Early expression changes in saul1-1

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.

With regard to hormone signaling the up-regulation of multiple genes encoding members of the ERF transcription factor family suggested a participation of ET in saul1 autoimmunity. ET is known to be associated with a broad spectrum of defense responses ²⁵³. On one hand, ET is essential for the JA-dependent repression of SA in case of necrotrophic pathogens, mediated by the TF ORA59 (OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59) ²⁴⁹. On the other hand, ET acts synergistically with SA in oil seed rape during an infection with a hemibiotrophic pathogen ²⁵⁴. This was in line with the results on gene expression changes in saul1-1. The importance of ET-mediated regulations of plant defense were supported by recent findings showing that the type III effector HoPAF1 from Pto DC3000 is targeting the ET synthesis pathway and suppresses plant immunity ²⁵⁵. With respect to the regulation of the ETI and HR it was shown, that ET levels are up-regulated at a very early time point, prior to the onset of the SA synthesis, and that this correlated with ROS detection ²⁵⁶. Genes that are associated with ROS were detected in saul1-1 as well, as part of the GO term response to oxygen-containing compound and response to oxidative stress (Cluster III). These GO terms were also detected in cluster I, although more genes were associated with them. Therefore, ROS-dependent gene regulation, which is initiated after 3 h, was clearly more predominant after 6 h and could regulate the progression of immune responses.

Figure 22: Transcriptional cluster analysis of early regulations in saul1-1. Heatmap of genes, which were differentially expressed (p ≤ 0.01) after 0 h, 1 h/2 h, 3 h and 6 h in saul1-1 in comparison to the segregating wild type. Expression values are depicted as normalized Z-scores. All seven expression clusters were used for further gene ontology analysis. Pathways which were significantly enriched (p ≤ 0.01) are depicted on the right side of the heatmap. Adjusted p values of each term are written in brackets next to the gene count.

ABA responsive pathways were up-regulated in saul1-1, too (Cluster III). Despite its developmental role and association with abiotic stress, increased ABA levels are known to lead to a higher susceptibility to certain pathogens, for example Pto and Blumeria graminis f. sp.

hordei ^257,258. These pathogens exploit this regulatory system by increasing the synthesis of ABA to dampen the SA and JA-ET answer ²⁵⁹. This is due to increased ABA levels leading to a reduction of JA, ET ²⁶⁰ and SA, directly reducing the expression of ABA-synthesizing enzymes ^261,262. Therefore, this hormone is mostly described as a negative regulator of the immune response ²⁶³. Contradictory to this, ABA also appears to play an important role in enhancing the defense response as well. In infection studies with necrotrophic pathogens such as Alternaria brassicicola higher ABA levels led to an increase in JA production and to a stronger resistance ²⁶⁴. In conclusion, detecting ABA responsive genes in saul1-1 plants could mean, that early regulations in ETI are ABA-dependent as well, as the ABA regulatory network is interwoven with nearly all defense pathways and might be a more subtle regulator of those ^263,265. In addition, ABA-dependent pathways were not detected in cluster I but only in cluster III. In conclusion, ABA-related pathways are not initiated after 6 h, but after 3 h. Therefore, ABA-dependent pathways are particularly involved in the early regulations of ETI. This seems to be quite reliable, because ABA has been linked to ETI before as well ^266,267.

Besides the described hormonal regulations, the GO term defense response by callose deposition with several linked DEGs was detected in Cluster III. This was especially interesting, as a cell wall thickening due to lignin and callose deposition has been described as part of the saul1-1 phenotype ¹³². Those changes in the cell wall composition are known to highly increase disease resistance in plants by acting as a mechanical barrier ^51,268. The importance of this defense mechanism is illustrated by the fact, that the pathway is involved in cell wall thickening in A. thaliana and is suppressed by the type III effector AvrPto from Pto DC3000 ²⁶⁹. The induction of cell wall thickening due to callose or lignin deposition is mostly described as PTI-related and takes place around 8 h after an infection or treatment with a PAMP, for example flg22 ⁵⁵. In saul1-1 autoimmunity, the first morphological changes associated with cell wall thickening were detectable 2 d after the temperature shift ¹³², whereas the initial genetic regulations take place even as early as 3 h. Interestingly, after 6 h (Cluster I), no regulations of callose-associated genes were detectable. Apparently, the thickening of the cell wall was initiated at a very early stage.

These results showed that cell wall thickening in A. thaliana occurs not only in association with PTI but also with ETI. This is supported by experiments in rice that revealed an induction of cell wall thickening due to an ectopic expression of the AvrXa27 effector from Xanthomonas oryzae pv.

oryzae ²⁷⁰. In an ETI context callose deposition and therefore thickening of cell walls could function as a physical barrier to prevent the spread of the pathogen and thus HR. This seems very reasonable, because during cell death a broad number of ROS, signaling compounds and vacuolar contents are released, which could drive adjacent cells into HR ⁸⁵. On a proteomic level it was shown that immune suppressing proteins are up-regulated in neighboring cells, which are thought to prevent an uncontrolled spread as well ²⁷¹. In conclusion, callose deposition in an ETI context seem to be quite reasonable. Future investigations on the saul1-1 autoimmune model could give valuable insights into this aspect of plant defense.

Down-regulated genes - Clusters V and VII

The Clusters V and VII contained almost exclusively down-regulated genes. These were linked to the GO terms glycosyl compound biosynthetic process and S-glycoside biosynthetic process. This may indicate that glycosylation was negatively regulated during the onset of the saul1-1 phenotype. By applying in silico analyses using Genevestigator it was possible to detect that these genes were also down-regulated during NLR-mediated defense responses and upon flg22 treatment 183,185,186. The down-regulation of those genes may thus generally be associated with immunity. However, glycosylation is mediated by a broad range of different pathways with diverging outcomes.

Remarkably, most detected genes are mainly involved in S- and not N-glycosylation. This is quite plausible, because N- in contrast to S-glycosylation is one of the major post-translational modifications, which is highly important for correct protein folding and trafficking. With respect

to the immune system an incorrect or missing N-glycosylation of the PRRs EFR (EF-TU RECEPTOR) and FLS2 (FLAGELLIN-SENSITIVE 2) has been shown to lead to non-functional receptors and retention in the endoplasmic reticulum ²⁷². Therefore, a down-regulation of N-glycosylation would be unexpected, since this posttranslational modification is vital for correct protein folding. In contrast, it is known that S-glycosylation is an important step in secondary metabolite synthesis ²⁷³. Although those pathways, which are down-regulated in saul1-1, are quite diverse, most of them have one characteristic in common: they are highly connected to the interplay between JA and SA ^274–276. For instance, the down-regulated TF MYB28 and MYB29, which promote glucosinolate biosynthesis, are known to be positively regulated by JA and suppressed by SA ^276,277. Additionally, repression of these TFs is known to lead to decreased growth ²⁷⁸. This is especially interesting, because a characteristic of the saul1-1 and other autoimmune-associated phenotypes is growth arrest ^123,134. Genes regulated by MYB28 or MYB29 comprise other TFs such as WRKY18 and WRKY40, which in turn regulate members of the CYP (Cytochrome P450) family ²⁷⁶. These, in particular CYP79F2 and CYP83A1, were found to be down-regulated in saul1-1 as well.

Interestingly, it has been reported that a mutant of CYP83A1 leads to a higher resistance against powdery mildew. This was due to an accumulation of camalexin, one of the major phytoalexins ⁴³. Phytoalexins are known to be expressed upon infection with necrotrophic pathogens and to be fungistatic ²⁷⁹. In context of ETI, they have not been excessively studied, but are thought to be expressed to prevent a secondary infection ²⁷¹. In addition, the regulations in cyp83a1 are linked to SA accumulation, because the increased resistance was found to be EDS1-, PAD4- and NDR1 (NON RACE-SPECIFIC DISEASE RESISTANCE 1)-dependent ⁴³. Other genes involved in S-glycosylation, like FMOGS-OX1 (Flavin Monooxygenase GS-OX1) and FMOGS-OX3, are known to be JA- and SA-responsive as well ²⁷⁵. Therefore, those diverse pathways connected to S-glycosylation, could be part of downstream regulations reacting to elevated SA levels in saul1-1 ¹³⁵.

In summary, the gene expression changes 6 h and especially 3 h after the temperature shift in saul1-1 appeared to be regulated in a quite complex manner involving an interplay of the hormones SA, JA, ET and ABA. These regulations seem to initiate all downstream regulations, since they were dominantly detected in cluster III and this cluster contained genes that were differentially expressed from 3 h onwards. This assumption is supported by the observation that after 6 h (Cluster I) fewer genes were differentially expressed for the first time. Consequently, the saul1-1 autoimmunity was defined by a large number of genes, which initiates after 3 h pathways that result in the saul1-1 phenotype. Therefore, clustering all DEGs in saul1-1 for the different time points was extremely helpful to get first insights into genes with similar behavior and into activation of pathways. However, genes that were down- and up-regulated were separated during this analysis. This may result in an insufficient amount of enough genes being detected to significantly enrich specific GO terms. In conclusion, some signaling pathways involved in saul1-1 may not be detected in GO analysis because they contain up- and down-regulated genes.