RNA-
DIRECTED TRANSCRIPTIONAL GENE SILENCING Selection of mutants using kanamycin resistance as indicatorIn order to identify mutants in which RNA-directed transcriptional gene silencing (RdTGS) of the ProNOS-NPTII reporter gene was released, and thus NPTII expression was reactivated, approx. 20,000 M2 seeds per obtained seed batch (see section 2.17) were germinated on growth medium (GM) containing 200 mg/l of kanamycin. All M2
batches yielded some kanamycin resistant (KanR) individuals, while no KanR plant was observed in the non-mutagenized C2 control. From each batch, ten KanR M2 plants (1-1 to 1-10, 2-1 to 2-10, and so on, till 32-1 to 32-10) were transferred to soil and allowed to self-pollinate. Resulting M3 seeds from individual M2 were collected and their kanamycin resistance was verified by germinating approx. 200 seeds per M2 line on GM containing 200 mg/l of kanamycin. This resulted in the identification of 104 KanR M3 lines that showed at least 95% of viable resistant seedlings in the presence of kanamycin. To avoid possible redundant siblings, initially one KanR M3 line per M2 batch was chosen for characterization.
As the first step of my thesis work, the presence and integrity of the K and the H transgene in KanR M3 lines was checked by PCR using transgene-specific primer combinations (Table S1). It revealed that all 32 initially selected KanR lines contained the K transgene, but only a single line contained the H transgene (Figure 3B and data not shown). As loss of the H transgene per se can result in the ProNOS-NPTII reactivation (Aufsatz et al., 2002), the presence of both transgenes is a de regieur prerequisite for the identification and later map-based cloning of gene loci essential for RdTGS. Thus, so far only KanR M3 line 2-5 was suitable for further work.
To obtain additional candidates suitable for further analysis and map-based cloning, the remaining 72 KanR M3 lines from the first screening were tested for the presence of full length K and H transgenes using transgene-specific PCR. Five additional lines were found to contain both transgenes and were included in further analysis (Figure 3C).
Selection of mutants using combined hygromycin and kanamycin resistance as indicator
As the above six confirmed KanR M3 lines were rather limited material to work with, it was attempted to isolate further candidate lines by repeating the screening of the M2 material.
Figure 3: Verification of transgene integrity in candidate mutant lines.
A) Transgene maps. Approximate positions of primers used for transgene-specific PCRs are indicated by arrows. The “H insert”
PCR only results in a product when the H transgene is absent or heterozygous. B) PCR test for 30 initial mutant candidates derived from the KanR screening. C) Transgene integrity in 6 lines showing at minimum 95% KanR in M3 generation obtained from KanR selection. D) Transgene integrity in mutant lines obtained from KanR HygR selection. PCR tests were performed using genomic DNA of original KanR HygR M2 plants. PCR product sizes are indicated B), C) Five M3 plants per line were tested to check for segregation of the transgenes, but all gave consistent results.
The result of the first round of screening implied that the seed material submitted to mutagenesis was contaminated with material that has lost the H transgene or contained it in a heterozygous manner. Therefore, the majority (98 of 104) of M2 lines obtained by screening for KanR phenotype were “false positive” plants that had lost the H transgene by segregation.
As a constitutively expressed HYGROMYCINE PHOSPHOTRANSFERASE (HPT) reporter gene that confers hygromycin resistance (HygR) is part of the H transgene, presence of the H can be selected for by germination on medium containing hygromycin in order to minimize the number of “false positive” KanR M2 plants. Thus, M2 seed stocks were rescreened for individuals that showed a KanR HygR phenotype by germination on GM containing 200 mg/l of kanamycin and 20 mg/l of hygromycin. No HygR KanR plant was observed in the non-mutagenized C2 control.
Surviving seedlings grown on agar medium containing hygromycin and kanamycin showed generally weak root growth. To evaluate whether the direct transfer of KanR HygR seedlings to soil was possible or a period of recovery at non-selective agar medium was necessary prior to the transfer, two sets of KanR HygR M2 seedlings, 12 per set, from batch no.1 were transferred either directly to soil (1-11 to 1-22) or first to non-selective medium (1-23 to 1-34). As plant viability was not compromised by the direct transfer to soil, plant recovery at non-selective medium was not further applied. Therefore, the maximum number of transferred KanR HygR M2 plants was reduced to 12 per individual batch (2-11 to 2-22; 3-11 and so on). Transferred plants were tested for the presence of both transgenes via PCR. Approximately 110 M3 seedlings of every obtained HygR KanR M2 plant were tested for KanR on GM supplied with 200 mg/l kanamycin. Thirteen M3
mutant lines (1-23, 2-11, 9-19, 9-20, 9-21, 11-11, 11-12, 11-13, 13-14, 14-12, 17-13, 18-15, 20-12) that showed at least 95% KanR plants were considered suitable and selected for further analysis.
3.2 T
HENPTII
PROTEIN LEVEL AS CRITERION FOR“
NORNA-DIRECTED TRANSCRIPTIONAL GENE SILENCING
”
MUTANTS Amount of NPTII protein in obtained mutant linesIn the transgene system used to perform the genetic screen, compromising of the RdTGS mechanism should result in a release of NPTII reporter gene expression. Hence, an increase in NPTII protein as cause for kanamycin resistance should be observed in
studies by others have shown that kanamycin resistance can also arise in A. thaliana loss-of-function mutants (Aufsatz et al., 2009; Conte et al., 2009) or gene overexpression lines (Mentewab and Steward, 2005) by affecting chloroplast-localized transporter proteins without any requirement for NPTII expression. Therefore, the amounts of the NPTII protein in relation to the total soluble protein in rosette leaves of mature M3 plants were determined by ELISA in comparison to control plants grown in parallel (Figure 4).
Figure 4: Quantification of NPTII protein levels by ELISA and growth phenotype of M3 lines
A) Amounts of the NPTII protein were measured in relation to total soluble protein in extracts from leaves of 8-week-old plants.
Results are indicated as relative NPTII levels relative to the mean value for non-silenced expression in (K/K;-/-) plants (set to 100%). Numbers in parentheses indicate numbers of individual M3 plants tested. The background signal obtained from non-transgenic control plants was subtracted prior to calculation. Column height represents mean values; error bars represent standard deviation. B) M3 seedlings of mutant lines showing released NPTII expression compared to mutant lines 19-7 and 30-2 displaying NPTII levels as low as wild-type K/K;H/H plants on GM supplied with 200 mg/l kanamycin.
The relative NPTII level was found reduced in non-mutagenized wild type plants harboring both (K/K;H/H) compared to those that only contained the K transgene (K/K;-/-), yet not completely abolished. The remaining NPTII might help to explain the high concentration of kanamycin necessary to suppress growth of K/K;H/H plants in my work in comparison to a previous study utilizing a similar transgene system involving a NPTII reporter gene (Aufsatz et al., 2002a; Aufsatz et al., 2002b; Aufsatz et al., 2004).
M3 plants from lines 2-5, 8-6, 26-5 and 29-8 from the KanR screen and lines 2-11, 9-20, 11-12, 13-14 and 20-12 from the KanR HygR screen clearly showed more NPTII protein than K/K;H/H plants, almost resembling K/K;-/- plants. Line 1-23 showed, however less pronounced, also a release of NPTII. Interestingly, despite their KanR phenotype, M3
plants from lines 9-19, 9-21, 11-11, 11-13, 14-12, 17-14, 18-15, 19-7 and 30-2 did not display a noticeable increase in the NPTII protein compared to wild type K/K;H/H plants.