Here we demonstrate broad and robust resistance-inducing activity of the currently best-studied and most active ascaroside, ascr#18, using a representative set of wheat cultivars and two Puccinia triticina races with very different virulence spectra. Recording Pt infection on infected leaves of wheat seedlings showed that spray-pretreatment of seedlings with ascr#18 significantly reduced the number and size of uredinia as compared to mock-pretreated Pt inoculated plants. In the few cases, small Pt uredinia could be detected in ascr#18-treated leaves, normal germ-tube elongation and branching of germinating spores were observed and fungal hyphae in the mesophyll close to the uredinia were seen (not shown). A dose-response analysis over the nano- and micromolar range revealed a broad optimum concentration for the control of Pt indicating that it is very potent. Moreover, microscopic analysis showed very early arrest of the fungus at the prehaustorial stage, with associated local accumulation of DAB-positive H2O2 at attacked stomata and abortive stoma penetration. Of note, no HR of epidermal of mesophyll cells could be detected at site of attempted penetration.
The morphology of infection structures of rust fungi and prehaustorial resistance in wheat and barley are well characterized (Bolton et a. 2008). The prehaustorial resistance against Pt found in the diploid wheat Einkorn (Triticum monoccocum) accession PI272560 also is race independent. It governs abortion of fungal development during the formation of haustorial mother cells and to increased hydrogen peroxide concentration in comparison to a susceptible accession (Triticum boeoticum ssp. Thaoudar var. reuteri). Consistent with our finding of a very early oxidative burst (see H2O2 accumulation at attacked stomata, Fig. 4), Serfling et al. (2016) found increased peroxidase activity in PI272560 within 6 hpi. Einkorn is closely related to Triticum urartu as a progenitor of wheat and generally shows a high level of resistance against wheat leaf rust. In contrast to the ascr#18-induced prehaustoral resistance we found in wheat cultivars, Einkorn accession PI272560 in addition to H2O2 accumulation exhibited a hypersensitive prehaustorial effector-triggered immune reaction, preventing the infection of Pt. Remarkably, this effector-triggered immune reaction turned out to be atypical as it is non-race-specific. Interestingly, further genetic dissection of the prehaustorial resistance in PI272560, identified a single gene located 3.4 Mbp from the peak marker within the major QTL that was upregulated (20- to 95-fold) after the inoculation in the resistant accession in comparison to the susceptible T. boeoticum accession. This gene belongs to a berberine bridge enzyme-like protein that is suspected to interact on the plant surface with glycoside hydrolases (GH) secreted by the fungus and to induce a hypersensitive defense reaction in the plant after fungal infections (Deblieck et al. 2023).
Partial resistance in barley to P. hordei was prehaustorial and nonhypersensitive, and its level could be assessed accurately by measuring the latent period of the fungus (Hoogkamp et al. 1998). The same authors showed that resistance to the inappropriate rust species (non-host resistance) is a combination of prehaustorial (nonhypersensitive) and posthaustorial (hypersensitive) mechanisms. The amount of nonhypersensitive, early abortion of P. recondita f. sp. tritici and P. hordei-murini sporelings in barley reflected the degree of prehaustorial defense to the two inappropriate rust species (Hoogkamp et al. 1998).
Previous reports showed the positive effect of ascr#18 in plant protection against a virus (Turnip Crinkle Virus), a bacterium (Pseudomonas syringae pv. tomato), a fungus (e.g. Blumeria graminis f sp. hordei), an oomycete (Phytophthora infestans) and two nematodes (Heterodera schachtii and Meloidogyne incognita) in four plant species (barley, potato, tomato, and Arabidopsis) (Manosalva et al. 2015). In another report, ascr#18 was applied to five crops (wheat, soybean, rice, maize, and tomato) against eight pathogens/pests, including one virus, bacteria, fungi, an oomycete, and a nematode. The results of this study showed that ascr#18 pretreatment provided partial to strong protection in seven of the eight plant-pathogen systems (except maize) (Klessig et al. 2019), suggesting that ascarosides are effective tools that can be used in crop production. Ascarosides consist of a fatty acid-derived side chain attached at the α position to the dideoxysugar ascarylose. Its structure bears some similarity to bacterial rhamnolipids, which consist of the lipophilic fatty acid side chain(s) attached at the α position to one or two rhamnose sugar moieties. The function of rhamnolipids is similar to MAMPs, through inducing cellular influx of Ca2+, generation of reactive oxygen species, activation of mitogen‐activated protein kinases, activation of salicylic acid- and jasmonic acid-mediated defense signaling pathways that stimulate the expression of genes involved in plant immunity (Ludewig and Schroeder 2013; Manohar et al. 2020; Yu et al. 2021).
In conclusion, the use of small-molecule signals, such as ascr#18, enables induction and modulation of different signaling pathways to activate immune responses in plants. Thus, ascr#18 and other small-molecule signals can be used as an alternative method to reduce disease damage and increase sustainable food security.