Are the four selected candidate genes casual genes?

Several in silico, in vitro and in planta analyses were performed to identify and to the most promising candidate genes. The presented results support the hypothesis that several of them were involved in the regulation of powdery mildew resistance responses and are in this sense resistance genes.

In respect of the annotated function and the functional in silico analysis (Table R 3 and Figure B 8), the candidate WB-CG_19 might correspond to the barley homolog of Arabidopsis’ dolichol phosphate mannose synthase 3 (DPMS3). DPMS3 is a binding partner of the DPMS2 and DPMS1 and it is required for the generation of isoprenly-linked glycans (Jadid et al., 2011). Glycans play an important role during infection with pathogens. As an example, human studies report that nearly all microbe and virus infections are linked to the binding of the pathogen to specific cell surface glycans (Hart & Copeland, 2010). In plants, it is proposed that glycans attached to plasma membrane recognition receptors are the initial contact site between cell and invading pathogen (Häweker et al., 2010). DPMS3 seems to represent the binding module between DPMS1 and DPMS2 in planta (Jadid et al., 2011). Additionally, yeast data revealed that DPMS3 tethers the protein complex to the endoplasmic reticulum membrane (Ashida et al., 2006). The functional complex of all three subunits generates dolichol-phosphate mannose which is an important intermediate during the N-glycosylation of proteins (Jadid et al., 2011). The correct attachment of N-glycans is essential for plant development and abiotic stress responses like salt stress (Kang et al., 2008; von Schaewen et al., 2008). Moreover, it was demonstrated that N-glycans are required for the plant innate immune responses of Arabidopsis (Saijo et al., 2009; Häweker et al., 2010).

Particularly, these responses were mediated by pattern recognition receptors in dependence of the receptor quality control in the endoplasmic reticulum (Saijo et al., 2009; Häweker et al., 2010).

These results suggest a crucial role of the candidate gene in respect of abiotic and biotic stress responses. Nevertheless, neither the transient overexpression nor the silencing of the tested alleles resulted in a significant alteration of the powdery mildew resistance (Table R 4 and R 5). In contrast to these negative results, the transcriptional analysis revealed that the gene is significantly upregulated by powdery mildew inoculation (Figure R 14). The transcript level spikes 24 h post inoculation corresponding to the time point when the haustorium is established in a compatible resistance interaction (Zhang et al., 2005). Interestingly, the transcript level is significantly reduced afterwards in the samples inoculated with the adapted powdery mildew fungus but not in the samples inoculated with the non-adapted fungus (Figure R 14). This observation suggests that only the adapted fungus could be able to manipulate the candidate transcription to probably supress an induced defence response. The results (Figure R 14) imply that the candidate gene is actually involved in the regulation of the defence or stress responses

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after powdery mildew attack. Furthermore, the results suggest that the candidate is involved in the basal and/or nonhost resistance. Theses hypotheses are supported by the reported extensive modifications of the plant glycan structure that occur after fungal attack (Chaliha et al. 2018). It is possible that the negative results of the transient assays (Table R 4 and R 5) are explained by the identical protein sequences of both candidate alleles (Figure B 8). In this regard, it is a plausible explanation that the causal SNP was not captured in the used constructs. The specific downregulation of the candidate transcript by the adapted powdery mildew fungus (Figure R 14), which could be necessary to circumvent the innate immunity of barley, might explain the negative results, too. A repetition of the transient assays after inoculation with the non-adapted powdery mildew fungus could shed light on this particular question.

The in silico functional analysis of WB-CG_17 (Table R 3 and Figure B 7) revealed that the protein with unknown function could encode an arabinogalactan protein (AGP). AGPs form highly complex families and the diversity of the attached O-glycan chains is astonishing (Ellis et al., 2010;

Nguema-Ona et al. 2014). AGPs can be found in the plasma membrane, the cell wall, the apoplastic space, multivesicular bodies and in secretions like wound exudates (Ellis et al., 2010;

Mareri et al., 2019). The functions of this protein class are still not fully understood, but they seem to be involved in various plant growth and developmental processes (Ellis et al., 2010; Nguema-Ona et al. 2014). Additionally, AGPs were involved in several abiotic and biotic stress responses (Mareri et al., 2019). The potential candidate homologs of the related grass species were classified as ‘UPF0664 stress-induced protein C29B12.11c’ (Table R 3) implying a general role in stress responses. This hypothesis is in accordance with previous observations that defence-related genes were involved in the regulation to various stresses like wounding and temperature stress (van Loon et al., 2006; Hua, 2013). Nevertheless, no further information is available for this protein family. The PH-GRAM-WBP2 domain, which is annotated in the potential candidate homologous proteins (Table R 3 and Figure B 7), represents a conserved motif in eukaryotes (https://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=275401). In humans, this domain is associated with various relevant disease and signalling pathways (Chen et al., 2017). In general, GRAM domain proteins are involved in plasma-membrane-associated processes like intracellular protein binding or lipid-binding signalling (Doerks et al., 2000). These results indicate that WB-CG_17 could be associated with membrane processes. This is further supported by the detection of the potential Arabidopsis homolog during the screening for membrane associated proteins (Marmagne et al., 2004 & 2007). Besides the regulatory functions named above, AGPs are also involved in the responses to microbes (Ellis et al., 2010; Nguema-Ona et al. 2014). These plant-microbe interactions were mainly studied in the root (Mareri et al., 2019). As an example, the production of extensins and AGPs is induced after pathogen attack probably to reinforce the

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cell wall via extensive oxidative cross-linking (Nguema-Ona et al. 2014). Additionally, it was proposed that AGPs might be involved in the recognition or attachment of pathogens (Mareri et al., 2019). Besides, there are indications that AGPs can form a ‘biofilm’ which show antimicrobial properties and that can act as physical barrier (Mareri et al., 2019). Gaspar et al. (2004) presented evidence for the specific function of AGP17 of Arabidopsis in the regulation of the response to Agrobacterium tumefaciens infection. They suggested mechanisms on how AGP17 can act as susceptibility factor in this interaction. The here presented results of the transient overexpression of the minor allele of WB-CG_17 (Table R 5) indicated that also the candidate may act as susceptibility factor or disease susceptibility gene. The observed increased susceptibility in the resistant as well as in the susceptible background (Table R 5) supports the general importance of this protein in the interplay between Bgh and barley. Liang et al. (2018) reported the specific expression increase of a plasma membrane bound AGP epitope in rice after infection with the biotrophic fungus Magnaporthe orzyae. A similar upregulation of WB-CG_17 transcript was detected in powdery mildew inoculated epidermis peels (Figure R 14). These results imply that the candidate is more likely participating in the regulation of defence responses than being involved as recognition site for the fungus during attack. Nevertheless, further investigation is necessary to specify the function of the candidate. In general, it is unclear if the detected expression alterations of AGPs after abiotic and biotic stresses are an indirect/direct result of the cell responses or just part of cell damages (Mareri et al., 2019). The transcriptional analysis of WB-CG_17 revealed further that the candidate could be regulated by the circadian clock (Figure R 14). The circadian clock is beside temperature, light and redox signalling one of the major regulators of defence responses to ensure the specific timing of the costly responses (Hua, 2013;

Sharma & Bhatt, 2015; Lu et al., 2017; Karapetyan & Dong, 2018). Wang et al. (2011) reported expression patterns of clock regulated defence genes after pathogen inoculation in Arabidopsis resembling the one of WB-CG_17 (Figure R 14). A further analysis of the candidate sequence, especially of the promoter region, for regulatory clock elements and a detailed assessment of the expression profile could shed light on the hypothesized circadian clock regulation. The observed transcription pattern of WB-CG_17 (Figure R 14) resembles in particular the pattern described for pathogen induced clock regulated defence genes in absence of a major R-gene (Wang et al., 2011).

This observation supports the hypothesized race-nonspecificity of the resistance response regulated by the candidate. Further supported is the hypothesis by the enhanced susceptibility mediated by the overexpression (Table R 5). This effect is achieved in the susceptible as well as in the resistant genotype. Particularly, this circumstance is interesting because the resistance is conferred in a race-specific manner (Figure R 5 and Table B 4, Hoseinzadeh et al., 2019).

Additionally, the altered expression of the potential Arabidopsis candidate homolog after

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Geminivirus infection (Ascencio-Ibánez et al., 2008) could indicate a general upregulation of the candidate after pathogen attack.

The third tested candidate WB-CG_23 is annotated as ‘Bric-a-Brac/-Tramtrack/-Broad Complex (BTB)/-POxvirus and Zinc finger (POZ)/ Kelch protein’ and the in silico functional analysis (Table R 3) implies that the candidate may act as barley homolog of Arabidopsis’ Light-Response BTB (LRB) 1 or 2. LRB proteins seem to be conserved between mono- and dicot plants, although they cluster in specific clades (Christians et al., 2012). In accordance with these results, the monocot LRB homologs of Brachypodium distschyon and rice show a higher sequence identity to the here presented barley homologs as compared to the dicot LRBs of Arabidopsis thaliana (At) (Figure B 9). In addition to LRB1 and LRB2, a third LRB is annotated in Arabidopsis which is presumed to be a pseudogene and absent in most species (Christians et al., 2012). The homologous proteins LRB1 and LRB2 were firstly described by Qu et al. (2010) as POB1 (POZ/BTB Containing Protein1) and POB2, respectively. They seem to negatively regulate the jasmonate mediated pathogen defence in Arabidopsis (Qu et al., 2010). LRB1 and LRB2 can homo- and dimerize to form together with Cullin3 ubiquitin E3 ligases (Christians et al., 2012). The protein complexes are involved in the regulation of diverse processes. On the one hand, these E3 ligases target FRIGIDA (a major regulator of flowering in Arabidopsis) for proteasomal degradation during vernalization and thus initiate flowering (Hu et al., 2014). On the other hand, they participate in the regulation of phytochromes (Christians et al., 2012; Ni et al., 2014). In Arabidopsis five phytochromes (PhyA-PhyE) have been annotated (Xu et al., 2015). They perceive the ratio of red to far-red light and modulate plant growth, development and immune responses (Xu et al., 2015;

Ballaré, 2014). In particular, phyB in combination with other phytochromes regulates defence responses like the activation of lipoxygenases (Zhao et al., 2014). This regulatory function of PhyB was demonstrated for different pathogens and the corresponding signalling pathways. As examples, the single phyB mutant of Arabidopsis is more susceptible against Fusarium oxysporum (Kazan & Manners, 2011) and Botrytis cinerea (Cerrudo et al., 2017) while the double mutant phyAphyB displays reduced resistance responses after infection with Pseudomonas syringae pv.

tomato (Genoud et al., 2002; Griebel & Zeier, 2008). In respect of the attacking pathogen, either the salicylic acid or the jasmonate signalling pathway is induced but both are regulated in a light dependent manner (Ballaré, 2014). These regulations rely mainly on PhyB and the ultraviolet-B photoreceptor UVR8 (Ballaré, 2014). Additionally to Arabidopsis, other plant species like rice display phytochrome regulated defence responses (Xie et al., 2011). There are also indications for a light dependent regulation of resistance responses against powdery mildews (Wang et al., 2010). The resistance of cucumber (Cumunis sativus L.) against Sphaerotheca fuliginea depends on salicylic acid and light quality (Wang et al., 2010). The described Culllin3-LRB1/2 E3 ligases of

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Arabidopsis promote the degradation of PhyB and PhyD in a light regulated manner (Christians et al., 2012). Additionally, the degradation depends on the phosphorylation and degradation of Phytochrome Interacting Factor 3 (PIF3, Ni et al., 2014). Moreover, the E3 ligases positively regulate the PhyA level (Christians et al., 2012). In monocot plants, only three phytochromes (PhyA to PhyC) were found (Mathews & Sharrock, 1997) and the same holds true for barley (Szücs et al., 2006). These results indicate a differentiated regulation of light signals in mono- and dicot plants. Nevertheless, the phytochrome dependent regulation mechanisms of salicylic acid and jasmonate dependent defence responses seem to be conserved. As an example, the rice phyAphyBphyC mutant displays enhanced susceptibility against Magnaporthe orzya (Xie et al., 2011). If the here presented candidate is actually involved in the regulation of phytochromes in barley needs further investigation. Nonetheless, the hypothesized function of WB-CG_23 as LRB homolog is supported by the high sequence identity of the functional domains between the homologous proteins (Figure B 9). Christians et al. (2012) presumed that the regions surrounding the BTB domain are involved in the target recognition. Particularly, the sequence following the BTB domain is conserved between the candidate and its potential homologs (Figure B 9). This finding supports the presumed function of WB-CG_23 as phytochrome regulator. In addition, the same rice proteins were identified as WB-CG_23 and AtLRB homologs (Gingerich et al., 2007;

Christians et al., 2012), although these rice proteins were annotated as ‘BTB with E1 subfamily conserved sequence’. The transient overexpression of the major candidate allele resulted in enhanced susceptibility against powdery mildew (Table R 5). This effect supports a function of the candidate in the regulation of these particular defence responses. Nonetheless, the detected effect is only observed in the resistant background (Table R 5). In this respect, further investigation is necessary to clarify whether the candidate acts in dependence of the genetic background. A repeated transient overexpression of both alleles in a moderately resistant genotype under various light conditions could shed light on this particular idea especially in respect of the possible regulation of phytochromes. Nevertheless, it has to be taken into account that the candidate and its potential homolog (HORVU5Hr1G116800, Table R 3) could act redundantly like it was reported for AtLRB1 and AtLRB2 (Christians et al., 2012). Nevertheless, none of the SNPs located in the barley homolog is significantly associated in the here reported GWAS (Table B 5). This could be related to genetic heterogeneity in the population or because the candidate WB-CG_23 acts specifically in the regulation of this particular defence responses. In comparison to the other candidate genes presented in this study, the expression of WB-CG_23 is rather low which complicates the results interpretation (Figure R 13, Figure R 14 and Figure B 6).

Nevertheless, it seems that the transcript level of the candidate gene is altered after powdery mildew inoculation (Figure R 14). This observation supports a defence related function. In case of

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AtLRB1/2, a broad expression in mainly all tissues and all developmental stages was reported by Christians et al. (2012). If the present candidate gene is also expressed in a wide variety of tissues or altered by different light conditions would need further investigation.

WB-CG_28 was the last candidate that was addressed in the functional validations. It was annotated as ‘flap endonuclease (FEN) 1-B’ which was confirmed by the in silico analysis (Table R 3 and Figure B 10). In general, FEN1 proteins form structure-specific endonucleases which are highly conserved among species (Balakrishnan & Bambara, 2013). They are essential for the processing of Okazaki fragments, long-patch base excision repair and telomere maintenance (Balakrishnan & Bambara, 2013). Most studies have been performed either in human, animal or yeast systems. Thus, the knowledge about plant FEN1 homologs is still limited. In Arabidopsis, FEN1 is a single copy gene (Schultz et al., 2007) which is essential for the maintenance of the genome stability and development (Zhang et al., 2016b) as well as transcriptional gene silencing (Zhang et al., 2016a). fen1 mutant plants have shorter telomers and altered DNA methylation (Zhang et al., 2016a). Additionally, their root apical meristem as well as the quiescent centre are defective (Zhang et al., 2016b). In contrast to most species, two functional homologs, FEN1-A and FEN1-B, were described in rice. Both proteins seem to be required for cell proliferation (Kimura et al., 2003). The here presented in silico analysis revealed further potential FEN1-B proteins in barley, Aegilops tauschii and Brachypodium distschyon (Table R 3 and Figure B 10). This circumstance could indicate that this protein type might be conserved in monocot plants. The further characterization of the rice FEN1-B led to the hypothesis that it fulfils distinct functions besides cell proliferation regulation. Firstly, only OsFEN1-A is able to complement the FEN1 null mutant of yeast (rad27, Reagan et al., 1995) whereas OsFEN1-B failed (Kimura et al., 2003).

Besides the expression of both homologs in proliferating tissue, OsFEN1-B seem to be specifically expressed in mature leaves (Kimura et al., 2003). Similar results were presented here for the barley FEN1-B homolog (WB-CG_28). The candidate expression was detected in mature first leaves and also in epidermis peels (Figure R 14 and Figure B 6). Regarding the significant transcript reduction in response to the adapted as well as the non-adapted powdery mildew fungus (Figure R 14), an involvement of the candidate in regulation of defence responses is hypothesized.

Unfortunately, it was not possible to retrieve an Escherichia coli clone expressing any of the full-length candidate alleles. In respect of the negative outcome of the transient silencing approach (Table R 4) and the missing results of the transient overexpression, it was not possible to further validate the candidate function regarding the powdery mildew resistance responses.

One aims of this study was the identification of race-nonspecific resistance genes (alleles) of barley involved in the response to powdery mildew. Several indications for the here presented candidates were discussed which allowed the hypothesis that the four selected candidates are

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involved in the regulation of powdery mildew resistance responses. If these genes act as resistance or susceptibility genes could not be determined finally. A function of these candidates in resistance responses is supported by the literature. Nevertheless, none of the genes was described until know in the context of powdery mildew resistance (in barley). In this sense, new insights in the barley-powdery mildew interaction could be obtained.

The results presented here allow the hypothesis that all four candidate genes may act indeed in a race-nonspecific manner. The major facts supporting this hypothesis are: (1) the selected candidates were significant in all three GWAS traits (Figure R 12 and Table B 5); (2) the annotated functions of the genes imply that they are involved in basic regulatory processes (Table R 3); (3) the transcript levels of all candidates were altered after inoculation with the adapted as well as the non-adapted powdery mildew fungus (Figure R 14); and (4) at least two of the candidates alter significantly the susceptibility against powdery mildew when overexpressed (Table R 5).

Furthermore, the obtained results indicate that these candidates act rather as susceptibility factor or disease susceptibility gene than as direct resistance genes.

The here presented approach was specifically designed to assess the differential effect of the candidate alleles on the powdery mildew response (Figure R 15, Table B 7 and B 8). The functional validations were performed as transient single cell transformations (Table R 4 and R 5). The assay design support further the race-nonspecific function of the candidates. As an example, a third powdery mildew isolate was selected for the evaluation of the allelic effects. Moreover, it has to be considered that the resistance of the resistant genotype selected for the functional validation is caused race-specifically by a major R-gene (Figure R 5 and Table B 4, Hoseinzadeh et al., 2019).

Nevertheless, this major R-gene may also mask the smaller effects of some of the candidate genes/alleles. The presumed small effects of the candidates could be one of the reasons why the transient silencing did not lead to clear results (Table R 4). In respect of the transient nature of the single cell analysis, it would be very difficult to assess if the generated hairpin structures of the constructs were actually processed to small interfering RNAs or how efficient they are. As alternative approach virus-induced gene silencing could be performed to analyse a knock-down of the candidates. Another factor which should be considered is a possible functional redundancy of the candidates and their potential homologous proteins. In case of the overexpression assay, it has to be taken into account that the natural candidate alleles were not knock-out prior the overexpression. In this regard, only dominant effects of the candidates could be observed.

Furthermore, is has to be kept in mind that the alleles were overexpressed under control of the 35S promoter. This promoter is weaker expressed in monocot as in dicot plants (McElroy et al., 1991), which was confirmed by the here presented analysis of different promoter_GUS constructs

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(Table R 6 and R 7). Probably a stronger overexpression of the candidate alleles under control of another promoter could further differentiate the allelic effects.

In respect of the presented results, interesting candidate genes were identified which could be further investigated by the production of stable transformed plants or an additional screening for further alleles. If these candidates were really the causal genes for the loci 13 and 14 could not be answered finally, but the reported results support the function of them in the regulation of powdery mildew resistance responses. In addition, they might act as resistance or as susceptibility genes.

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