3. Results
3.4 Reverse genetic analysis of anac071
3.5.7 AT5G24080-Venus fusion protein accumulates after ABA treatment
A B
without the stop-codon for C-terminal tagging (promAt5g24080::At5g24080-Δstop) by PCR.
TheVenus fluorescent protein gene was amplified without start-codon (Δstart-Venus) using the binary vector pC3 as template (Ghareebet al., 2016). Both PCR products were then assembled into the pC3 expression vector backbone using the Gibson Assembly® protocol (New England Biolabs) Therefore, 5´ and 3´ overlapping overhangs were introduced into promAt5g24080::At5g24080-Δstop and Δstart-Venusby PCR primer design. A linker sequence between the At5g24080 gene and Venus fluorescent protein was introduced as well. The generated vectors were transformed into E. coli TOP 10 cells (Invitrogen). Subsequently, transformants were plated on selective LB Agar plates and successful transformation verified by colony-PCR. Here, primer pairs binding in the pC3 backbone and At5g24080 orVenus and pC3 backbone were used, only yielding a PCR product when promAt5g24080::At5g24080-Δstop and Δstart-Venus were inserted into pC3 backbone in the correct orientation. Vectors were verified by digestion as well as Sanger sequencing and subsequently transformed into Agrobacterium tumefaciens strain GV3101 pMP90RK. Transformants were plated on selective LB Agar plates and successful transformation verified by colony-PCR.
The promAt5g24080::At5g24080::Venus construct was introduced into the at5g24080-1 and at5g24080-2 mutant using the floral dip protocol for Agrobacterium mediated transformation ofA. thaliana. T1 transgenic plants were selected with glufosinate ammonium, as they carried a resistance to this herbicide. To test expression of the transgene, 12 independent T1 transgenic lines of each genotype were screened for expression of the AT5G24080-Venus fusion protein by confocal laser scanning microscopy (CLSM). Yet, Venus fluorescence was not detectible in either of the lines (data not shown). Since in silico and expression analyses demonstrated inducibility ofAt5g24080 by ABA, AT5G24080-Venus accumulation was analysed by CLSM in leaf discs of the stable transgenicA. thaliana T1 plants infiltrated with 50 µM ABA. Line #1,
#2, #3, #4, #5 and #6 of at5g24080-1 transformed with promAt5g24080::At5g24080::Venus were selected for subsequent analyses, as Venus fluorescence was detectable in these lines (data not shown). In case ofat5g24080-2transformed with promAt5g24080::At5g24080::Venus, line
#1, #2, #4 and #12 were chosen for subsequent analyses.
In order to confirm inducibility of the promAt5g24080::At5g24080::Venusconstruct by ABA, leaf discs of stable transgenic A. thaliana T1 plants were infiltrated with 50 µM ABA and At5g24080 as well as At5g24080::Venus transcript abundance analysed by semi-quantitative RT-PCR. At5g24080 transcript levels were increased in Col-0 wild-type and all tested independent stable transgenic lines after treatment with 50 µM ABA overnight as compared to
abundance was higher in several mock treated stable transgenic lines transformed with promAt5g24080::At5g24080::Venusas compared to mock treated Col-0 wild-type (Fig. S15A and S16A, upper panel), indicating that the transgene was inserted at a transcriptionally more active locus. When usingAt5g24080::Venus specific primer pair, which binds in theAt5g24080 andVenusgene, an increase in transcript abundance was observed after 50 µM ABA treatment as compared to mock, suggesting that the introduced promAt5g24080::At5g24080::Venus construct is inducible by abscisic acid (Fig. S15A and S16A, middle panel). In addition to semi-quantitative RT-PCR, At5g24080 transcript levels in stable transgenic T1 lines were assessed by qPCR. Results of the qPCR analyses corresponded to the results obtained in semi-quantitative RT-PCR (Fig. S15B and S16B).
Thereafter, immunoblot analyses were performed in order to test whether full length AT5G24080-Venus protein accumulates after ABA treatment. For this purpose leaf discs of stable transgenic A. thaliana T1 plants were infiltrated with 50 µM ABA and total protein extraction was carried out. In immunoblot analyses A. thaliana Col-0 wild-type was used as a negative control. In the experiment shown in Fig. 24A a transgenic line overexpressing AT5G24080-Venus was included as a positive control. The calculated molecular weight of AT5G24080-Venus fusion protein corresponds to ca. 125 kDa.
Interestingly, AT5G24080-Venus protein band corresponded to a size of ca. 140 kDa (Fig. 24), suggesting posttranslational modification of the protein. In case of at5g24080-1 transformed with promAt5g24080::At5g24080::Venus, AT5G24080-Venus fusion protein accumulated in the independent stable transgenic T1lines #2, #3, #4 and #5 after 50 µM ABA treatment as compared to the mock and untreated controls (Fig. 24A). The strongest AT5G24080-Venus protein accumulation was observed in line #2 and #4. In at5g24080-2 transformed with promAt5g24080::At5g24080::Venus, AT5G24080-Venus fusion protein accumulated after 50 µM ABA treatment in independent stable transgenic T1line #1, #2, #4 and #12 as compared to mock (Fig. 24B). The strongest AT5G24080-Venus accumulation was observed in line #4.
An AT5G24080-Venus protein band was not detectable in the Col-0 wild-type.
In summary, semi-quantitative RT-PCR analyses demonstrated inducibility of the promAt5g24080::At5g24080::Venusconstruct by ABA in in stable transgenic T1at5g24080-1 and at5g24080-2 lines. Subsequent qPCR analyses supported results of the semi-quantitative
Figure 24. Immunoblot analysis of independent transgenic Arabidopsis thaliana T1 lines stably expressing At5g24080::Venus under the control of its native promoter after ABA treatment. Upper panels show α-GFP immunoblot detecting Venus, whereas lower panels represent 2,2,2-Trichloroethanol (TCE) in-gel total protein visualisation as control for equal loading. Eight leaf discs (∅ 0.55 cm) from 10-week-old plants were pressure infiltrated with 50 µM ABA (+) or mock (-) treated overnight. “S” represents the size standard. The experiments were repeated with similar results. (A) AT5G24080 protein levels in A. thaliana at5g24080-1 expressing promAt5g24080::At5g24080::Venus. The upper panel displays A. thalianaCol-0 and transgenic lines #1 to #3.
The lower panel displays transgenic lines #4 to #6. 35S::AT5G24080::Venus was used as positive control. “u”
represents untreated control. Protein extraction was performed using the total protein extraction protocol. (B) AT5G24080 protein levels in A. thaliana at5g24080-2 expressingpromAt5g24080::At5g24080::Venus line #1,
#2, #4 and #12. Since unspecific bands and strong background signal was observed in the experiment shown in (A), protein extraction was performed using the receptor-like kinase optimised protocol.