Leaf rust caused by the biotrophic fungus Puccinia hordei is a serious disease of barley, leading to yield losses from 15–25% (Whelan et al., 1997) up to 62% (Park et al., 2015). Visual symptoms vary from small chlorotic lesions to large orange-brown pustules of about 0.5 mm in size, often surrounded by green leaf areas (Clifford, 1985). A major strategy in controlling leaf rust epidemics is breeding of resistant barley cultivars. While several resistance genes (Rph1 to Rph28, and Rph _MBR1012) have been identified in recent years (Singh et al., 2015; Fazlikhani et al., 2019; Chen et al., 2021), most of these are already overcome. Breakdown of resistance by mutations in effector genes of the pathogen frequently lead to the occurrence of new virulent races (Park, 2003), and thus a rapid decrease of the number of effective Rph genes (Kavanagh et al., 2017). Other effective management practices include the application of chemical fungicides, which can cause negative environmental (Ballabio et al., 2018) and public health effects (Perlin et al., 2017) if applied incorrectly. In this regard, decision-based management strategies may help to reduce their environmental impact considerably in the coming years (Lázaro et al., 2021). However, to promote more sustainable agricultural systems establishing alternative strategies to manage leaf rust epidemics is of prime importance, besides the ongoing genetic mapping of resistance genes.

Novel approaches for sustainable and economical crop production under ever-changing climate conditions include microbiome-facilitated crop management strategies (Liu et al., 2020; Trivedi et al., 2021). Beneficial plant-associated microbes include predominantly rhizobacteria and fungi as well as viruses, actinomycetes, cyanobacteria, archaea (Mauch-Mani et al., 2017; Mahapatra et al., 2020). The barley-microbiome was reported to largely vary between the different plant organs, developmental stages, origin, as well as between genotypes (Yang et al., 2017; Alegria Terrazas et al., 2020; Bziuk et al., 2021). Reported positive effects of such interactions in barley include reduced virulence of plant pathogenic fungi (Wehner et al., 2019; Karlsson et al., 2021), higher tolerance to abiotic stress (Caddell et al., 2019; Yang et al., 2020), as well as plant growth promotion and nutrient uptake under adverse conditions (Trivedi et al., 2020; Ribaudo et al., 2020).

The activation of induced defense mechanisms by various stimuli, such as from pathogens, beneficial microbes, arthropods, as well as chemicals and abiotic cues, is generally regarded as priming (Mauch-Mani et al., 2017; Desmedt et al., 2021). Upon priming, plants respond stronger and faster to a biotic or abiotic stress factor, resulting in resistance and securing yield. The use of priming for enhancing resistance has a long tradition, and the phenomenon called “sensitization” or “preformed defense” was investigated and used since the 1930s (Chester, 1933; Starkey, 1958). In this respect, it has to be noted, that microbial priming-induced plant responses vary between species and depend on the composition of the soil-microbiome as well as on the genotype and vice versa (Jack et al., 2019; Morella et al., 2020; Alegria Terrazas et al., 2020; Si et al., 2021).

Various organic compounds, for instance salicylic acid (SA), benzothiadiazole, β-aminobutyric acid, and azelaic acid are known to induce priming (Conrath et al., 2002; Jung et al., 2009; Mauch-Mani et al., 2017). In addition, many recent reports are highlighting bacterial quorum sensing (QS) molecules as priming-inducers in plants. Related to plant-pathogen interactions, the best-studied QS molecules are N-acyl homoserine lactones (AHL), which are produced by numerous gram negative bacteria to communicate and to monitor the density of populations (Kaplan and Greenberg, 1985; Fuqua et al., 1994). Plants generally respond to short-chained AHL with modifications in growth, while long-chained AHL induce AHL-priming for enhanced resistance against pathogen infection (Shrestha et al., 2020).

In tomato plants, root colonization with AHL-producing Serratia liquefaciens MG1 and Pseudomonas putida IsoF lead to increased systemic resistance against the fungal leaf pathogen Alternaria alternate, likely in an SA- and ethylene-dependent manner (Schuhegger et al., 2006). The effect has been related to the four AHLs N-(3-oxo-hexanoyl)-L-homoserine lactone (3-oxo-C6-HSL), 3-oxo-C8-HSL, 3-oxo-C10-HSL, and 3-oxo-C12-HSL produced by P. putida IsoF (Gantner et al., 2006), and to the two AHLs butanoyl-homoserine lactone (C4-HSL) and 3-oxo-C6-HSL produced by S. liquefaciens MG1. Plants inoculated with the AHL-negative mutant strain S. liquefaciens MG44 exhibited a susceptible phenotype (Schuhegger et al., 2006).

Another rhizobacterium with known AHL-priming capacity is Ensifer meliloti, a naturally occurring gram negative soil bacterium (Teplitski et al., 2003; Fagorzi et al., 2020). By studying AHL-priming with E. meliloti as a model in the laboratory, different authors tested resistance against various pathogens, for instance Pseudomonas syringae in Arabidopsis thaliana (Zarkani et al., 2013; Shrestha et al., 2020), Blumeria graminis (Shrestha et al., 2019), Puccinia hordei in barley (Hernández-Reyes et al., 2014; Wehner et al., 2019; Shrestha and Schikora, 2020) and the pest Pratylenchus penetrans in soybean (Adss et al., 2021). Notably, barley genotypes showed different sensitivity to AHL-priming when treated with E. meliloti, indicated by diverse resistance responses of seven barley accessions to P. hordei infection (Wehner et al., 2019) and of eight barley accessions to B. graminis infection (Shrestha et al., 2019). These results suggest that AHL-priming is highly influenced by the genetic background of the respective accession, and strongly propose the utilization of priming-efficiency as a future breeding goal. However, the genetic background underlying the variability of AHL-priming-induced resistance in plants has not yet been investigated.

In order to map genetic loci underlying differences in response to AHL-priming, genome wide association studies (GWAS) are a powerful tool since high throughput marker technologies like the 9k iSelect chip (Comadran et al., 2012), genotyping by sequencing (Poland and Rife, 2012) or exome capture (EC) sequencing (Mascher et al., 2013) are available in barley. While family-based linkage analysis searches for associations within populations developed from bi-parental crosses, association mapping utilizes historic patterns of recombination that have occurred within a sample of individuals. Association mapping is based on the principle that over multiple generations of recombination, correlations only with markers tightly linked to the trait of interest will be present. As a predominantly inbreeding species, cultivated barley is an attractive target for association mapping as its genome contains extensive blocks of chromatin in linkage disequilibrium (Rostoks et al., 2006), providing a well-defined haplotype structure from which marker-trait associations can be identified (Cockram et al., 2008).

In this study, we aim to investigate the genetic background of resistance priming in barley. Thus, the objective is identifying barley varieties with an enhanced potential to respond to E. meliloti treatment with an increased resistance to a P. hordei infection by screening a set of barley genotypes with a diverse genetic background. A set of 198 spring barley accessions was challenged with either the 3-oxo-C14-HSL accumulating E. meliloti strain expR+ch or with the control E. meliloti strain attM, not being able to accumulate AHL (Zarkani et al., 2013). Responses after P. hordei infection were monitored and relative infection values were used as a target for GWAS. Identified primable accessions and QTL are discussed regarding the molecular mechanisms of AHL-priming and breeding applications.

Within this three-year trial, a positive effect of priming with the gram negative soil bacterium E. meliloti strain expR+ch in relation to resistance to P. hordei was demonstrated for spring barley. The effect on relative infection and thus priming efficiency varied among the set of 198 accessions, of which 87 accessions showed significant (p<0.05) treatment effects and therefore have been considered highly primable ( Figure 2 ). Nevertheless, in practical applications the priming effect might be influenced by environmental conditions, such as soil type and the presence of other microorganisms. To our knowledge, the presented study is the first investigation on the effects of the priming inducer E. meliloti strain expR+ch on cereal resistance against fungal pathogens including a larger set of genetically differentiating accessions. While several recent reports are suggesting that the microbiome varies between the different plant organs, developmental stages, origin, as well as between genotypes in wheat (Gdanetz and Trail, 2017; Kuźniar et al., 2020; Žiarovská et al., 2020) and barley (Yang et al., 2017; Alegria Terrazas et al., 2020; Bziuk et al., 2021), studies on genetic variation of priming efficiency are rare. In a previous experiment, we observed significant differences between seven spring barley accessions, indicating genotypic differences in the response to priming by E. meliloti strain expR+ch towards P. hordei resistance (Wehner et al., 2019). Similarly, using a set of eight genetically diverse spring barley accessions, Shrestha et al. (2019) have demonstrated differences in their ability to be primed by E. meliloti strain expR+ch with respect to resistance against B. graminis f. sp. hordei. Other root associated microorganisms, for which genotype dependent responses have been shown, include arbuscular mycorrhiza fungi (AMF), reviewed in Parke and Kaeppler (2000) and Sawers et al. (2010). Varying abilities to respond to AMF were reported for wheat (Hetrick et al., 1993), maize (Kaeppler et al., 2000; An et al., 2010), and onion (Powell et al., 1982; Tawaraya et al., 2001; Galván et al., 2011). Consistently, investigation of a diverse set of 94 bread wheat genotypes (Triticum aestivum) regarding root colonization by AMF identified significant genotypic differences (p<0.001) for mycorrhizal colonization and detected 30 significant genetic markers associated with root colonization (Lehnert et al., 2017). Within the same set of genotypes, drought stress tolerance was significantly increased in the presence of AMF colonization compared to drought stress tolerance in the absence of AMF colonization (Lehnert et al., 2018).

In our results, we observed a trend to non-European origin and six-rowed ear type being correlated with higher priming efficiency ( Table S2 ). However, no clear relation to genetically determined phenotypic traits could be made. The model cultivar Morex was among the ten accessions with highest priming efficiency. This is concordant with our previous finding, where Morex was identified as a well primable accession (Wehner et al., 2019). In this study, the accessions BCC768 and HOR7985 were also assigned as well-primable, a result which was confirmed in the recent study. While the resistance gene Rph8 was mapped in the Morex reference genome on chromosome 5H at 12.7 Mb (Mascher et al., 2017), we observed high values for relative infection under attM control treatment in this accession ( Table S2 ). This can be explained by breakdown of resistance gene Rph8 by the P. hordei I-80 isolate (Niks et al., 2000), which was used in the present study. Notably, some QTL identified during the control treatment were in the genomic region of known resistance genes, for example Rph19 (Park and Karakousis, 2002) on the barley chromosome 7H (see Figure 3 ). Together, results suggest a high genetic determination of priming responses and thus highlight the importance of screening large diversity sets for detecting quantitative trait loci or candidate genes involved in priming.

In our study, of the 129 MTA identified, 17 MTA, which cluster in 11 QTL, were associated to improved resistance to P. hordei after priming with the oxo-C14-HSL-producing E. meliloti expR+ch, with a hot spot on the short arm of barley chromosome 6H ( Figure 3 , Table 3 , and Table S3 ). Except two markers, all markers were assigned to positions in the genome within annotated barley coding genes. On chromosome 6H, the marker chr6H_16152203EC (QTL 18, LOD 4.23) is localized in the gene HORVU6Hr1G008870 encoding for a pentatricopeptide repeat-containing protein. This gene product belongs to the class of zinc finger proteins involved in DNA binding, which may have regulatory function, and which was shown to be induced by drought stress in barley in a previous study (Guo et al., 2009). Another significant marker on chromosome 6H (SCRI_RS_121633, QTL 22, LOD 4.33) is localized in the gene HORVU6Hr1G018610 encoding for the protein FLUORESCENT IN BLUE LIGHT. In A. thaliana, this gene product was reported as a negative regulator of chlorophyll biosynthesis acting in a light dependent manner (Hou et al., 2019). How both gene products mentioned above are involved in the process of induced systemic resistance by AHL priming remains speculation. However, limitation of photosynthetic activity has been assumed one possible strategy of plants to increase pathogen resistance. For instance, the accumulation of oxylipin in distal tissues during AHL-priming in Arabidopsis promoted stomatal closure. The closed stomata enhanced plant resistance to bacterial pathogens (Schenk and Schikora, 2015). In this line, a very promising QTL (21) on chromosome 6H comprises five significant markers from two different marker types (SCRI_RS_182367, chr6H_44247682, chr6H_44247749, chr6H_44247752, chr6H_44247776), all localized in the same gene HORVU6Hr1G018050, which encodes for a esterase/lipase/thioesterase family protein. In barley, this protein was not characterized functionally yet, but a BLAST search with the protein sequence in the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) revealed highest similarity to the chloroplastic phytyl ester synthase 1 (PES1) from A. thaliana (At1g54570). It exhibits phytyl ester synthesis and diacylglycerol acyltransferase activities with broad substrate specificities, and can employ acyl-CoAs, acyl carrier proteins, and galactolipids as acyl donors. PES1 was shown to be involved in fatty acid phytyl ester synthesis in chloroplasts, a process required for the maintenance of the photosynthetic membrane integrity during abiotic stress and senescence (Lippold et al., 2012). Accordingly, deposition of free phytol and free fatty acids in the form of phytyl esters in chloroplasts was reported for etiolated barley seedlings after 6 to 8 h irradiation (Liljenberg, 1977), and in primary leaves of barley during methyl jasmonate induced leaf senescence (Springer et al., 2015). Notably, HORVU6Hr1G018050 was also found in a QTL region on chromosome 6H in the interval between 37-76 Mb associated with resistance against net blotch causing Pyrenophora teres f. teres in a barley diversity set (Novakazi et al., 2019). On chromosome 5H, the marker BOPA1_8215-496 (QTL 14, LOD 4,73) is localized in the gene HORVU5Hr1G065220 encoding for a PDZ domain-containing protein, which is likely involved in the regulation of protein translation via the proteasome with unknown contribution to pathogen resistance. Interestingly, the marker SCRI_RS_205235 (QTL 13, LOD 4,25) is localized in the ATP-dependent zinc metalloprotease FtsH coding gene 2HORVU5Hr1G063340. FtsH is the major thylakoid membrane protease required for photosynthetic pathways in plants (Kato and Sakamoto, 2018). This gene was recently shown to be relatively highly expressed in all grain organs suggesting its crucial role in the accumulation of the micronutrient metals (i.e., Cu, Fe, Mn, and Zn) in barley grains (Thabet et al., 2022). It may be speculated, that FtsH represents another component of the mechanisms activated to limit photosynthetic activity to increase pathogen resistance. However, despite the upregulation observed during infection and wounding ( Table 3 ), the involvement of FtsH in improved resistance against P. hordei after AHL-priming needs further molecular validation. Two other markers on chromosome 5H (chr5H_5584131EC, chr5H_5584216EC) cluster in QTL 11 with LOD 4.3 and 4.35, respectively. Both are located in the same gene HORVU5Hr1G001990, which encodes for a laccase. Laccases constitute a multi-gene family of multi-copper glycoproteins and have diverse and overlapping physiological functions in plants, including involvement in redox metabolism, responses to wound healing, defense against pathogens or insects, synthesis of lignin and suberin, and cross-linking of cell wall components; cf. Janusz et al. (2020) and Tomková et al. (2012). While several laccases have been annotated in the barley genome on all chromosomes recently (https://plants.ensembl.org/Hordeum_vulgare/Search/Results?page=3;q=laccase;species=Hordeum_vulgare;collection=all;site=ensembl), only HvLac1 on chromosome 4H has been characterized in detail so far with unclear molecular function (Tomková et al., 2012). However, we suggest that the laccase, identified in our study is likely involved in cell wall fortification representing one of the processes leading to increased resistance to P. hordei by AHL priming. Consistently (Shrestha et al., 2019) showed that the principal mechanism of AHL-induced priming in barley seems similar to the mechanism in Arabidopsis, i.e., it is associated with the activation of MAPKs, enhanced expression of various defense-related genes and of genes involved in the remodeling of cell wall structure. Additionally, laccases were also identified among a group of genes that were associated with Fusarium graminearum resistance in barley (Huang et al., 2016). In the same line, the two markers identified on chromosome 3H (chr3H_15470342EC, chr3H_15470343EC, QTL7, LOD 4.84), which are both localized in the gene (HORVU3Hr1G006440) annotated as a xylanase inhibitor, represent interesting candidates with potential function in the fortification and remodeling of cell wall structure. This gene was also found to be upregulated after infection and wounding in barley ( Table 3 ). Most cereals contain arabinoxylan as a structural component in their cell walls (Henry, 1985). As xylanases have a general role in the depolymerization of plant and fungal cell walls (Hrmova et al., 1997), the xylanase inhibitor identified in our study could help to reduce or overcome the effect of plant and fungal related xylanases during infection with P. hordei and thus promote resistance. A marker with high potential in translation regulatory processes was identified on chromosome H2 (chr2H_44543906, LOD 4.13) localized in the gene HORVU2Hr1G017720, which is annotated as WRKY transcription factor 4. The WRKY transcription factor gene family is one of the largest, and it is known to be involved in a wide range of plant developmental and physiological processes. Although, more than 60 unique barley genes have been annotated containing the WRKY domain, WRKY proteins of barley are not yet fully annotated and most of them are not functionally characterized (Liu et al. (2014), http://www.barleygfdb.com/second/family.php?xu=77). The data of Dey et al. (2014) suggest that bacteria-induced systemic immunity in barley is associated with the local and/or systemic induction of transcript accumulation of Ethylene Responsive Factor (ERF)-like transcription factors (HvERF-like, HvERF44411) as well as HvWRKY22 and HvWRKY38/1. Similarly, Gao et al. (2018) proposed Nonexpressor of Pathogenesis-Related Genes1 (NPR1) homologs and WRKY transcription factors as the master regulators of systemic acquired resistance in wheat and barley. Furthermore, they showed that transient expression of HvWRKY6, HvWRKY40, and HvWRKY70, in wheat leaves by Agrobacterium-mediated infiltration enhanced the resistance to Puccinia triticina. In another study, HvWRKY10, HvWRKY19, and HvWRKY28 were shown to positively regulate the response of barley to B. graminis infection (Meng and Wise, 2012). On the other hand, the wound and pathogen-inducible HvWRKY1 and HvWRKY2 are known as negative defense regulators repressing the activity of the powdery mildew-induced promoter of HvGER4c, a germin-like defense-related protein (Liu et al., 2014). However, WRKY transcription factor 4 (HORVU2Hr1G017720) was not functionally characterized yet, despite the induced expression of HORVU2Hr1G017720 after fungal infection and wounding ( Table 3 , http://www.barleygfdb.com/second/PRJNA728113.php?xu=77). It also needs to be considered that WRKY functions may be genotype specific, such as shown for WRKY transcription factors in barley cultivars infected with Fusarium culmorum (Uluhan et al., 2019).

In conclusion, our results provided clear evidence for the improved resistance of barley against P. hordei infection by AHL-mediated priming. Priming responses were genotype specific and most significant genetic markers for improved resistance to P. hordei after priming identified in our study were related to limiting photosynthetic activity, cell wall fortification, and regulation of transcription and translation. Our findings are important, as they open doors to study the mechanisms and the interactions between the plant genetic background and AHL-priming as well as supporting novel breeding approaches for priming efficient accessions and thus sustainable crop protection after validation.

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

The initial research idea was developed by FO and GW. The study conception and experimental design was established by AS and GW. Material preparation and data collection was performed by GW, and data analysis was performed by GW and AM. The first draft of the manuscript was written by GW, amended by AM, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.