Gene ontology (GO) analysis based on Redundancy Analysis (RDA)

We identified the GO categories contributing to the differences in RDA analyses by averaging the RDA loadings of the genes in each of the categories, and tested whether the values significantly differ from random scores obtained by permutation. Genes contributing to the separation between inoculation and control were enriched for ABA and cytokine signaling, primary metabolism and chloroplast activity. ABA induces resistance to powdery mildew in barley (Wiese, Kranz, & Schubert, 2004), and repression of ABA biosynthesis as well as genes regulated by ABA, such as cold/dehydration/salinity responsive genes, are associated with mildew resistance in nonhost plants in general (Jensen et al., 2008). Cytokinin suppresses programmed cell death and plays a role in the synthesis and maintenance of chlorophyll (Walters & McRoberts, 2006); both processes were enriched among the differentially expressed genes. Additionally, cytokinin levels regulate cell division together with auxin. Interestingly, in Arabidopsis, Golovinomyces orontii inoculation induced cell cycle related genes and endoreduplication, possibly due to increased metabolic demands of the pathogen (Chandran, Inada, Hather, Kleindt, & Wildermuth, 2010). On the other hand, Choi, Choi, Lee, Ryu, and Hwang (2011) have shown that plant based cytokinins systematically induce plant resistance against pathogens by cytokinin and salicylic acid signaling.
Genes associated with the differences between the phenotypes showed GO enrichments for kinase activity, carbohydrate metabolism, plant cell wall organization, photosystem II and response to cold GO categories, whereas the genes contributing to the differences between genotypes were enriched for tryptophan metabolism, plant cell wall and chloroplast (Figure 4a and Figure 2d/e; Supplementary Figure 3). In Arabidopsis (Chandran et al., 2010), the expression of cold/drought responsive genes were decreased together with ABA biosynthesis after inoculation withG. orontii . Together with the observed induction of ABA during inoculation, this suggests that the phenotypes may differ in how strongly ABA activates its targets such as cold responsive genes.
The responses to infection start to play a role in genotype-by-experiment effect where the genes are mainly enriched for immune and defense responses, illustrating that the genes in these processes highly differ by the transcription levels between genotypes. Overall, the enriched GOs show a clear activation of defense responses in general, and defense responses to fungi in particular. The GO category with highest positive average of RDA loadings (and therefore, high contribution to separation) is aldose 1−epimerase activity (GO:0004034) which, based on Sheshukova et al. (2017), is activated because of the mechanical damage inflicted by the pathogen and results in methanol emission and priming of the non-infected leaves. The presence of hydrogen peroxide metabolic process and salicylic acid mediated signaling pathway are both well-established pathogen-induced defense mechanisms (Hua, 2009; Kuniak & Urbanek, 2000; Niu & Liao, 2016; Sheshukova et al., 2017), further demonstrating the activation of the defense processes due to the pathogen infection. The GO category with most negative average RDA loadings is RNA splicing, via endonucleolytic cleavage and ligation (GO:0000394). An emerging field in plant molecular biology, it is becoming increasingly clear that plants use alternative RNA splicing extensively as a means to respond to their environment and defend against pathogens (Shang, Cao, & Ma, 2017; Staiger, Korneli, Lummer, & Navarro, 2013). Within the signaling-specific GOs (Figure 4b) the genotype by experiment effect showed the activation of jasmonic acid (JA) and abscisic acid signaling (as expected, (Yang et al., 2019)), but again, in a genotype-specific manner. Further inspection of putative orthologs of marker genes for different hormonal signaling pathways showed activation of auxin biosynthesis and signaling, as well as differences in the activation of JA signaling and NLR signaling through EDS1 ortholog (Supplementary Table 4). In R1, specific induction of the SA biosynthesis and systemic acquired resistance was also detected.
The most significant contributor to phenotype-by-experiment is photosystem II activity. The GO category with highest average RDA loadings for phenotype by experiment effect is oligopeptide transmembrane transporter activity (GO:0035673). The perception and transduction of fungal oligopeptides will trigger multiple defense responses (Hahlbrock et al., 1995; Nürnberger et al., 1994). Multitude of photosynthetic processes were also enriched; their role in defense was discussed above. The categories with most negative average loadings were response to fungus, and cytokinin biosynthetic process (GO:0009691).