Hanns Ulrich Seitz

Phytotoxicity and Innate Immune Responses Induced by Nep1-Like Proteins

We show that oomycete-derived Nep1 (for necrosis and ethylene-inducing peptide1)–like proteins (NLPs) trigger a comprehensive immune response in Arabidopsis thaliana, comprising posttranslational activation of mitogen-activated protein kinase activity, deposition of callose, production of nitric oxide, reactive oxygen intermediates, ethylene, and the phytoalexin camalexin, as well as cell death. Transcript profiling experiments revealed that NLPs trigger extensive reprogramming of the Arabidopsis transcriptome closely resembling that evoked by bacteria-derived flagellin. NLP-induced cell death is an active, light-dependent process requiring HSP90 but not caspase activity, salicylic acid, jasmonic acid, ethylene, or functional SGT1a/SGT1b. Studies on animal, yeast, moss, and plant cells revealed that sensitivity to NLPs is not a general characteristic of phospholipid bilayer systems but appears to be restricted to dicot plants. NLP-induced cell death does not require an intact plant cell wall, and ectopic expression of NLP in dicot plants resulted in cell death only when the protein was delivered to the apoplast. Our findings strongly suggest that NLP-induced necrosis requires interaction with a target site that is unique to the extracytoplasmic side of dicot plant plasma membranes. We propose that NLPs play dual roles in plant pathogen interactions as toxin-like virulence factors and as triggers of plant innate immune responses.

A common toxin fold mediates microbial attack and plant defense

Many plant pathogens secrete toxins that enhance microbial virulence by killing host cells. Usually, these toxins are produced by particular microbial taxa, such as bacteria or fungi. In contrast, many bacterial, fungal and oomycete species produce necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) that trigger leaf necrosis and immunity-associated responses in various plants. We have determined the crystal structure of an NLP from the phytopathogenic oomycete Pythium aphanidermatum to 1.35Å resolution. The protein fold exhibits structural similarities to cytolytic toxins produced by marine organisms (actinoporins). Computational modeling of the 3-dimensional structure of NLPs from another oomycete, Phytophthora parasitica, and from the phytopathogenic bacterium, Pectobacterium carotovorum, revealed a high extent of fold conservation. Expression of the 2 oomycete NLPs in an nlp-deficient P. carotovorum strain restored bacterial virulence, suggesting that NLPs of prokaryotic and eukaryotic origins are orthologous proteins. NLP mutant protein analyses revealed that identical structural properties were required to cause plasma membrane permeabilization and cytolysis in plant cells, as well as to restore bacterial virulence. In sum, NLPs are conserved virulence factors whose taxonomic distribution is exceptional for microbial phytotoxins, and that contribute to host infection by plasma membrane destruction and cytolysis. We further show that NLP-mediated phytotoxicity and plant defense gene expression share identical fold requirements, suggesting that toxin-mediated interference with host integrity triggers plant immunity-associated responses. Phytotoxin-induced cellular damage-associated activation of plant defenses is reminiscent of microbial toxin-induced inflammasome activation in vertebrates and may thus constitute another conserved element in animal and plant innate immunity.

Biosynthesis of p-hydroxybenzoic acid in elicitor-treated carrot cell cultures

Carrot (Daucus carota L.) cells respond to treatment with fungal elicitors by synthesizing wallbound p-hydroxybenzoic acid (p-HBA). The biosynthetic pathway to p-HBA is still hypothetical. Tracer experiments with l-phenylalanine indicate the involvement of the general phenylpropanoid pathway. 3,4 (Methylenedioxy) innamic acid, an inhibitor of hydrocycinnamate CoA ligase, inhibits the accumulation of anthocyanins in carrot, while it does not interfere with p-HBA synthesis. Thus p-HBA biosynthesis does not appear to involve CoA thioesters. In the present report the sequence of enzymic reactions leading to p-HBA was investigated in vitro using protein preparations from cells treated with a fungal elicitor from Pythium aphanidermatum (Edson) Fitzp. The side-chain degradation from p-coumaric acid to p-HBA is not analogous to the β-oxidation of fatty acids and involves p-hydroxybenzaldehyde as an intermediate. The final step from p-hydroxybenzaldehyde to p-HBA is catalyzed by an NAD-dependent p-hydroxybenzaldehyde dehydrogenase (EC 1.2.1.-). This reaction was characterized with regard to cofactor requirements, pH and temperature optima. The in-vitro formation of p-HBA from p-coumaric acid and the activity of the hydroxybenzaldehyde dehydrogenase are moderately elicitor-induced but to a much lesser extent than phenylalanine ammonialyase, which is the starting enzyme of the general phenylpropanoid pathway.

The uptake of acylated anthocyanin into isolated vacuoles from a cell suspension culture of Daucus carota

Anthocyanin-containing vacuoles were isolated from protoplasts of a cell suspension culture of Daucus carota. The vacuoles were stable for at least 2 h as demonstrated by the fact that they showed no efflux of anthocyanin. The uptake of radioactively labelled anthocyanin was time-dependent with a pH optimum at 7.5, and could be inhibited by the protonophore carbonylcyanide m-chlorophenylhydrazone. Furthermore, the transport was specific, since vacuoles from other plant species showed no uptake of labelled anthocyanin, and strongly depended on acylation with sinapic acid, as deacylated glycosides were not taken up by isolated vacuoles. Hence, it is suggested that the acylation of anthocyanin, which is also required for the stabilization of colour in vacuoles, is important for transport, and that acylated anthocyanin is transported by a selective carrier and might be trapped by a pH-dependent conformational change of the molecule inside the acid vacuolar sap.

Elicitor-Induced Changes in Ca2+ Influx, K+ Efflux, and 4-Hydroxybenzoic Acid Synthesis in Protoplasts of Daucus carota L

Suspension-cultured carrot cells (Daucus carota) and their protoplasts respond to a fungal elicitor prepared from the culture medium of Pythium aphanidermatum by accumulating 4-hydroxybenzoic acid (4-HBA). Protoplasts release the compound into the culture medium. Using 45CaCl2 as a tracer, we were able to demonstrate that the secretion of 4-HBA is preceded by a rapid increase in the Ca2+ influx and a concomitant K+ efflux. If the increased Ca2+ influx was prevented by ethyleneglycol-bis([beta]-aminoethylether)-N,N[prime]-tetraacetic acid, 4-HBA synthesis was inhibited by 70%. These results are discussed with regard to signal transduction from the plasma membrane to the nucleus of carrot protoplasts.

Regulation of enzymes involved in anthocyanin biosynthesis in carrot cell cultures in response to treatment with ultraviolet light and fungal elicitors

Abstract. The accumulation of anthocyanins in cell cultures of Daucus carota L. and the enzymes involved in their biosynthesis were investigated under growth in the dark, continuous irradiation with UV light, incubation with elicitors from Pythium aphanidermatum, and elicitor treatment of UV-irradiated cells. Upon UV irradiation, anthocyanin accumulation was strongly enhanced, and the enzymes of the phenylpropanoid and flavonoid pathways, including the “late” enzymes cyanidin galac-tosyltransferase, cyanidin galactoside xylosyltransferase, cyanidin triglycoside sinapoyltransferase and sinapic acid glucosyltransferase, all showed transient increases in their activities. The time courses of the enzyme activities exhibited successive maxima with an ordered sequence corresponding to their position in the biosynthetic pathway, suggesting a coordinated induction of the entire set of enzymes. The key enzymes phenylalanine ammonia-lyase and chalcone synthase are regulated on a transcriptional level. Incubation of dark-grown carrot cells with fungal elicitors led to a rapid and transient induction of phenylalanine ammonia-lyase corresponding to the formation of 4-hydroxybenzoic acid, but the amount of anthocyanin did not increase and there was no enhancement of any of the enzyme activities which are part of the anthocyanin pathway, including the enzymes catalyzing glycosylation and acylation reactions. Treatment with UV light and elicitors resulted in a rapid induction of the phenylpropanoid pathway, whereas the inducing effect of UV light on the anthocyanin content, on chalcone synthase and on the enzymes catalyzing the final steps of anthocyanin biosynthesis was suppressed. These results indicate a coordinated regulation of the enzymes involved in anthocyanin biosynthesis, an independent inducibility of the phenylpropanoid pathway, and a hierarchy of the different effectors, as shown by the dominating role of the elicitor-signal over the UV stimulus.

Anthocyanin accumulation and PAL activity in a suspension culture of Daucus carota L.

Cells of Daucus carota grown in a liquid medium produced large amounts of cyanidin as the only flavonoid aglycon. After inoculation in fresh medium a maximum activity of phenylalanine ammonia lyase (PAL; EC 4.3.1.5) was observed within 24 h. L-α-aminooxy-β-phenylpropionic acid (L-AOPP), thought to be a competitive inhibitor of PAL, inhibited cyanidin accumulation up to 80%. In order to study the regulatory role of PAL, the effects of L-AOPP and t-cinnamic acid, the product of the deamination of phenylalanine, were investigated. Cinnamic acid, applied in vivo (10-4 M), was not able to compensate for the inhibition of cyanidin production caused by L-AOPP (10-4 M) in the same sample. Carrot cells treated with L-AOPP exhibited a “super-induction” of PAL already described for gherkin hypocotyls (Amrhein and Gerhardt 1979). This effect was not influenced by t-cinnamic acid. L-AOPP seems to be a very specific inhibitor since it affected neither growth nor soluble protein content, whereas t-cinnamic acid inhibited both. Investigations on the content of soluble amino acids in L-AOPP-treated cells revealed a specific accumulation of soluble phenylalanine, whereas treatment with t-cinnamic acid led to an increase of amino acids in general, thus indicating that the latter compound has a rather unspecific effect on cellular metabolism. In vitro studies with PAL isolated from Daucus carota revealed that L-AOPP inhibited the enzyme at very low doses (KI=2.4·10-9), whereas t-cinnamic acid, by comparison, affected the enzyme at high concentrations (KI=1.8·10-4).

Inhibition of flavonoid biosynthesis by gibberellic acid in cell suspension cultures of Daucus carota L.

In carrot cells (Daucus carota L.), cultured in the presence of gibberellic acid, anthocyanin synthesis is blocked at the level of chalcone synthase. By feeding suitable precursors for anthocyanins (naringenin, eriodictyol, dihydroquercetin) biosynthesis of cyanidin glycosides can be restored. After addition of these substrates to the culture medium in the presence of gibberellic acid, the activity of chalcone synthase remained as low as in the control without precursors. The highest increase in anthocyanin content was achieved using dihydroquercetin as the added precursor. The time course of this supplementation showed a rapid response; within 4 h a substantial increase in anthocyanin could be observed. In contranst, the flavonol quercetin is not a precursor for cyanidin. The fact that naringenin was also accepted for cyanidin synthesis leads to the conclusion that hydroxylation in 3′-position of ring B in Daucus carota takes place at the flavonoid stage.

Acylation of anthocyanins with hydroxycinnamic acids via 1-O-acylglucosides by protein preparations from cell cultures of Daucus carota L.

Protein preparations from cell-suspension cultures of an Afghan cultivar of Daucus carota (carrot) catalyzed the formation of acylated anthocyanins from a cyanidin triglycoside isolated from the carrot cultures using (1-O-sinapoyl-, 1-O-feruloyl-) and 1-O-(p-coumaroyl)-β-glucose as acyl donors. The enzyme activities can be classified as 1-O-hydroxycinnamoyl-β-glucose: cyanidin 3-O-(2″-O-xylosyl-6″-O-glucosylgalactoside) 6‴-O-hydroxycinnamoyltransferases (EC 2.3.1.-).

Elicitor-induced cell death and phytoalexin synthesis in Daucus carota L.

Abstract. Suspension-cultured carrot cells and intact leaves respond to crude and purified protein elicitors from the non-host fungus Pythium aphanidermatum by activating the general phenylpropanoid pathway and incorporating de-novo-synthesized 4-hydroxybenzoic acid (4-HBA) into the cell wall. The cultured cells undergo a very rapid elicitor-induced cell death. Both reactions are directly correlated in their time course and their dose dependency. Cell death in elicitor-treated protoplasts resulted in early membrane damage and the digestion of DNA into oligonucleosomal fragments. The same pattern of DNA degradation could be induced in protoplasts by the G-protein activators Mas-7 or mastoparan. In cell cultures, both activators induced a rapid loss of viability without the activation of the general phenylpropanoid pathway. The elicitor-induced reactions, the loss of viability and the induction of 4-HBA biosynthesis were blocked by the calcium-channel blocker nifedipine. Neomycin and U73122, two inhibitors of phospholipase C, blocked the induction of 4-HBA biosynthesis but did not affect the loss in viability. The injection of the elicitor into the leaves of intact carrot plants confirmed the results obtained with cell cultures with regard to the induction of the hypersensitive response. The purification of the active compound revealed a 25-kDa protein which triggers both cell death and 4-HBA synthesis. The signalling pathways to both reactions could be independently blocked or induced.