Elicitor-mediated Induction Research Articles

Elicitor-mediated induction of Cajanus cajan defences altered nutritional indices of Maruca Vitrata (Fab.) (Lepidoptera: Crambidae)

Elicitor-mediated induction of Cajanus cajan defences altered nutritional indices of Maruca Vitrata (Fab.) (Lepidoptera: Crambidae)

Correction to: Fungal Elicitor-Mediated Induction of Innate Immunity in Catharanthus roseus Against Leaf Blight Disease Caused by Alternaria alternata

Correction to: Fungal Elicitor-Mediated Induction of Innate Immunity in Catharanthus roseus Against Leaf Blight Disease Caused by Alternaria alternata

Fungal Elicitor-Mediated Induction of Innate Immunity in Catharanthus roseus Against Leaf Blight Disease Caused by Alternaria alternata

Fungal Elicitor-Mediated Induction of Innate Immunity in Catharanthus roseus Against Leaf Blight Disease Caused by Alternaria alternata

The Leucine-Rich Repeat Receptor-Like Kinase BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 and the Cytochrome P450 PHYTOALEXIN DEFICIENT3 Contribute to Innate Immunity to Aphids in Arabidopsis

The importance of pathogen-associated molecular pattern-triggered immunity (PTI) against microbial pathogens has been recently demonstrated. However, it is currently unclear if this layer of immunity mediated by surface-localized pattern recognition receptors (PRRs) also plays a role in basal resistance to insects, such as aphids. Here, we show that PTI is an important component of plant innate immunity to insects. Extract of the green peach aphid (GPA; Myzus persicae) triggers responses characteristic of PTI in Arabidopsis (Arabidopsis thaliana). Two separate eliciting GPA-derived fractions trigger induced resistance to GPA that is dependent on the leucine-rich repeat receptor-like kinase BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1)/SOMATIC-EMBRYOGENESIS RECEPTOR-LIKE KINASE3, which is a key regulator of several leucine-rich repeat-containing PRRs. BAK1 is required for GPA elicitor-mediated induction of reactive oxygen species and callose deposition. Arabidopsis bak1 mutant plants are also compromised in immunity to the pea aphid (Acyrthosiphon pisum), for which Arabidopsis is normally a nonhost. Aphid-derived elicitors induce expression of PHYTOALEXIN DEFICIENT3 (PAD3), a key cytochrome P450 involved in the biosynthesis of camalexin, which is a major Arabidopsis phytoalexin that is toxic to GPA. PAD3 is also required for induced resistance to GPA, independently of BAK1 and reactive oxygen species production. Our results reveal that plant innate immunity to insects may involve early perception of elicitors by cell surface-localized PRRs, leading to subsequent downstream immune signaling.

Isolation and characterisation of a protein elicitor from Sclerospora graminicola and elicitor-mediated induction of defence responses in cultured cells of Pennisetum glaucum.

Sclerospora graminicola (Sacc.) Schroet., an oomycete pathogen of Pennisetum glaucum (L.) R.Br. infects the meristematic tissues of young seedlings. The motile zoospores from the sporangia encyst, germinate and penetrate the plant tissue. Resistance to the invading pathogen is governed by the specific recognition of conserved pathogen-associated proteins or elicitors. In the present study, a zoospore protein was isolated and purified to homogeneity by a combination of size exclusion and high-performance liquid chromatography (HPLC). The crude fractionated protein was able to elicit an array of defence responses in resistant and susceptible cells of pearl millet. Treatment of cultured cells of pearl millet with partially purified elicitor protein resulted in a rapid loss of cell viability in the resistant cells and the percentage of cell death was higher in the resistant than in the susceptible cells. Cultures of resistant cells showed a sharp increase in the extra cellular pH compared with susceptible cells when treated with the crude elicitor. Increased oxidative burst was also recorded in the cells treated with the crude elicitor. The purified elicitor showed unique properties. The purified protein was acidic with a pI of 5.6 as revealed by isoelectric focusing (IEF) and matrix-assisted laser desorption ionisation (MALDI) analysis showed that the elicitor had a molecular mass of 7040 daltons. The primary structure determined by N-terminal Edman degradation and searches with BLAST did not reveal similarities to any known plant pathogenic or oomycete elicitor. Higher activities of the important defence-related enzymes phenylalanine ammonia lyase (PAL) and peroxidase in the resistant cell cultures than in the susceptible cell cultures treated with the purified elicitor were clearly evident. Studies of gene expression by northern blotting with heterologus peroxidase, PAL and oxalate oxidase probes showed that the mRNA transcripts were strongly up-regulated in resistant cell cultures within 30 min of elicitor treatment. The purified elicitor also demonstrated a very strong concentration-dependent sterol binding. The purified elicitor protein belongs to a class of low molecular weight oomycete elicitors with sterol carrier properties. The identified low molecular weight protein elicitor displays unique properties that can be exploited for synthesis of novel molecules for eco-friendly crop protection.

A new class II rice chitinase, Rcht2, whose induction by fungal elicitor is abolished by protein phosphatase 1 and 2A inhibitor.

Among the four classes of chitinase, a class II chitinase had not yet been reported for rice. We have isolated and characterized a class II acidic chitinase, Rcht2, from rice (Oryza sativa L. cv. Cheongcheongbyeo). The protein consists of a single polypeptide chain of 261 amino acid residues and includes a putative signal sequence of 29 amino acids at its N-terminus. It has a calculated molecular mass of 27,642 Da and an isoelectric point of 5.56. The Rcht2 chitinase lacks the cysteine-rich and hinge domains in the N-terminal region of the protein, which is the criterion for its classification as a class II chitinase. Comparison of the genomic and the cDNA sequence revealed that the coding region of Rcht2 consist of three exons of 301, 112, and 370 bp separated by two introns of 89 and 984 bp. In suspension-cultured rice cells, the transcript level of Rcht2 was dramatically increased by treatment with both glycol chitin and fungal elicitor. The application of protein phosphatase 1 and 2A inhibitors, calyculin A and okadaic acid, effectively abolished the induction of Rcht2 in response to fungal elicitor. In contrast, the activation of Rcht2 transcript was not inhibited by both cycloheximide and protein kinase inhibitors. These results demonstrate that protein dephosphorylation events play a crucial role in the elicitor-mediated induction of Rcht2 in rice cells, while de novo protein synthesis is not required for induction.

Elicitor-mediated induction of anthraquinone biosynthesis and regulation of isopentenyl diphosphate isomerase and farnesyl diphosphate synthase activities in cell suspension cultures of Cinchona robusta How.

Treatment of a Cinchona robusta How. cell suspension culture with a homogenate of Phytophthora cinnamomi resulted in cessation of growth and a rapid induction of the biosynthesis of anthraquinone-type phytoalexins. The strongest induction of anthraquinone biosynthesis was obtained when the elicitor was added in the early growth phase of the growth cycle. The accumulation of anthraquinones was accompanied by a tri-phasic response in the activity of isopentenyl diphosphate (IPP) isomerase (EC 5.3.3.2): phase I was characterised by a rapid induction of activity, reaching a maximum at 12 h after elicitation. During phase II, IPP isomerase rapidly decreased to levels below those found in untreated cells. At phase III, IPP isomerase activity increased again, reaching a second maximum at about 72 h after elicitation. During phase I, the activity of farnesyl diphosphate synthase (EC 2.5.1.10) was found to be suppressed. Extraction and assay conditions were optimised for IPP isomerase. The presence of Mn2+ in the incubation buffer resulted in a marked increase in the activity of the enzymes obtained from cells in phase I. The induction of IPP isomerase in combination with a concomitant inhibition of farnesyl diphosphate synthase might result in an efficient channeling of C5-precursors into phytoalexin biosynthesis.

Elicitor-mediated induction of isochorismate synthase and accumulation of 2,3-dihydroxy benzoic acid in Catharanthus roseus cell suspension and shoot cultures

After elicitation, cell suspension cultures of Catharanthus roseus accumulate phenolic compounds. The major phenolic compound produced was isolated and identified as 2,3-dihydroxybenzoic acid (DHBA). The accumulation of this compound is a rapid response to the addition of elicitor; within 6 h after the addition of elicitor, DHBA concentration reached 6.3 mg/l cell suspension. DHBA was not detected in non-elicited cells. The formation of DHBA in elicited cells was correlated with the induction of the enzyme isochorismate synthase (ICS). Shoot cultures of C. roseus also presented a strong induction of ICS after elicitation. Due to its biological activity, DHBA could play a role in the defence mechanism of C. roseus.

Enzymological aspects of the redirection of terpenoid biosynthesis in elicitor-treated cultures of Tabernaemontana divaricata

The elicitor-mediated induction of pentacyclic triterpenoid phytoalexin accumulation in cells of five-day-old suspension cultures of Tabernaemontana divaricata is accompanied by: a rapid and transient increase in HMG-CoA reductase (EC 1.1.1.34) activity; an increase in IPP isomerase (EC 5.3.3.2), prenyl transferase (EC 2.5.1.1) and squalene synthetase (EC 2.5.1.21) activity; a rapid inhibition of squalene 2,3-oxide:cycloartenol cyclase activity (EC 5.4.99.8), and a rapid and relatively transient appearance of squalene 2,3-oxide:amyrin cyclase (EC 5.4.99.-) activity. These findings are entirely consistent with an elicitor-induced redirection of the cytosolic-microsomal pathway of terpenoid biosynthesis away from phytosterol biosynthesis and towards pentacyclic triterpenoid phytoalexin biosynthesis. The switch being mediated as a direct result of the rapid inhibition of squalene 2,3-oxide:cycloartenol cyclase activity just prior to the de novo synthesis of squalene 2,3-oxide:amyrin cyclase and the other enzymes on the post-squalene 2,3-oxide span of the pentacyclic triterpenoid phytoalexin pathway. The increased activities of the enzymes common to both pathways reflects the fact that the rate of accumulation of pentacyclic triterpenoid phytoalexins in elicited cultures is more rapid than the rate of phytosterol biosynthesis in control cultures. The very rapid and transient increase in HMG-CoA reductase activity points to the microsomal form(s) of this enzyme having a key regulatory role in controlling the flux of carbon into the cytosolic-microsomal pathway of terpenoid biosynthesis.

Elicitor-mediated induction of enzymes of lignin biosynthesis and formation of lignin-like material in a cell suspension culture of spruce (Picea abies)

Incubations of photomixotrophic suspension culture cells of spruce (Picea abies) (L.) (Karst) with an autoclaved cell wall preparation of Rhizosphaera kalkhoffii as elicitor led to a rapid increase of the activity of a number of enzymes involved in lignin biosynthesis. l-phenylalanine ammonia-lyase (EC 4.3.1.5) was induced about 10-fold, feruloyl-Coenzyme A reductase (ED 1.2.1.44) 4-fold, cinnamyl alcohol dehydrogenase (NADP+) (EC 1.1.1.195) 2-fold and peroxidase (EC 1.11.1.7) about 1.5-fold. The induction of the enzymes, with the exception of the peroxidase, was transient, showing maximal activity within 3 days after elicitation. Extracellular peroxidase activity, determined in the culture medium, rapidly decreased on initiation of elicitation.

Inhibition of phytosterol biosynthesis in elicitor-treated cultures of Ammi majus

The elicitor-mediated induction of coumarin phytoalexin production by cell-suspension cultures of Ammi majus is accompanied by the inhibition of both biomass production and phytosterol biosynthesis [from mevalonic acid (MVA)]. However, cell-free preparations of cells from elicitor-treated cultures are able to support the synthesis of squalene from MVA, isopentenyl diphosphate (IPP) or (2 E, 6 E)-farnesyl diphosphate (FPP), even though radioactive squalene is not found following administration of [2- 14C]MVA to elicitor-treated cultures. The preparations, as expected, also exhibit umbelliferone:O-dimethylallyl transferase activity. These results indicate that in vivo there is a specific inhibition of one of the enzymes on the MVA to dimethylallyl diphosphate (DMAPP) span of the cytosolic microsomal pathway of terpenoid biosynthesis in elicitor-treated cultures, and that in cell-free preparations this deficiency is made good by the corresponding plastid enzyme. The finding that the coumarin phytoalexins are not labelled on feeding either [2- 14C]acetate or [2- 14C]MVA to elicitor-treated cultures precluded us from establishing whether the inhibition of phytosterol biosynthesis is a means of increasing the input of cytosolic IPP into the plastid for coumarin phytoalexin biosynthesis (in which case cytosolic IPP isomerase would be the enzyme inhibited in elicitor-treated cultures) or whether the cytosolic and plastid pathways of terpenoid biosynthesis are not linked and the site of inhibition (between MVA and DMAPP) is unrelated to the provision of plastid IPP for phytoalexin biosynthesis. The feeding experiments with [2- 14C]acetate suggested that, somewhat unexpectedly, polar lipid biosynthesis is not repressed in elicitor-treated cultures.

Increased Accumulation of Acridone Alkaloids by Cell Suspension Cultures of Ruta graveolens in Response to Elicitors

Summary Two dark-grown cell suspension cultures of Ruta graveolens L. were challenged with fungal elicitors. The treatment of cell line R-4 resulted in increased accumulation of hydroxy rutacridone epoxide, rutacridone epoxide and gravacridontriol. The latter alkaloid was not detectable in non-elicited cells. Another response was observed in cell line R-15. Besides rutacridone epoxide, for the first time rutacridone accumulation was stimulated either by a yeast, Phytophthora or Colletotrichum elicitor. Using cell line R-15, an elicitor-mediated induction of 5-adenosyl-L-methionine: anthranilic acid N-methyltransferase, N-methylanthranilic acid «activating» enzyme and rutacridone synthase was recorded. Enzyme activities were influenced by incubation time and amount of elicitor.

Elicitor-mediated induction of phenylalanine ammonia lyase and tryptophan decarboxylase: Accumulation of phenols and indole alkaloids in cell suspension cultures of Catharanthus roseus

Cell suspension cultures (cell line No 615) of Catharanthus roseus cv. Little Delicata responded to elicitor treatment by accumulating monoterpenoid indole alkaloids and phenolic compounds. The excretion of phenols into the culture medium resulted from the induction of the branch-point enzyme phenylalanine ammonia lyase. The accumulation of alkaloids, however, occurred several hours earlier than the elicitor-mediated induction of tryptophan decarboxylase through which shikimate pathway intermediates are channelled into tryptamine and related indole alkaloids. The results indicate that both pathways for phenol and indole alkaloid biosynthesis responded to elicitor treatment and that no obvious causal relationship between pathways could be deduced from this study.

Elicitor-mediated induction of tryptophan decarboxylase and strictosidine synthase activities in cell suspension cultures of Catharanthus roseus

Treatment of one cell line (No. 615) of Catharanthus roseus c.v. Little Delicata with an elicitor preparation of autoclaved and homogenized Pythium aphanidermatum culture resulted in rapid accumulation of indole alkaloids. Alkaloid formation was preceded by rapid transient increases in the extractable activities of the enzymes tryptophan decarboxylase and strictosidine synthase. The induction of these two enzyme activities occurred when cells were transferred to alkaloid production medium or treatment with fungal elicitors. Treatment of this cell line with translational or transcriptional inhibitors prevented the Pythium-induced increases of enzyme activity as well as alkaloid accumulation. When cells were transferred to alkaloid production medium the induction of strictosidine synthase activity preceded that of tryptophan decarboxylase by many hours even when cells were also treated with Pythium elicitor. Results suggested that tryptophan decarboxylase induction proceeds only when endogenous tryptamine levels were decreased by two-third. The internal cellular level of tryptamine, therefore, could regulate expression of tryptophan decarboxylase, whereas induction of strictosidine synthase or of another enzyme in the biosynthetic pathway could control channeling of tryptamine into alkaloids. The results demonstrate that fungal elicitors can be used to facilitate studies of the factors which regulate expression of indole alkaloid pathway enzymes and their ultimate pathway products.

Elicitor-mediated induction of chalcone isomerase in Phaseolus vulgaris cell suspension cultures

Approximately fourfold increases in the extractable activity of the enzyme chalcone isomerase (CHI, EC 5.5.1.6) were observed within 24 h of treatment of cell suspension cultures of Phaseolus vulgaris with a crude elicitor preparation heatreleased from the cell walls of the bean pathogen Colletotrichum lindemuthianum. The induction of CHI activity was highly dependent upon elicitor concentration, with maximum induction occurring in two discrete concentration ranges. A basal half-life for CHI>32 h in control cultures was determined by labelling with (2)H from (2)H2O followed by analysis of the equilibrium distribution of enzyme activity in CsCl density gradients. Comparative density labelling indicated that at both the lower and higher effective elicitor concentrations, the induced appearance of CHI activity was the result of an apparent initial activation of pre-existing enzyme followed by an increase in the rate of de-novo synthesis of the enzyme as compared with non-elicited controls. The increased appearance of the enzyme over the first 8 h in elicitor-treated cultures was inhibited by cycloheximide, cordycepin and actinomycin D. The results are discussed in relation to the mechanisms of co-ordinate enzyme induction operating in French-bean cell cultures exposed to fungal elicitors.