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).