Analysis of Transposon Insertion Lines

The reverse genetic approach was used to study the function of the GRX480. A "RIKEN Arabidopsis Transposon mutant" line, in which a Ds transposon was inserted 45bp upstream of the start codon in an Arabidopsis thaliana Nössen/Landsberg cross- ecotype was used (Fedoroff et al., 1993, Smith et al., 1996). Sequence analysis downstream from the H (19S-Hyg)-edge of the Ds transposon insertion gave a hit for the GRX480 coding sequence comparable with that known for A. thaliana Col-O ecotype. The sequence upstream from the G (GUS)-edge of the Ds-transposon insertion was also comparable to the promoter of the Colombia ecotype, except for a few base pair insertions and exchanges which differ from the sequence published from the Colombia ecotype (see appendix 10.29). The as-1 like elements (TGAGC motives), found in the promoter are nevertheless conserved.

A combination of primers that anneal on the Ds-transposon (Ds3-2a anneals 150bp upstream of the D-edge), primers annealing upstream of the transposon insertion site (LP2GRX480 and LP1GRX480 annealing 409 and 193bp upstream respectively) and a primer annealing downstream of the Ds-insertion site (RP480 annealing 272bp downstream) were used to analyze the transposon insertion lines, in order to distinguish wild-type from heterozygous and homozygous mutant lines (Figure 6.6D).

In WT plants (Columbia, Nössen and Landsberg erecta), product sizes of 680bp and 464bp were expected from using upstream/downstream primers LP2GRX480/RPGX480 and LP1GRX480/RPGX480 respectively. In each case, no product was expected if the plant line was homozygous for the transposon insertion mutation. In case the plants are

heterozygous, two products of 680bp and 422bp were expected when the primer combination of LP2GRX480/DS3-2a/RPGX480 were used

At1g28480

RP GX480 LP1 GRX480

LP2GRX480

GRX promoter

3' UTR 5' UTR

BamHI (251) EcoRI (148)

G-Edge H-Edge Ds3-2a

Ds-transposon Insertion site

Figure 6.6D. Schematic diagram showing primer design for the analysis of At1g28480 transposon insertion mutants.

The transposon contains a 5´G-edge and a 3´ H-edge. Primers were designed (blue arrows) to analyze the genomic DNA of the plants, so that by the size of the PCR product, a distinction can be made between WT, homozygous and heterozygous mutant lines. Primers flanking the transposon insertion site would not produce a product in a homozygous mutant, due to the large size of the transposon insertion.

The illustrated promoter is truncated. The illustrated transposon insertion is not to scale.

.

Seven lines analyzed were detected to be homozygous for containing the Ds-transposon insertion, and therefore produced no PCR product when primer combinations flanking the transposon insertion site were used, unlike in the controls (Colombia, Nössen and Landsberg erecta) where PCR products were obtained. A product was only detectible with the transposon insertion mutants when a primer annealing on the transposon was used in combination with a downstream primer downstream of the insertion site. (Figure 6.6E) Mutant lines were further confirmed by their inability to accumulate GRX480 transcripts even after induction with salicylic acid. (Figure 6.6F)

The knockout lines were further characterized to determine if there was any phenotype observed in the ability for the plants to repress JA induced PDF1.2 in the presence of SA (Figure 6.6G). The plants were still able to repress the accumulation of PDF1.2 after induction with JA in the presence of exogenously applied SA. The amounts of PDF1.2 transcript levels accumulating in the knockout lines after JA induction were about 3-fold less in the presence of SA, compared to about 7-15 fold reduction in the control lines.

680bp

Figure 6.6E. PCR-analysis of GRX480 transposon insertion mutant lines.

Genomic DNA was prepared from leaves of WT plants (Col-O, Nös and Ler) and seven “RIKEN transposon insertion lines”. These were used as template for 21 PCR reactions. The primer combinations and the expected outcome are illustrated in the table. All the reaction products separated on a 1% agarose gel, after loading in the slots indicated 1-21. PCR 21 was used as a control. A DNA ladder (M) was also loaded to access the band sizes of the products obtained.

This amount was nevertheless about 2 fold more than in the Nössen ecotype, and about 8-fold more than in the Colombia and Landsberg ecotypes under the same conditions. These results were reproducible when individual induction experiments were compared side by side between the knockout lines and Landsberg lines at the same time point.

PR1 GRX

RNA Col-o

Nössen Landsberg

1 2 3 4 5 6 7 8 9 10 11 12 GRX At1g28480 knockout lines

Transcript accumulation after 2Hrs, SA

Figure 6.6F. Northern blot analysis of GRX480 transposon insertion mutant lines.

Four-week old GRX480 transposon insertion lines and WT control lines (Col-O, Nös and Ler) were treated for 2hrs with 1mM SA by spraying. 100mg of leaf material (1-2 leaves) were harvested and used for RNA preparation. 20µg each of RNA was separated on a denaturing agarose gel. After northern blotting, the membrane was hybridized with radioactively labeled probes for detecting the At1g28480 (GRX) and PR1 transcripts. The RNA loading control is based on EtBr staining.

5Hrs

O SA JA S/J EH O SA JA S/J EH O SA JA S/J EH O SA JA S/J EH

Col-o Landsberg Nössen grx knockout

PDF1.2

PR1

GRX

RNA

Figure 6.6G. Analysis of GRX480 dependent cross talk in GRX480 transposon insertion lines.

Three week old GRX480 transposon insertion lines #1 and wild type control lines (Col-O, Nös and Ler) were induced by floating in phosphate buffer with either 1mM SA/Ethanol (SA), 20µM meJA (JA), SA/JA (SJ) or 0.01% Ethanol (EH) for 5hours. Un-induced plants (0) were also collected. After pooling and collecting an average of 15 plants per time point, 20µg each of prepared RNA was separated on a denaturing agarose gel. After northern blotting, the membrane was hybridized with radioactively labeled probes for detecting the At1g28480 (GRX), PR1 and PDF1.2 transcripts. The RNA loading control is based on EtBr staining.

The repression of the JA pathway in the presence of SA at later time points (after 12 hours) was comparably the same in the wild type and the controls (results not shown).

It could be concluded from the above observations that GRX480 is necessary but not sufficient on its own in repressing the JA pathway. The above results suggest the fact that there might be a GRX-independent pathway actively complementing the absence of GRX480, possibly involving NPR1. The generation of a double mutant between npr1-1 and grx480 by crossing, and analyzing these for their phenotype would give a better idea about the function of GRX480. The two proteins might function in parallel pathways to repress parts of the JA pathway, since in the npr1-1 mutant which is compromised in its ability to repress the JA pathway, there still occurs about a 2 fold repression of the PDF1.2 transcript in the presence of SA.

Alternatively, these results might be due to a redundancy in GRX480 protein by other GRX480-like proteins. The glutaredoxin coded by the gene AT1G03850, which also has the closest homology to GRX480 (Figure 6.1B) and is also pathogen inducible (Table 6.2A) might be a good candidate worth investigating for its ability to interact with TGA2 transcription factor, its effect after over-expression and its phenotype in a double mutant with GRX480.

The interpretation of results was also made difficult by the fact that the mutant is present in a background of a cross between two ecotypes. Some factors and characteristics which are randomly inherited from the different ecotype-backgrounds make it hard to compare with accuracy, the effects observed in the mutant lines with that observed in the controls.

As an example, the induction of PDF1.2 by JA was always weaker in the Landsberg ecotype, while the nössen ecotype in many cases had more background of PDF1.2.