Over 30 years ago branched β-1→3-glucans and the EP – AA and EPA – were characterized as potent oomycete elicitors of innate immune responses in plants. These and the Phytophthora elic itin proteins with activities in a somewhat narrower host range figured prominently in the literature in subsequent years, and were used to examine physiological, biochemical and molecular events associated with the HR and induced resistance. Intriguing is that β-glucans and EP are important in modulating innate immunity and inflammation in animals, although these cross-kingdom parallels are likely not fully appreciated by the plant and animal research communities. Oomycetes are among the most important plant pathogens, responsible for devastating plant diseases worldwide. New Phytophthora species, in particular, are continually being discovered, with the number of species identified nearly double that of only a decade ago . Downy mildew pathogens and the diseases they cause are also current threats to U.S. and world agriculture, with two listed as Select Agents as serious threats to U.S. agriculture . The Phytophthora research community is attuned to the need and urgency to develop novel control strategies that are broadly applicable yet sustainable, with vigorous research programs studying population genetics, genomics, effector biology, host resistance, and disease epidemiology and management. Within this research portfolio, determining how β-glucans and EP are perceived and act in plants could be useful for enhancing disease resistance against oomycetes and possibly other attackers. In this review, we highlight early studies of β-1→3- glucans and EP, discuss their roles as evolutionarily conserved signals,strawberry grow pot and consider their action in relation to current models of MAMP1-triggered immunity.

Arachidonic acid and eicosapentaenoic acid are 20-carbon, all-cis PUFAs containing four and five double bonds, respectively . In mammals, AA and EPA undergo enzymatic oxidation to oxylipins, referred to as eicosanoids, which serve crucial signaling functions in stress responses . Examples of these eicosanoids include prostaglandins and thromboxanes, formed via the action of cyclooxygenases, and leukotrienes, formed via the action of LOXs. Eicosanoid-mediated stress responses include pain, inflammation and fever , platelet aggregation and vasoconstriction , and allergic responses and asthma . Although higher plants do not contain AA and EPA, AA and EPA are found in oomycete pathogens and plants are exposed to these fatty acids during infection . Many molecules of microbial pathogens identified as elicitors in earlier studies have been reclassified as MAMPS to conform to terminology used in animal immunity. MAMPs are motifs in essential molecules such as proteins, lipids, and polysaccha rides that are present in entire classes of microbes . These molecular motifs are generally absent from hosts and can be recognized by plants and animals, such as in response to attempted infection or colonization. Studies of PTI have focused on the bacterial pep tides flagellin and EF-Tu and their action in Arabidopsis. These peptides are perceived by PRRs, receptor-like kinases that are crucial for perception of flagellin/EF-Tu and activation of PTI. However, unlike flagellin and EF-Tu, many of the historical elicitors that stimulate well-characterized defense responses in plants have not been sufficiently investigated to resolve their modes of action . The elicitors AA and EPA conform to the definition of MAMPs: they are not present in higher plants, are essential components in oomycete cells, are largely absent from other classes of microbes, and elicit similar defense responses in plant species where they have been studied . Eicosapolyenoic acid elicitor activity in plants was first dis covered in the interaction between Phytophthora infestans and potato. Mycelial extracts of P. infestans induced sesquiterpenoid phytoalexins, lignin deposition and cell death in potato tissue in a reaction similar to a HR to incompatible races of the pathogen. Purification and analysis of all active fractions in these extracts identified AA and EPA, without exception, either free or esterified to other molecules .

Elicitation was specific to AA and EPA. Treatment with 15 other fatty acids, including LA and ALA , the primary unsaturated fatty acids found in higher plants , as well as structurally similar eicosatrienoic acid and arachidonyl alcohol, did not elicit defense responses. Treatment of tuber disks with AA also protected them from subsequent P. infestans infection .Eicosapolyenoic acids induce systemic resistance in potato as well as in other plant species to various pathogens. Although the mechanisms remain unresolved, EP have been shown to elicit SA, JA, and ET in different experimental systems. Colonization of avocado seedling roots by P. cinnamomi was reduced in roots treated with AA prior to inoculation . Pearl millet seedlings were protected to a greater degree against infection by the downy mildew pathogen, Sclerospora graminicola, following seed treatment with AA or EPA, in contrast to seedlings emerging from seeds treated with LA, ALA, DHA or water . EP elicit SAR or SAR-like responses in tobacco, potato, and tomato. Treatment of lower leaves of tobacco plants with AA induced local and SAR to TMV . EP treatment of the lower leaves of potato plants protected the upper leaves from infection by P. infestans, a systemic resistance that developed within 5 days of the inducing treatment . Plants treated with LA, ALA, oroleic acid displayed partial protection but not to the level of EP-treated plants. AA also induced resistance in potato leaves to the early blight pathogen, Alternaria solani, with levels of SA and a PR1-like protein elevated in the AA-treated leaves . AA-treatment of tomato leaves induced localized accumulation of transcripts for P4 , a PR-1 family member and SAR marker in tomato , but did not induce expression of the proteinase inhibitor gene PI-2. The latter is strongly induced by wounding and JA treatment and serves as a markerfor JA-mediated resistance in tomato . Although the studies in tobacco, potato,hydroponic grow table and tomato indicate that EP-induced resistance may operate through SA, recent research suggests EP action is more complex . Treatment of tomato and Arabidopsis leaves with AA increased JA levels, reduced SA levels, and increased resistance to Botrytis cinerea.

Arabidopsis plants transformed to produce small amounts of EPs were less susceptible to P. capsici, B. cinerea, and feeding by aphids. However, these plants were more susceptible to Pseudomonas syringae pv. tomato . The EP plants had constitutively elevated levels of JA and JA marker gene expression and reduced levels of SA and SA-marker gene expression relative to wild-type plants. The differential effect of EP on disease and pest outcomes corresponds to EP’s impact on SA and JA defense signaling, and this effect is dependent upon JA as demonstrated with a JA-deficient aos mutant line. Salicylic acid and JA can be mutually antagonistic , making it difficult to reconcile these different findings. AA treatment elicits ET production in both pepper and potato , and ET can modulate SA- and JA-defense networks . The different experimental outcomes may result in part from differences in EP concentrations used in the various studies. Higher concentrations of EP can induce an intense, localized necrosis at the site of application, particularly in solanaceous plants. This strong phenotype could trigger or result from phytohormone changes different from those induced by low con centrations. Also, it is possible that all three phytohormones are important in establishing EP-induced resistance through a process of transitional signaling . A study in potato indicates that both SA and JA are important in PTI responses , and a study of PTI in Arabidopsis using signal allocation analysis of mutants deficient in ET, SA, and JA signaling indicated that PTI depends on synergy among ET, SA, and JA . Further research is needed to fully elucidate the interactions among SA, JA, and ET in their involvement in EP-induced resistance and defense responses.Eicosapolyenoic acids have been useful in dissecting aspects of secondary metabolism in plants, with a focus on sesquiterpenoid phytoalexins in solanaceous plants. However, EP elicits produc tion of defense metabolites in other plant families as well. The isoflavanoid phytoalexins phaseollin and coumestrol accumulate in leaves of French bean following infiltration with AA . Phenol-2,4-bis , a defense compound in avocado, is induced in roots treated with AA as well as with SA . Among solanaceous plants EP elicit sesquiterpenoid phytoalexin synthesis in thorn apple, eggplant, chili pepper, green pepper, potato, and tomato . In potato tuber, AA elicits sesquiterpenoid phytoalexin biosynthesis with strong expression of sesquiterpene cyclase, a committed step in the pathway. Concurrent with this is a complete suppression of wound-induced squalene synthase and steroid glycoalkaloid accumulation . HMGR catalyzes the first step in the synthesis of stress-induced isoprenoids from mevalonate in potato. Three isoforms of HMGR are differentially induced by wounding and AA treatment , and a similar expression pattern of the corresponding HMGR isoforms occurs in tomato .In addition to potato, EP have been shown to elicit PCD, characteristic of the HR, in other plant species.

Pearl millet seedlings treated with AA displayed a HR similar to that induced by the oomycete, S. graminicola, the causal agent of downy mildew. Following treat ment with AA, the HR developed more quickly in pearl millet seedlings with genotypes rated as resistant versus susceptible to S. graminicola . Tomato protoplasts treated with AA underwent PCD with characteristic DNA fragmentation and laddering, while LA and ALA treatment had no PCD-inducing effects . In both potato and pepper, AA was found to induce ROS in a similar manner. AA treatment of potato tuber disks elicited a biphasic oxidative burst peaking at 1 and 6–9 h after treatment and increased expression of StRBOHB, a homolog of gp91, which encodes a subunit within the neutrophil NADPH oxidase complex . As in potato, treatment of pepper fruit with AA elicited an immediate, rapid ROS burst. When DPI, an inhibitor of NADPH-dependent oxidases, was applied to the fruit prior to application of AA, ROS generation decreased as the concentration of DPI was increased .The mode of action of EP in PTI is unresolved, although the structural requirements of EP as elicitors are well characterized. These include at least a 20 carbon backbone with all cis-1,4-pentadiene unsaturation beginning at the 5 position and at least four dou ble bonds in the chain . While this specificity could pro vide evidence for involvement of a receptor that recognizes these structural features, previous studies of EP in potato indicate that initial perception by plant cells may be quite different than other MAMPs. Initial recognition of AA and EPA may occur by specific disruption of host membrane integrity and/or perturbation of oxylipin metabolism, with the possibility that plant cells produce novel oxylipins from EP . Studies in potato showed that U-14C radiolabeled AA was quickly incorporated into neutral lipids and polar lipids . A small fraction, ∼2–5% of the AA, was oxidized . Also, sporangia of P. infestans readily incorporated exogenous 14C-AA into phospholipids , diglycerides and TGs. By 12–14 h after inoculation, microautoradiographic studies revealed that the radioactivity from sporangia was released into the epidermal and palisade mesophyll cells adaxial to the inoculated leaf surface and distant from fungal structures . Plant phospholipases are activated following attack by pathogens or treatment of plants with elicitors . This could create an opportunity for any EP incorporated into plant lipids during infection to be released and accessible to plant oxylipin enzymes. Research in potato and tomato indicates that the 9-LOX path way may play an important role in EP action. The first step in the enzymatic formation of phyto-oxylipins involves the action of LOX . Plant LOXs act on PUFA containing a cis– pentadiene system, inserting an oxygen molecule to form hydroperoxy fatty acids. These are further metabolized to various oxylipin families by members of CYP74 cytochrome P450s: AOSs, HPLs, and DESs, or by less well-characterized POX or PXG and EASs . The importance of LOX, in particular a 9-LOX2, in EP elicitor activity is supported by fatty acid structure-activity requirements and studies of LOX expression. The carboxyl function of EP is critical, a feature consistent with the substrate requirement of plant LOXs . A 5 double bond at the beginning of a methylene-interrupted series with at least four double bonds provides the highest elicitor activity . AA stimulates LOX expression in potato and tomato , with 5-HPETE a principal LOX product formed after treatment of tissue with AA .