphenicol as selectable markers for bacteria which led us to construct the vector pPZP3425 . In this vector the CaMV35S promoter for the plant selectable marker (kanamycin) was replaced by the weaker nos promoter and a kanamycin resistance gene for selection in bacteria was included in the vector backbone.
Although pPZP3425 proved useful for our purposes in producing promoter:: GUS lines (by replacing the CaMV promoter in the GUS cassette with a promoter of interest) or overexpression lines (by replacing GUS in the GUS cassette with a gene of interest) , selection of homozygous transgenic lines with a strong expression of the gene of interest was still a lengthy procedure. Since we were interested to produce a large number of transgenic Arabidopsis overexpression lines with a strong expression level for putative antimicrobial peptides we set out to construct a pPZP vector that would have three important features: First, it should allow us to use selection or screening on soil to avoid growing Arabidopsis under sterile conditions. Second, we wanted to easily select lines with a strong expression level. Third, the vector should be used for the expression in syncytia using specific promoters that would not be active in seedlings or leaves.
The first precondition excluded the use of antibiotic resistance such as kanamycin or hygromycin or the use of metabolite resistance genes such as the E. coli-derived phosphomannose isomerase which allows growth on mannose  or the Streptomyces rubiginosus xylose isomerase (xylA) gene which allows growth on xylose  as a plant selectable marker for which sterile conditions would have to be used. Herbicide resistance would avoid the need for sterile growth but all T1 seedlings have to be grown for selection. In contrast, if using a fluorescent marker that is expressed in seeds, only the transgenic seeds obtained after transformation would have to be grown further and no additional treatment would be needed. Such an approach is especially important if a large number of transgenic lines have to be produced. An example is the work of  who conducted a high-throughput screen in Arabidopsis for castor genes that would lead to changes in hydroxy fatty acid composition in seeds. We decided to try the DsRed gene as a screenable marker [13–17]. This leaves the possibility to use in addition GFP or other most frequently used fluorescent markers as reporter genes. Furthermore, DsRed has a rather weak fluorescence which might seem to be a disadvantage. However, this weak fluorescence makes it easier to identify seeds with a different level of fluorescence.
Expression of the gene of interest varies largely in independent transgenic lines  due mainly to position effects. One way of reducing this variation is the inclusion of matrix attachment regions in the T-DNA. However, this only worked with gene silencing mutants, which limits the use of these vectors . We have demonstrated here that the use of the vector pMAA-Red allows an easy and efficient selection of transgenic lines with a strong expression of the gene of interest in wild-type Arabidopsis plants. Of course, mutants could also be used, if needed. Pre-selection of lines with a strong DsRed expression in seeds according to their fluorescence reduces the number of lines that have to be tested at the transcript or protein level.
Our third precondition was that the vector should allow the use of syncytium-specific promoters instead of the CaMV promoter which is active in most tissues of Arabidopsis plants. We have recently shown that several genes are expressed in syncytia which are normally expressed in pollen , such as MIOX4 and MIOX5, or in seeds, such as Pdf2.1 whose promoter is used here to drive the expression of DsRed in seeds. Using the MIOX4 or MIOX5 promoter would allow a specific expression in syncytia which could be useful for genes with a negative effect on plant growth. However, screening such transgenic lines would require the analysis of expression of the gene of interest in syncytia. Such a screening is very time consuming because syncytia have to be cut out from infected roots. In this case a pre-screening that would reduce the number of lines would lead to a significant reduction of time and effort. Thus, the pMAA-Red vector that we have constructed is especially useful for the syncytium-specific expression of transgenes or for similar cases where the expression of the transgene would be restricted to tissues that could not be easily screened. Promoter::reporter constructs and overexpression lines can be produced from this vector as from pPZP3425 . The CaMV promoter can be replaced by a promoter of interest using NcoI and one of the unique sites in the polylinker. The GUS sequence can be replaced with a sequence for overexpression of a gene of interest by using NcoI and BamHI. If the sequence to be cloned contains NcoI or BamHI site, it is usually possible to use a restriction enzyme that produces compatible cohesive ends with NcoI and BamHI. For instance, BspHI, PciI, and FatI produce cohesive ends that are compatible with NcoI.
After transformation of Arabidopsis plants it takes about 3–4 weeks until T1 seeds can be harvested and inspected for fluorescent seeds. A big advantage of transformation with the vector pMAA-Red is that only these selected seeds have to be grown on soil to produce the T2 generation. After another 4 weeks, the first siliques of these plants can be screened for a 3:1 segregation of fluorescent seeds which can then be used to produce homozygous T3 seeds for further analysis. Again, the first siliques of these plants can be used to select homozygous lines and only those will be grown for maturity, which will take a total of 6 to 8 weeks. Depending on the growth conditions, the whole procedure from transformation to harvesting homozygous seeds could be completed within four month.
We have constructed compact pPZP vectors without NotI sites having either bacterial spectinomycin or kanamycin resistance (pPZP500 and pPZP600, respectively) and a vector (pMAA-Red) which allows an easy production of transgenic Arabidopsis overexpression lines with strong expression levels of the gene of interest.
Standard procedures were used for restriction enzyme mediated DNA digestion, ligation, and transformation . Restriction enzymes and T4 DNA ligase were from Fermentas -Thermo Fisher Scientific and New England Biolabs. The E coli strain DH10B was used throughout this work. PCR was done using Eppendorf Mastercycler Gradient.
Construction of pPZP500
Vectors used in this study
pPZP200 (Additional file 1: Figure S1)
Spectinomycin resistance gene for bacterial slection
CaMV 35 S 2X promoter with omega element, LacZ operon, GUS, CaMV terminator (GUS cassette), kanamycin resistance gene for bacterial and plant selection
pPZP500 (Figure 1)
pPZP200 modified backbone without NotI sites
pPZP5025 (Additional file 2: Figure S2)
pPZP500 with GUS cassette
pPZP3425 containing pPDF2.1::DsRed instead of the 35 S::GUS cassette
pPZP600 (Additional file 2: Figure S2)
pPZP500 with kanamycin resistance gene for bacterial selection
pPZP650 (Additional file 2: Figure S2)
pPZP600 with PDF2.1 promoter, DsRed gene, and 35 S terminator
pPZP653 (Additional file 2: Figure S2)
pPZP650 with new polylinker
pPZP6535 (Additional file 2: Figure S2)
pPZP653 with GUS cassette
pMAA-Red (Figure 3)
pPZP6535 with PDF2.1 promoter, DsRed gene, nos terminator and GUS cassette
Construction of pPZP600
The spectinomycin resistance gene of pPZP500 was replaced by the nptII gene. The nptII gene (~1 kb) was amplified from pPZP3425 using KanforMph and KanrevMph primers. The pPZP500 vector backbone was amplified by using PZP500Mphfor and PZP500Mphrev primers. Both primers had a Mph1103I site at their ends. Insert and vector fragment were digested with Mph1103I, ligated, and transformed into E. coli to yield pPZP600 (Additional file 2: Figure S2).
N. benthamiana and Arabidopsis plants were grown in a growth chamber at 25 ± 1°C, with a 16 hour light/8 hour dark photoperiod and approximately 65% humidity.
Binary plasmids were transformed into A. tumifaciens strain GV3101 by the freeze-thaw method . Agrobacteria were selected on YEB plates with appropriate antibiotics which included 25 μg/ml gentamycin and 35 μg/ml rifampicin for Agrobacteria. Selection for binary plasmids was done with 200 μg/ml spectinomycin (for pPZP5025) or 50 μg/ml kanamycin (for pMAA-Red).
Agroinfiltration of N. Benthamiana
Agrobacteria were grown to an OD600 of 0.8 overnight in an incubator/shaker at 28°C. Bacteria were harvested by centrifugation at 5000 rpm for 6 min in a table top centrifuge at room temperature and suspended in infiltration medium (10 mM MES pH 5.6, 10 mM MgCl2 and 100 μM acetosyringone) to obtain bacterial suspensions of the an OD600 of 1.0. After incubation for 2 hr at room temperature, Agrobacterium suspensions were infiltrated in the abaxial side of leaves by using a 1 ml syringe without a needle. Infiltrated plants were kept under the same growth conditions as mentioned above. For co-infiltration of the RNA silencing inhibitor P19, an equal volume of a bacterial suspension harbouring pBin61P19  was added prior to infiltration.
The vector construct pMAA-Red was transformed into Arabidopsis ecotype Col by the floral dip method . Seeds of transformed plants were harvested and photographed under an inverse microscope equipped with a DsRed fluorescence filter (Axiovert 200 M; Zeiss, Hallerbergmoos, Germany) and an integrated camera (AxioCam MRc5; Zeiss). Fluorescent seeds (T1) were selected and put on soil to grow the next generation (T2), of which the first mature silique was detached from the plant and examined under the microscope for fluorescent seeds to check for 3:1 segregation. These seeds were then used to produce homozygous lines. Seeds that were kept in the dark at 4°C still showed fluorescence after several years.
GUS staining of the N. benthamiana leaves was done by overnight incubation in X-Gluc solution (50 mM sodium phosphate buffer pH 7.0, 10 mM EDTA pH 8.0, 0.1% (v/v) Triton X-100 and 0.5 mg/ml X-Gluc) at 37°C  followed by several washings with 70% ethanol to remove the chlorophyll from leaf tissues.
GUS activity assay
GUS activity was measured according to Jefferson et al.  with some modifications in black 96-well Greiner plates. To 100 μl of total protein extract 50 μl of 4 mM 4-MUG was added. The reaction was incubated at 37°C for 5 min and then 50 μl of 0.5 M Na2CO3 was added to stop the reaction. Fluorescence was measured at 355 nm excitation and 460 nm emission in a FLUOstar Omega micro plate reader (BMG Labtech) using 4-MU standards (10 mM stock in ethanol and diluted in GUS extraction buffer) in the range of 1–100 μM.
Nematode infection and examination of fluorescence in syncytia
The seeds of a pMAA-Red transgenic Arabidopsis line were surface-sterilized for 7 min in 10% Chlorox (v/v), submerged for 5 min in 70% (v/v) ethanol, and then washed three times in sterile water. The sterilized seeds were placed on a modified 0.2 concentrated Knop agar medium supplemented with 2% sucrose . H. schachtii was multiplied in vitro on mustard (Sinapsis alba cv. Albatros) roots growing on 0.2 concentrated Knop medium supplemented with 2% sucrose . Hatching of J2 larvae was stimulated by soaking the cysts in sterile 3 mM ZnCl2. The juveniles were washed four times in sterile water and resuspended in 0.5% (w/v) Gelrite for inoculation. Roots of 12-day-old Arabidopsis plants were inoculated with about 40–50 juveniles under sterile conditions. At 5 and 10 dpi the fluorescent syncytia were examined using an inverse microscope.
Resistance tests and size measurement of syncytia and nematodes
H. schachtii (Schmidt) infection was done in the same way as described above. Roots of 12-day old Arabidopsis plants were inoculated under axenic conditions with about 50 juveniles per plant. The number of male and female nematodes per cm of root length were counted at 14 dpi. The data were analysed using single factor ANOVA (P < 0.05). As the F-statistic was greater than F-critical, a Least Significance Test (LSD) was applied. At 14 dpi, pictures of male syncytia, female syncytia and females nematodes (30 photos for each) were taken. Syncytia and nematodes were measured using an inverse microscope (Axiovert 200 M; Zeiss, Hallerbergmoos, Germany). The data were analyzed using single factor ANOVA (P < 0.05).
GenBank accession numbers
This research was supported by grants P20471-B11 and P21896-B16 of the Austrian Science Fund (FWF). Muhammad Amjad Ali and Kausar Hussain Shah were supported by Higher Education Commission (HEC) of Pakistan. The authors are thankful to Shahid Siddique, Amjad Abbas, Bachar Almaghrabi, Martina Niese and Stephan Plattner for their support. We thank Dr. David Baulcombe for pBin61P19.
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