Characterisation of at5g24080 knockout mutants

In order to obtain anat5g24080 knock-out mutant, three T-DNA insertion lines were ordered from NASC. Lines SALK_086625 and SAIL_551_D12, which both carry a T-DNA insertion in the second exon of the At5g24080 gene were designated at5g24080-1 and at5g24080-3 respectively (Fig. 14A). In addition, the line SALK_147104, which is annotated to carry a

Express Arabidopsis Gene Mapping Tool (O’Malleyet al., 2015) was designatedat5g24080-2.

However, Sanger sequencing of homozygous mutants demonstrated that the T-DNA insertion in the at5g24080-2 line was located in the fifth exon of the At5g24080 gene (Fig. 14A and Fig. S8). The amino acid sequences, which correspond to ORFs resulting from the T-DNA insertions inat5g24080 mutants, are shown in Fig. S9.

Homozygous T-DNA insertion lines were isolated by PCR based genotyping in two steps as described for therd17 mutants in section 3.3.1 using genotyping-specific primers designed with the T-DNA Express iSect tool (O’Malley et al., 2015). The respective primer sequences are shown in Section 2.1.7. at5g24080-1line #7, #8, #13 and #27, at5g24080-2 line #2, #9, #17 and #28 as well asat5g24080-3 line #1, #3 and #26 were kept as homozygous T-DNA insertion lines for further analyses (Fig. S5, S6 and S7). F2 progeny was generated by selfing of at5g24080-1, at5g24080-2 and at5g24080-3 homozygous T-DNA insertion lines and analysed for loss of functionalAt5g24080 transcripts by semi-quantitative RT-PCR. RNA was extracted from plants infected with theVerticillium chlorosis-inducing isolate c-VL43 and c-V76 and the wilting-inducing isolate w-JR2 at 21 dpi or mock treated plants. Verticillium challenged at5g24080 mutants were tested, since At5g24080 transcripts were hardly detectable in mock treated samples in initial RT-PCR analyses (Fig. 4). Furthermore, analysis of Verticillium challenged at5g24080 mutants allowed to assess whether T-DNA insertion had an effect on gene induction duringVerticillium infection. At5g24080transcript abundance was tested using a RT-PCR-specific primer combination binding at the transcriptional start and stop, thus flanking all three T-DNA insertion sites (F1 and R2 primer in Fig. 14A). Moreover, in order to test for residual 5´- as well as 3´-transcripts, primer combinations were used that bind in the first and second exon of the gene (F1 and R1 primer in Fig. 14A) and in the fourth and fifth exon of the gene (F4 and R2 primer in Fig. 14A).

Consistent with initial expression analyses, At5g24080 transcript abundance increased during chlorosis-inducing isolate c-VL43 and c-V76 infection of A. thaliana Col-0 wild-type as compared to mock treatment or wilting isolate w-JR2 infection (Fig. 14B). No At5g24080 transcripts were present in all three tested at5g24080 mutants using the F1 and R2 primer combination (Fig. 14B, upper panel). Yet, when using the F1 and R1 primer combination residual 5´-transcripts were detected in the at5g24080-1, at5g24080-2 and at5g24080-3

Figure 14. Characterisation of at5g24080-1, at5g24080-2 and at5g24080-3 T-DNA insertion mutants.

(A) Schematic representation ofAt5g24080 gene structure. Exons are represented as blue boxes, whereas introns are shown as black lines. Positions of T-DNA insertions determined experimentally by Sanger sequencing of homozygous mutants (Figure S8) are shown as triangles. Arrow shows the orientation of the T-DNA (left border → right border). Forward (F) and reverse (R) primers used in B and C are represented as black boxes.

(B) Semi-quantitative RT-PCR analysis ofAt5g24080 transcript abundance inA. thaliana Col-0 wild-type and at5g24080 T-DNA insertion mutants duringV. longisporum isolate c-VL43 as well asV. dahliae c-V76 and w-JR2 infection. The housekeeping geneActin was amplified as a control. A genomic DNA (gDNA) control was included to monitor potential contamination by gDNA. A reverse primer which binds two exon borders and spans an intron sequence was used in case of the Actin gene to exclude gDNA amplification. The Actin cDNA PCR product corresponds to 302 bp. First panel shows full length At5g24080 transcript amplified with primers F1 and R2.

Expected sizes of F1+R2 PCR products are 2619 bp cDNA and 3058 bp gDNA. Second panel shows5´-At5g24080 transcript part amplified with primers F1 and R1. Expected sizes of F1+R1 PCR products are 514 bp cDNA and 684 bp gDNA. Third panel shows 3´-At5g24080 transcript part amplified with primers F4 and R2. Expected sizes of F4+R2 PCR products are 580 bp cDNA and 681 bp gDNA. (C) Semi-quantitative RT-PCR analysis for 3´-At5g24080 transcript inat5g24080-1 and at5g24080-3arising due to transcriptional activation by the SAIL or SALK T-DNA. PCR was performed as described in B using same cDNA. Left panel shows T-DNA activated 3´-At5g24080 transcript inat5g24080-1amplified with primers F2 and R2. Expected sizes of F2+R2 PCR products are 2110 bp cDNA and 2378 bp gDNA. Right panel shows T-DNA activated 3´-At5g24080 transcript in at5g24080-3amplified with primers F3 and R2. Expected sizes of F3+R2 PCR products are 1662 bp cDNA and 1930 bp gDNA. The experiment was performed once.

A

B

C

the cDNA band whereas the upper band represents gDNA contamination. In order to assess, whether residual 3´-transcripts in at5g24080-1 and at5g24080-3 mutants may arise due to transcriptional activation by the SAIL or SALK T-DNA construct, a RT-PCR using the T-DNA binding F2 or F3 forward primer respectively and the At5g24080 binding R2 reverse primer was performed. Indeed, transcripts were obtained with the F2 and R2 primer combination in at5g24080-1as well as the F3 and R2 primer combination inat5g24080-3(Fig. 14C).

Taken together, RT-PCR analyses demonstrated that at5g24080-1, at5g24080-2 and at5g24080-3 mutants lack full-length At5g24080 transcripts. Moreover, 5´-transcripts were present in all tested at5g24080 mutants, indicating that a truncated version of At5g24080 mRNA is produced. Furthermore, 3´-transcripts were detected in at5g24080-1 and at5g24080-3, suggesting transcriptional activation by the SAIL or SALK T-DNA construct.

3.5.3 Analysis of at5g24080 disease phenotype during Verticillium chlorosis