Differential induction of oxylipin pathway in potato and tobacco cells by bacterial and oomycete elicitors

Key message

Potato and tobacco cells are differentially suited to study oxylipin pathway and elicitor-induced responses.

Abstract

Synthesis of oxylipins via the lipoxygenase (LOX) pathway provides plant cells with an important class of signaling molecules, related to plant stress responses and innate immunity. The aim of this study was to evaluate the induction of LOX pathway in tobacco and potato cells induced by a concentrated culture filtrate (CCF) from Phytophthora infestans and lipopolysaccharide (LPS) from Pectobacterium atrosepticum. Oxylipin activation was evaluated by the measurement of LOX activity and metabolite quantification. The basal levels of oxylipins and fatty acids showed that potato cells contained higher amounts of linoleic (LA), linolenic (LnA) and stearic acids than tobacco cells. The major oxylipin in potato cells, 9(S),10(S),11(R)-trihydroxy-12(Z),15(Z)-octadecadienoic acid (9,10,11-THOD), was not detected in tobacco cells. CCF induced a sharp increase of LA and LnA at 8 h in tobacco cells. In contrast they decreased in potato cells. In CCF-treated tobacco cells, colneleic acid increased up to 24 h, colnelenic acid and 9(S)-hydroxyoctadecatrienoic acid (9(S)-HOT) increased up to 16 h. In potato cells, only colneleic acid increased slightly until 16 h. A differential induction of LOX activity was measured in both cells treated by CCF. With LPS treatment, only 9,10,11-THOD accumulation was significantly induced at 16 h in potato cells. Fatty acids were constant in tobacco but decreased in potato cells over the studied time period. These results showed that the two elicitors were differently perceived by the two Solanaceae and that oxylipin pathway is strongly induced in tobacco with the CCF. They also revealed that elicitor-induced responses depended on both cell culture and elicitor.     

Phosphatidic acid production in chitosan-elicited tomato cells, via both phospholipase D and phospholipase C/diacylglycerol kinase, requires nitric oxide

Nitric oxide (NO) and the lipid second messenger phosphatidic acid (PA) are involved in plant defense responses during plant-pathogen interactions. NO has been shown to be involved in the induction of PA production in response to the pathogen associated molecular pattern (PAMP) xylanase in tomato cells. It was shown that NO is critical for PA production induced via phospholipase C (PLC) in concerted action with diacylglycerol kinase (DGK) but not for the xylanase-induced PA via phospholipase D (PLD). In order to study whether this is a general phenomenon during PAMP perception or if it is particular for xylanase, we studied the effect of the PAMP chitosan in tomato cell suspensions. We observed a rapid NO production in tomato cells treated with chitosan. Chitosan induced the formation of PA by activating both PLD and PLC/DGK. The activation of either phospholipase-mediated signaling pathway was inhibited in cells treated with the NO scavenger cPTIO. This indicates that NO is required for PA generation via both the PLD and PLC/DGK pathway during plant defense response in chitosan elicited cells. Responses downstream PA were studied. PLC inhibitors neomycin and U73122 inhibited chitosan-induced ROS production. Differences between xylanase and chitosan-induced phospholipid signaling pathways are discussed.     

A role in immunity for Arabidopsis cysteine protease RD21, the ortholog of the tomato immune protease C14

Secreted papain-like Cys proteases are important players in plant immunity. We previously reported that the C14 protease of tomato is targeted by cystatin-like EPIC proteins that are secreted by the oomycete pathogen Phytophthora infestans (Pinf) during infection. C14 has been under diversifying selection in wild potato species coevolving with Pinf and reduced C14 levels result in enhanced susceptibility for Pinf. Here, we investigated the role C14-EPIC-like interactions in the natural pathosystem of Arabidopsis with the oomycete pathogen Hyaloperonospora arabidopsidis (Hpa). In contrast to the Pinf-solanaceae pathosystem, the C14 orthologous protease of Arabidopsis, RD21, does not evolve under diversifying selection in Arabidopsis, and rd21 null mutants do not show phenotypes upon compatible and incompatible Hpa interactions, despite the evident lack of a major leaf protease. Hpa isolates express highly conserved EPIC-like proteins during infections, but it is unknown if these HpaEPICs can inhibit RD21 and one of these HpaEPICs even lacks the canonical cystatin motifs. The rd21 mutants are unaffected in compatible and incompatible interactions with Pseudomonas syringae pv. tomato, but are significantly more susceptible for the necrotrophic fungal pathogen Botrytis cinerea, demonstrating that RD21 provides immunity to a necrotrophic pathogen.     

Beta-1,3 glucan sulfate, but not beta-1,3 glucan, induces the salicylic acid signaling pathway in tobacco and Arabidopsis

Sulfate substituents naturally occurring in biomolecules, such as oligosaccharides and polysaccharides, can play a critical role in major physiological functions in plants and animals. We show that laminarin, a beta-1,3 glucan with elicitor activity in tobacco (Nicotiana tabacum), becomes, after chemical sulfation, an inducer of the salicylic acid (SA) signaling pathway in tobacco and Arabidopsis thaliana. In tobacco cell suspensions, the oxidative burst induced by the laminarin sulfate PS3 was Ca2+ dependent but partially kinase independent, whereas laminarin triggered a strickly kinase-dependent oxidative burst. Cells treated with PS3 or laminarin remained fully responsive to a second application of laminarin or PS3, respectively, suggesting two distinct perception systems. In tobacco leaves, PS3, but not laminarin, caused electrolyte leakage and triggered scopoletin and SA accumulation. Expression of different families of Pathogenesis-Related (PR) proteins was analyzed in wild-type and mutant tobacco as well as in Arabidopsis. Laminarin induced expression of ethylene-dependent PR proteins, whereas PS3 triggered expression of ethylene- and SA-dependent PR proteins. In Arabidopsis, PS3-induced PR1 expression was also NPR1 (for nonexpressor of PR genes1) dependent. Structure-activity analysis revealed that (1) a minimum chain length is essential for biological activity of unsulfated as well as sulfated laminarin, (2) the sulfate residues are essential and cannot be replaced by other anionic groups, and (3) moderately sulfated beta-1,3 glucans are active. In tobacco, PS3 and curdlan sulfate induced immunity against Tobacco mosaic virus infection, whereas laminarin induced only a weak resistance. The results open new routes to work out new molecules suitable for crop protection.     

Activation of defence reactions in Solanaceae: where is the specificity?

When a potential pathogen attempts to infect a plant, biochemical and molecular communication takes place and leads to the induction of plant defence mechanisms. In the case of efficient defence, visible symptoms are restricted and the pathogen does not multiply (incompatible interaction); when defence is inefficient, the plant becomes rapidly infected (compatible interaction). During the last 30 years, a growing body of knowledge on plant-pathogen interactions has been gathered, and a large number of studies investigate the induction of various plant defence reactions by pathogens or by pathogen-derived compounds. However, as most papers focus on incompatible interactions, there is still a lack of understanding about the similarities and differences between compatible and incompatible situations. This review targets the question of specificity in Solanaceae-pathogen interactions, by comparing defence patterns in plants challenged with virulent or avirulent pathogens (or with pathogen-associated molecular patterns from these). A special emphasis is made on analysing whether defence reactions in Solanaceae depend primarily on the type of elicitor, on the plant genotype/species, or on the type of interaction (compatible or incompatible).     

The jasmonate pathway is involved differentially in the regulation of different defence responses in tobacco cells

Jasmonic acid, a product of the lipoxygenase (LOX) pathway, has been proposed to be a signal transducer of defence reactions in plants. We have reported previously that methyl jasmonate (MJ) induced accumulation of proteinase inhibitors in tobacco cell suspensions (Rickauer et al., 1992, Plant Physiol Biochem 30: 579–584). The role of this compound in the induction of this and of other defence reactions is further studied in this paper. Treatment of tobacco cell suspensions with an elicitor from Phytophthora parasitica var. nicotianae induced a rapid and transient increase in jasmonic acid levels, which was abolished when cells were preincubated with eicosatetraynoic acid (ETYA), an inhibitor of LOX. Pretreatment with ETYA also inhibited the induction of proteinase inhibitors by fungal elicitor, but not by MJ. Linolenic acid, a precursor of jasmonate biosynthesis, induced this defence response, whereas linoleic acid had no effect. Expression of defence-related genes encoding proteinase inhibitor II, hydroxyproline-rich or glycine-rich glycoproteins, glucanase and chitinase, was induced in a basically similar manner by fungal elicitor or MJ. However, ETYA did not inhibit, or only partially inhibited, the elicitation of these defence genes. Expression of the sesquiterpene cyclase (5-epi-aristolochene synthase) gene was not induced by MJ, but only by fungal elicitor, and ETYA pretreatment had no effect on this induction. The obtained results indicate that synthesis of jasmonate via the LOX pathway seems to be only part of a complex regulatory mechanism for the onset of many, but not all, defence reactions. Received: 4 July 1996 / Accepted: 23 November 1996     

Regulation of tobacco lipoxygenase by methyl jasmonate and fatty acids

Lipoxygenase (LOX) activity and gene expression have been described previously to be induced in tobacco by fungal infection and elicitor treatment. We now report that LOX activity is induced in tobacco cell suspensions by treatment with methyl jasmonate (MeJa). This compound had no effect on the in vitro activity of tobacco LOX. Induction of LOX activity is a dose-dependent response with a maximum around 890 μM MeJa. Linolenic acid, the precursor for jasmonate synthesis, also induces LOX activity. When applied together with fungal elicitor, linolenic acid drastically increases and prolongs the induction of LOX activity. LOX activity and gene expression in elicited tobacco cells are partially inhibited by pretreatment with eicosatetraynoic acid (ETYA), a potent inhibitor of tobacco LOX in vitro. The induction by methyl jasmonate, in contrast, was not inhibited by ETYA pretreatment. These data suggest that induction of LOX gene expression and activity upon elicitation are regulated at least partially by LOX products. © Académie des Sciences/ Elsevier, Paris     

Molecular and biochemical basis of the interaction between tomato and its fungal pathogen Cladosporium fulvum

The interaction between the biotrophic fungal pathogen Cladosporium fulvum and tomato complies with the genefor-gene model. Resistance, expressed as a hypersensitive response (HR) followed by other defence responses, is based on recognition of products of avirulence genes from C. fulvum (race-specific elicitors) by receptors (putative products of resistance genes) in the host plant tomato. The AVR9 elicitor is a 28 amino acid (aa) peptide and the AVR4 elicitor a 106 aa peptide which both induce HR in tomato plants carrying the complementary resistance genes Cf9 and Cf4, respectively. The 3-D structure of the AVR9 peptide, as determined by 1H NMR, revealed that AVR9 belongs to a family of peptides with a cystine knot motif. This motif occurs in channel blockers, peptidase inhibitors and growth factors. The Cf9 resistance gene encodes a membrane-anchored extracellular glycoprotein which contains leucine-rich repeats (LRRs). 125I labeled AVR9 peptide shows the same affinity for plasma membranes of Cf9+ and Cf9- tomato leaves. Membranes of solanaceous plants tested so far all contain homologs of the Cf9 gene and show similar affinities for AVR9. It is assumed that for induction of HR, at least two plant proteins (presumably CF9 and one of his homologs) interact directly or indirectly with the AVR9 peptide which possibly initiates modulation and dimerisation of the receptor, and activation of various other proteins involved in downstream events eventually leading to HR. We have created several mutants of the Avr9 gene, expressed them in the potato virus X (PVX) expression system and tested their biological activity on Cf9 genotypes of tomato. A positive correlation was observed between the biological activity of the mutant AVR9 peptides and their affinity for tomato plasma membranes. Recent results on structure and biological activity of AVR4 peptides encoded by avirulent and virulent alleles of the Avr4 gene (based on expression studies in PVX) are also discussed as well as early defence responses induced by elicitors in tomato leaves and tomato cell suspensions.     

Mechanisms of xylanase-induced nitric oxide and phosphatidic acid production in tomato cells

The second messenger nitric oxide (NO), phosphatidic acid (PA) and reactive oxygen species (ROS) are involved in the plant defense response during plant–pathogen interactions. NO has been shown to participate in PA production in response to the pathogen-associated molecular pattern xylanase in tomato cell suspensions. Defense responses downstream of PA include ROS production. The goal of this work was to study the signaling mechanisms involved in PA production during the defense responses triggered by xylanase and mediated by NO in the suspension-cultured tomato cells. We analyzed the participation of protein kinases, guanylate cyclase and the NO-mediated posttranslational modification S-nitrosylation, by means of pharmacology and biochemistry. We showed that NO, PA and ROS levels are significantly diminished by treatment with the general protein kinase inhibitor staurosporine. This indicates that xylanase-induced protein phosphorylation events might be the important components leading to NO formation, and hence for the downstream regulation of PA and ROS levels. When assayed, a guanylate cyclase inhibitor or a cGMP analog did not alter the PA accumulation. These results suggest that a cGMP-mediated pathway is not involved in xylanase-induced PA formation. Finally, the inhibition of protein S-nitrosylation did not affect NO formation but compromised PA and ROS production. Data collectively indicate that upon xylanase perception, cells activate a protein kinase pathway required for NO formation and that, S-nitrosylation-dependent mechanisms are involved in downstream signaling leading to PA and ROS.