4.2. Interaction with the necrotrophic seedborne fungi Alternaria brassicicola
Because seeds contribute to reproductive success, it is hypothesis that seed protection mechanisms would be part of their adaptive traits [
24]. At the time of dissemination, the mature seed is already equipped with protection systems [
5,
24] that could contribute to non-host resistance. This could be illustrated by the accumulation in the mature seed of secondary metabolites such as phenolic compounds [
25] and by the development of physical barriers such as the seed coat [
5,
24]. Also, pending seed germination and seedling establishment, these properties enable seeds to maintain their survival when challenged by abiotic and biotic pressures in their environment [
8,
24,
25,
58,
59].
The endosperm acts not only in the control of seed germination but also as a protective barrier during germination [
5,
60]. Genes encoding detoxifying enzymes, such as glutathione S-transferases and peroxidases, play a crucial role in protecting embryo against ROS that could result from pathogen interaction during seed germination. Also, genes related to plant-fungus interactions and hormonal metabolism hold crucial roles in enhancing immunity and regulating plant growth. A transcriptomic study showed an over-accumulation of genes responsible for synthesizing salicylic acid (SA), indole-3-acetic acid (IAA), and amino synthetases (GH3) [
60]. At the transcriptome level, the activation of secondary metabolites and defense response in the endosperm illustrates the activation of defense pathways during germination [
60]. It is quite remarkable that certain mechanisms in seeds seem to differ from those in plants. Nevertheless, there are also instances of similar defense mechanisms being observed. For example, this includes plant defense through polyphenol oxidase (PPO), a group of enzymes that catalyze the oxidation of hydroxy phenols, yielding products with antimicrobial properties. In dormant wild oat (
Avena fatua L.) seeds attacked by
Fusarium avenaceum trigger post-translational activation of the PPO, demonstrating the seed ability to induce enzymatic biochemical defenses against pathogenic fungi. This PPO activity was also found to be induced in non-living caryopsis cover tissues, such as lemma and pale [
25].
The mature seed has developed resistance properties to face biotic and abiotic stress after dispersal. However, the immune responses of seeds to biotic interactions during germination have been little studied, although this step is crucial to control seed-borne pathogen transmission to the new plant [
21,
24]. The model pathosystem
Arabidopsis thaliana/Alternaria brassicicola has been recently used to describe gene regulation induced by the necrotrophic interaction during germination and seedling establishment [
1,
21,
61].
RNAseq data from
Alternaria-infected and healthy
Arabidopsis seeds were compared at different time points of the germination kinetics until the stage of seedling establishment. Although a transcriptomic study carried out on inoculated rosette leaves
23 highlighted an induction by
Alternaria of the jasmonate pathway and camalexin metabolism (
Figure 1b), the response of the germinating seed rather reflected an activation of the SA and ET pathways to the detriment of the jasmonate pathway [
1]. It is documented that this type of unexpected response could constitute a diversion towards inappropriate defense pathways of the plant by the fungus to induce a SR favoring its development [
38,
39]. Additionally, genes relate to response to hypoxia and indole-derived metabolites such as GSL were induced by
Alternaria during germination [
1]. Gene ontology analysis showed a stronger and more significant association among genes related to the response to hypoxia at an early development stage. Their fold enrichment decreased as seedling establishment progressed (3 Days After Sowing (DAS): 7.08, 6 DAS: 4.29 and 10 DAS: 3.07) [
1]. This response is surprising, but it illustrates a competition for oxygen which benefits the physiology of the early imbibed seed. Seed germination is well adapted to low oxygen levels [
62] while these low oxygen levels are limiting the growth of the fungus. Either the induction at the RNA level of indole GSL metabolism that was observed in
Arabidopsis seeds [
1] is well described [
63].
Phenotyping analyses of mutant seeds deficient for the identified pathways showed that the mutants deficient for ethylene response
ein2 and
etr1 exhibited a lower rate of necrosis, respectively 21.9% and 55.4% lower than the WT control. Also, GSL-deficient mutants (
qk0 and
gtr1gtr2) exhibited a lower rate of necrosis, respectively 65.9% and 73.6% lower than those in the control group. Noticeably, the GSL-deficient mutants displayed also a reduced
Alternaria overgrowth [
1], indicating that the lack of necrosis, usually mediated by
Alternaria-induced GSL pathway in WT, would be limiting for the colonization by the necrotrophic fungus. In this context, the induction of GSL metabolism could also contribute to the SR of the infected seed. The identified changes in gene regulation induced by
Alternaria in germinating seed provide an original working model illustrating a simultaneous existence of SR and competition strategy (
Figure 2).
This study reinforces the hypothesis that the interaction of the seed with the pathogen differs from the models described for whole plants. It is notable that the transcriptome of infected tissues differed drastically between 6-day-old seedlings and 10-day-old seedlings [
1,
61]. As an additional illustration, necrosis symptoms (
Figure 3) in newly germinated seedlings (4 days after sowing) show much more delineated patterns than in older seedlings (after 10 days), where necrosis is more diffuse and widespread in the infected tissue.
We hypothesize that at the germinative stage, the young seedling reacts to infection with a HR whose profile is generally well circumscribed. The fact that GLS-deficient mutants did not show any necrosis symptoms at the young seedling stage (4 days after sowing) suggests that the observed HR would be mediated by activation of the plant's GSL pathway, whereas after 6 and 10 days of development, new necrosis were observed, including in the GSL mutants. The diffused appearance of the latter would result from an effect of the fungus as it penetrates the plant tissue.