Gene ontology (GO) analysis based on differential expression

To look for common pathways that are activated by inoculation in all plants, we carried out GO enrichment analysis of the differentially expressed genes. The different responses by the genotypes were visible also at the level of activated pathways (Figure 3; Supplementary Figure 2; Supplementary Table 3). This may be due to differences in plant defense responses or manipulation of the plant defense mechanisms by the pathogen.
To explore molecular underpinnings between susceptible and resistant genotypes we searched for differential activation of defense response pathways by identifying the GOs with decreased average expression levels in susceptible phenotypes and elevated levels in resistant phenotypes. In resistant phenotypes, genes encoding photosynthesis related proteins and NAD(P)H dehydrogenase complex had increased transcript levels. This could contribute in defense against the pathogen, as it has been shown that photosynthesis plays an important role in plant defense against biotic stress (Gohre, 2015). Genes assigned to photosynthesis functions showed elevated transcript levels in susceptible phenotypes as well but not to the same extent. Chlorosis is a hallmark sign of powdery mildew infection and biotrophic fungi are known to reduce photosynthetic rate and possibly damage chloroplast structure (Perez-Bueno, Pineda, & Baron, 2019), thus the upregulation could be either compensation, plant defense mechanism or induced by pathogen. In addition, uroporphyrinogen decarboxylase activity (GO:0004853) was upregulated in resistant phenotypes. Involved in chlorophyll biosynthesis, it also points towards acting against the chlorosis induced by the pathogen (Mock, Keetman, Kruse, Rank, & Grimm, 1998).
Among both susceptible phenotypes, the GO category with most decreased expression levels was induction of programmed cell death (GO:0012502), suggesting that as a biotrophic pathogen, P. plantaginis may have disabled the programmed cell death and is keeping the host cells alive. However, also the resistant phenotypes showed reduced expression levels, possibly due to successful manipulation by the pathogen, and therefore the comparison between susceptible versus resistant did not identify this process as significantly different between phenotypes (P=0.0559).
In addition to the shared responses, the genotypes showed individual enrichment of various disease resistance pathways. In susceptible genotype 1 (S1), the processes with most decreased average expression levels were tripeptide transporter activity (GO:0042937), tripeptide transport (GO:0042939) and delta12−fatty acid dehydrogenase activity (GO:0016720), whereas S2 demonstrated decrease in Oxazole or thiazole biosynthetic process (GO: 0018131) and Low−affinity nitrate transport (GO:0080054 & GO:0080055). Fatty acids play a direct role in modulating the plant defense response to pathogens (Kachroo & Kachroo, 2009), and thiazole or thiamine has been shown to play a crucial role in activation of the defense responses, callose/lignin deposition and stomatal closure (Zhou, Sun, & Xing, 2013).
Tripeptide transport includes also nitrate transporters. Interestingly, powdery mildew causative agent Erysiphe necator elevates the expression levels of nitrate transporters in grapevine and Arabidopsis (Pike et al., 2014), possibly to acquire nutrients from the host. In addition to decreased levels of the GO categories related to nitrate transport in both S1 and S2, we identified homolog of Arabidopsis nitrate transporter (AtNRT1.5) to be upregulated after inoculation in susceptible vs resistant comparison. In Arabidopsis, the protein is responsible for nitrate transport from roots to shoots, and in this context suggests towards manipulation of host nutrient distribution by the pathogen. Mur, Simpson, Kumari, Gupta, and Gupta (2017) have argued that nitrogen and nitrates and their transportation to different tissues in the plant during the pathogen infection could be the “silver bullet” of the plant defense. Besides nitrate transport, tripeptide transport activities also play an important role for defense against biotic and abiotic stress (Karim et al., 2007), suggesting a reason for the decreased expression of the tripeptide transporters as a whole in the susceptible phenotypes.
In resistant phenotypes, the glucosyltransferase (GO:0050284) upregulation in R1 is a sign of early preparation for pathogen response (Le Roy, Huss, Creach, Hawkins, & Neutelings, 2016), and in R2 genotype, the activation of NADH dehydrogenase complex assembly (GO:0010258) has been shown to be involved in defense signaling (Wallstrom et al., 2014).