Exploring early-regulated genes in saul1-1

The regulation of biological processes often involves a very early induction of key regulatory genes that may act high in the hierarchy of the signaling cascade ³⁸. Therefore, the molecular genetic analyses were at first focused on selected candidate genes among the 19 genes, which

were changed in their expression after 1 h and 2 h, to identify possible key regulators of ETI in saul1-1 mutants. Their selection was dependent both on availability of mutants and on a previously described connection to plant immunity. The five genes, which were selected, were TRX5 (Thioredoxin H-Type 5), AT5G52760, AT3G28580, AT4G16260 and WRKY46.

The TRX5 gene appeared to be highly interesting, because the encoded thioredoxin is involved in the SA-dependent monomerization and therefore activation of NPR1 (NONEXPRESSER OF PR GENES 1) ²³⁴. NPR1 is the key regulator of SA-dependent defense downstream signaling, and accordingly TRX5 could be one of the earliest regulators during ETI (see chapter 1.1.2) ⁷⁴. In case of AT5G52760, it has been reported that this gene is differentially expressed during flg22 treatment ¹⁸³. Thus, this gene may also function in the early regulation of ETI. AT3G28580 is known to respond selectively to reactive oxygen species (ROS) ^235,236 and to be regulated during the immune response ^186,187. Since the ROS burst is one of the first events in ETI (see chapter 1.1.2) ²⁵, AT3G28580 could be one of the earliest regulators. AT4G16260 and WRKY46 are both known to be involved in the immune response, although both have been associated with the basal immune response. WRKY46 is known to interact with other WRKY TFs in the regulation of PTI ²³⁷. AT4G16260 is a target of pathogenic effectors and acting as a positive regulator of plant immunity ²³⁸. Since PTI and ETI are highly connected, both genes could as well be involved in the early regulation of ETI.

Figure 19: T-DNA insertion sites in different mutant lines of early regulated genes in saul1-1^156,157: Schematic representation of five early regulated genes with the positions of the T-DNA insertions (indicated by a triangle) in the mutant lines. Exons are depicted as grey boxes. (A) TRX5 with the T-DNA insertion being located +976 bp. (B) AT5G52760 with the T-DNA insertion being located +436 bp. (C) AT3G28580 with the T-DNA insertion being located -273 bp. (D) AT4G16260 with the T-DNA insertion being located +1299 bp. (E) WRKY46 with the T-DNA insertion being located +733 bp. Positions are in respect to the transcription start site (TSS). In case of different splice variants being known, both are depicted. TSS and transcription termination sites (TTS) are marked with a bar, respectively. Scale bars represent 200 bp.

To analyze their function and characterize their putative position in the signaling cascade, double mutants should be generated between saul1-1 and the respective single mutants to screen for phenotypical changes compared to saul1-1 single mutants. It was hypothesized that in case the

rescued or at least partially changed. The generation of three double mutants was performed in context of the bachelor theses of Sally Marusoi and Simon Peter Meyer ^156,157. Additional two mutant lines were generated by Teresa Wulf. All five mutant lines were genotyped, and T-DNA insertion sites were determined by sequencing (Figure 19, Table S4). Whereas the mutation was detected in the exon and intron of two lines, respectively, the insertion was detected to be located 273 bases upstream of the transcription start site (TSS) in the at3g28580 mutant.

Figure 20: Genotyping and RT-PCR of double mutants of early regulated genes in saul1-1^156,157. RT-PCR of single mutants with an expected product size of (A) trx5 of 232 bp, (C) at5g52760 of 345 bp, (E) at3g28580 of 413 bp, (G) at4g16260 of 1010 bp and 120 bp (I) wrky46 of 843 bp and 289 bp. As a marker (M) the GeneRuler™ 1kb Plus DNA Ladder was used. Genotyping of double mutants of saul1-1 with (B) trx5, (D) at5g52760, (F) at3g28580, (H) at4g16260 and (J) wrky46. Depicted is for each gene the mutant (MT) and the wild type (WT) genotyping with the DNA ladder (M), the corresponding double mutant (MT) and the matching mutant or wild type control (C), negative control (N) or wild type sample (WT).

To investigate, whether these insertions indeed led to a loss of expression for the gene of interest (GOIs), transcript levels were monitored by reverse transcription PCR (RT-PCR) experiments.

These could show that full-length transcripts of the GOI were not detectable in any of the mutant lines, because no corresponding band was observed in contrast to the WT samples (Figure 20A,C,E,G,I). In case of at4g16260 and wrky46 a partial and smaller transcript was detected. This corresponded in case of at4g16260 to the first exon, as the T-DNA insertion was determined to reside in the first intron (Figure 19D). For wrky46 the partial transcript corresponded to the first and second exons, in accordance with the T-DNA insertion localized in the second intron (Figure 19E). Although AT4G16260 and WRKY46 were still partially expressed, it was likely that such aberrant transcripts get degraded. This is either done by nonsense-mediated decay in case of a premature stop codon or by non-stop decay if the stop codon is missing due to the T-DNA insertion ²³⁹.

Consequently, all five lines were used to generate double mutants with saul1-1. Therefore, homozygous single mutants were used for crossings with saul1-1, and the F1-generation was self-pollinated. Afterwards, the F2-generation was genotyped and screened for double homozygous plants (Figure 20B, D, F, H, J). For each of the double mutants used, corresponding T-DNA insertion bands were detected for both affected genes (MT) and no bands in case of the control PCRs determining the WT gene status. Consequently, in all double mutants homozygous T-DNA insertions were detected, and thus the double mutants were used for further experiments.

It was assumed that these genes regulate the early onset of saul1-1autoimmunity and therefore hypothesized that in the double mutants the saul1-1 phenotype could be reversed. Double mutants were grown together with saul1-1 for 12 days at 25 °C and afterwards shifted to 20 °C.

After five days, a point of time at which the autoimmune phenotype is clearly detectable in saul1-1 plants (Figure 13A), all investigated double mutants displayed completely yellow leaves. In addition, a growth arrest, comparable to the one of saul1-1 single mutant plants, was detected (Figure 21). All double mutants did not differ in their appearance from saul1-1 plants and did thus not revert the phenotype.

Figure 21: Phenotype of mutants of early regulated genes and double mutants with saul1-1. Control (WT), saul1-1, single mutants and double mutants, which were all crossed with saul1-1, were grown on soil for 12 days at 25 °C and shifted to 20 °C for 5 days.

In conclusion, the selected genes may not be essential or solely involved in the observed regulations of the ETI-like autoimmunity in saul1-1. Nevertheless, each of the investigated genes has two or more homologs in A. thaliana with a sequence identity of at least 50 % (Table S5). A sequence identity of 50 % and more is sufficient in a related protein to potentially result in a similar function ²⁴⁰. Especially since TRX5, AT5G52760 and AT3G28580 have homologs with a sequence identity of more than 70 % that are highly likely to have a similar function (Table S5).

Therefore, homologs of the investigated genes may take over their function, and a reversal of the saul1-1 autoimmune phenotype would not occur. One particular example is TRX3, which is a homolog of TRX5, and is involved as well in the regulations of NPR1 oligomerization and therefore

the early onset of ETI ²³⁴. In summary, it can be said that only mutants generated between saul1-1 and all homologs may make it possible to identify potential early regulators of the ETI observed in saul1-1. Consequently, further experiments will focus on the generation of these multiple mutants to overcome redundancy.

In addition, there may be good candidates among the remaining 15 DEGs such as the two TF genes ERF2 (Ethylene Response Factor 2) and ZAT7 (Zinc Finger of Arabidopsis thaliana 7).Both genes are highly related to immune responses (see chapter 4.1.8) ^241–243 and are thus good candidates to be involved in the regulation of the early regulatory mechanisms in saul1-1 and possibly ETI. Their potential function will be discussed in following chapters. Although the mutant line for ZAT7 is currently being investigated, no mutant lines were available for the ERF2 gene. In the future, however, knock-out lines could be generated by using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9) approach. This method allows for specific generation of erf2 mutants ²⁴⁴, without potential additional insertions that are found in around 13 % of T-DNA-derived mutant lines ²⁴⁵. This would help to determine the function of ERF2 in context of the ETI-based autoimmune phenotype in saul1-1.