Felix Mauch

Plant chitinases are potent inhibitors of fungal growth

The antimicrobial arsenal of plants is thought to consist mainly of secondary metabolites, among which the phytoalexins are the best-studied1–3. But plants may also possess antimicrobial proteins4,5: it has been reported that wheat-germ agglutinin, a chitin-binding lectin from wheat embryos, inhibits growth of the fungus Trichoderma viride4. This has led to the notion that plant lectins, with their intriguing biochemical similarity to animal antibodies, have an antibody-like antimicrobial function4,6,7. We report here that the main proteinaceous inhibitor of fungal growth in bean leaves is chitinase, an enzyme that can be induced by the plant hormone ethylene, or by pathogen attack. Among commercial preparations of purified chitin-binding lectins (from wheat germ, tomato, potato, pokeweed and gorse), only those containing contaminating chitinase activity inhibit fungal growth. Our data indicate that plant chitinases, but not chitin-binding lectins, are important antifungal proteins in plants.

Chitinase in bean leaves: induction by ethylene, purification, properties, and possible function.

Ethylene induced an endochitinase in primary leaves of Phaseolus vulgaris L. The enzyme formed chitobiose and higher chitin oligosaccharides from insoluble, colloidal or regenerated chitin. Less than 5% of the total chitinolytic activity was detected in an exochitinase assay proposed by Abeles et al. (1970, Plant Physiol. 47, 129–134) for ethylene-induced chitinase. In ethylene-treated plants, chitinase activity started to increase after a lag of 6 h and was induced 30 fold within 24 h. Exogenously supplied ethylene at 1 nl ml−1 was sufficient for half-maximal induction, and enhancement of the endogenous ethylene formation also enhanced chitinase activity. Cycloheximide prevented the induction. Among various hydrolases tested, only chitinase and, to a lesser extent, β-1,3-glucanase were induced by ethylene. Induction of chitinase by ethylene occurred in many different plant species. Ethylene-induced chitinase was purified by affinity chromatography on a column of regenerated chitin. Its apparent molecular weight obtained by sodium dodecyl sulfate-gel electrophoresis was 30,000; the molecular weight determined from filtration through Sephadex G-75 was 22,000. The purified enzyme attacked chitin in isolated cell walls of Fusarium solani. It also acted as a lysozyme when incubated with Micrococcus lysodeikticus. It is concluded that ethylene-induced chitinase functions as a defense enzyme against fungal and bacterial invaders.

The PP2C-Type Phosphatase AP2C1, Which Negatively Regulates MPK4 and MPK6, Modulates Innate Immunity, Jasmonic Acid, and Ethylene Levels in Arabidopsis

Wound signaling pathways in plants are mediated by mitogen-activated protein kinases (MAPKs) and stress hormones, such as ethylene and jasmonates. In Arabidopsis thaliana, the transmission of wound signals by MAPKs has been the subject of detailed investigations; however, the involvement of specific phosphatases in wound signaling is not known. Here, we show that AP2C1, an Arabidopsis Ser/Thr phosphatase of type 2C, is a novel stress signal regulator that inactivates the stress-responsive MAPKs MPK4 and MPK6. Mutant ap2c1 plants produce significantly higher amounts of jasmonate upon wounding and are more resistant to phytophagous mites (Tetranychus urticae). Plants with increased AP2C1 levels display lower wound activation of MAPKs, reduced ethylene production, and compromised innate immunity against the necrotrophic pathogen Botrytis cinerea. Our results demonstrate a key role for the AP2C1 phosphatase in regulating stress hormone levels, defense responses, and MAPK activities in Arabidopsis and provide evidence that the activity of AP2C1 might control the plants response to B. cinerea.

Differential Induction of Distinct Glutathione-S-Transferases of Wheat by Xenobiotics and by Pathogen Attack

We have previously characterized a pathogen-induced gene from wheat (Triticum aestivum L.) that was named GstA1 based on sequence similarities with glutathione-S-transferases (GSTs) of maize (R. Dudler, C. Hertig, G. Rebmann, J. Bull, F. Mauch [1991] Mol Plant Microbe Interact 4: 14–18). We have constructed a full-length GstA1 cDNA by combinatorial polymerase chain reaction and demonstrate by functional expression of the cDNA in Escherichia coli that the GstA1-encoded protein has GST activity. An antiserum raised against a GstA1 fusion protein specificially recognized a protein with an apparent molecular mass of 29 kD on immunoblots of extracts from bacteria expressing the GstA1 cDNA and extracts from wheat inoculated with Erysiphe graminis. The GstA1-encoded protein was named GST29. RNA and immunoblot analysis showed that GstA1 was only weakly expressed in control plants and was specifically induced by pathogen attack and by the GST substrate glutathione, but not by various xenobiotics. In contrast, a structurally and antigenically unrelated GST with an apparent molecular mass of 25 kD that was detected with an antiserum raised against GSTs of maize was expressed at a high basal level. This GST25 and an additional immunoreactive protein named GST26 were strongly induced by cadmium and by the herbicides atrazine, paraquat, and alachlor, but not by pathogen attack. Compared with the pathogen-induced GST29, GST25 and GST26 showed a high affinity toward glutathione-agarose and were much more active toward the model substrate 1-chloro-2,4-dinitrobenzene. Thus, wheat contains at least two distinct GST classes that are differentially regulated by xenobiotics and by pathogen attack and whose members have different enzymic properties. GST25 and GST26 appear to have a function in xenobiotic metabolism, whereas GST29 is speculated to fulfill a more specific role in defense reactions against pathogens.

Colorimetric assay for chitinase

Publisher Summary This chapter describes the colorimetric assay for chitinase, which is applicable to the various types of chitinase present in microorganisms, animals, and plants. The chapter evaluates it with particular reference to plant chitinases, because they may function as a defense against chitin-containing pathogens. The most widely used colorimetric assay for plant chitinases has been an exochitinase assay, based on the determination of monomeric N-acetylglucosamine (GlcNAc) released from colloidal chitin. However, plant chitinases generally are endochitinases and produce chitooligosaccharides as principal products. Therefore, measurements of plant chitinases with the exochitinase assay should be viewed with caution. For accurate determination, it is essential to measure the chitooligosaccharides produced in the assay. This can be accomplished by the enzymatic hydrolysis of the reaction products to monomeric GlcNAc prior to the colorimetric measurement. The chapter compares the assay with other chitinase assays.

The glutathione‐deficient mutant pad2‐1 accumulates lower amounts of glucosinolates and is more susceptible to the insect herbivore Spodoptera littoralis

Summary Plants often respond to pathogen or insect attack by inducing the synthesis of toxic compounds such as phytoalexins and glucosinolates (GS). The Arabidopsis mutant pad2-1 has reduced levels of the phytoalexin camalexin and is known for its increased susceptibility to fungal and bacterial pathogens. We found that pad2-1 is also more susceptible to the generalist insect Spodoptera littoralis but not to the specialist Pieris brassicae. The PAD2 gene encodes a gamma-glutamylcysteine synthetase that is involved in glutathione (GSH) synthesis, and consequently the pad2-1 mutant contains about 20% of the GSH found in wild-type plants. Lower GSH levels of pad2-1 were correlated with reduced accumulation of the two major indole and aliphatic GSs of Arabidopsis, indolyl-3-methyl-GS and 4-methylsulfinylbutyl-GS, in response to insect feeding. This effect was specific to GSH, was not complemented by treatment of pad2-1 with the strong reducing agent dithiothreitol, and was not observed with the ascorbate-deficient mutant vtc1-1. In contrast to the jasmonate-insensitive mutant coi1-1, expression of insect-regulated and GS biosynthesis genes was not affected in pad2-1. Our data suggest a crucial role for GSH in GS biosynthesis and insect resistance.

Crosstalk and differential response to abiotic and biotic stressors reflected at the transcriptional level of effector genes from secondary metabolism

Plant secondary metabolism significantly contributes to defensive measures against adverse abiotic and biotic cues. To investigate stress-induced, transcriptional alterations of underlying effector gene families, which encode enzymes acting consecutively in secondary metabolism and defense reactions, a DNA array (MetArray) harboring gene-specific probes was established. It comprised complete sets of genes encoding 109 secondary product glycosyltransferases and 63 glutathione-utilizing enzymes along with 62 cytochrome P450 monooxygenases and 26 ABC transporters. Their transcriptome was monitored in different organs of unstressed plants and in shoots in response to herbicides, UV-B radiation, endogenous stress hormones, and pathogen infection. A principal component analysis based on the transcription of these effector gene families defined distinct responses and crosstalk. Methyl jasmonate and ethylene treatments were separated from a group combining reactions towards two sulfonylurea herbicides, salicylate and an avirulent strain of Pseudomonas syringae pv. tomato. The responses to the herbicide bromoxynil and UV-B radiation were distinct from both groups. In addition, these analyses pinpointed individual effector genes indicating their role in these stress responses. A small group of genes was diagnostic in differentiating the response to two herbicide classes used. Interestingly, a subset of genes induced by P. syringae was not responsive to the applied stress hormones. Small groups of comprehensively induced effector genes indicate common defense strategies. Furthermore, homologous members within branches of these effector gene families displayed differential expression patterns either in both organs or during stress responses arguing for their non-redundant functions.

A pathogen-induced wheat gene encodes a protein homologous to glutathione-S-transferases.

Winter wheat (Triticum aestivum) shows local, induced resistance against the plant-pathogenic fungus Erysiphe graminis f. sp. tritici following exposure to the nonpathogen E. g. f. sp. hordei. The onset of this resistance has been shown to be correlated with the activation of putative defense genes, and cDNA clones representing transcripts of induced genes have been obtained (P. Schweizer, W. Hunziker, and E. Mösinger, Plant Molecular Biology 12:643-654, 1989). We have cloned and sequenced a gene corresponding to one of these cDNAs, WIR5. Sequence analysis indicated that this gene contains three exons and encodes a protein of 229 amino acids. S1 mapping showed that transcripts homologous to this gene are at least 20 times more abundant in leaves infected 14 hr earlier with E. g. f. sp. hordei than in control leaves. Sequence comparison showed that the WIR5 gene product is highly homologous to glutathione-S-transferases (GSTs; EC 25.1.18) of maize. This, together with the fact that the intron positions of both the wheat gene and the maize GSTI gene are conserved, suggests that the cloned pathogen-induced gene, named GstA1, encodes a wheat glutathione-S-transferase.

Quantification of induced resistance against Phytophthora species expressing GFP as a vital marker: beta-aminobutyric acid but not BTH protects potato and Arabidopsis from infection.

SUMMARY Induced resistance was studied in the model pathosystem Arabidopsis-Phytophthora brassicae (formerly P. porri) in comparison with the agronomically important late blight disease of potato caused by Phytophthora infestans. For the quantification of disease progress, both Phytophthora species were transformed with the vector p34GFN carrying the selectable marker gene neomycine phosphotransferase (nptII) and the reporter gene green fluorescent protein (gfp). Eighty five per cent of the transformants of P. brassicae and P. infestans constitutively expressed GFP at high levels at all developmental stages both in vitro and in planta. Transformants with high GFP expression and normal in vitro growth and virulence were selected to quantify pathogen growth by measuring the in planta emitted GFP fluorescence. This non-destructive monitoring of the infection process was applied to analyse the efficacy of two chemical inducers of disease resistance, a functional SA-analogue, benzothiadiazole (BTH), and beta-aminobutyric acid (BABA) which is involved in priming mechanisms of unknown nature. BABA pre-treatment (300 microm) via soil drench applied 24 h before inoculation completely protected the susceptible Arabidopsis accession Landsberg erecta (Ler) from infection with P. brassicae. A similar treatment with BTH (330 microm) did not induce resistance. Spraying the susceptible potato cultivar Bintje with BABA (1 mm) 2 days before inoculation resulted in a phenocopy of the incompatible interaction shown by the resistant potato cultivar Matilda while BTH (1.5 mm) did not protect Bintje from severe infection. Thus, in both pathosystems, the mechanisms of induced resistance appeared to be similar, suggesting that the Arabidopsis-P. brassicae pathosystem is a promising model for the molecular analysis of induced resistance mechanisms of potato against the late blight disease.

Disease resistance of Arabidopsis to Phytophthora brassicae is established by the sequential action of indole glucosinolates and camalexin.

We have analysed the role of tryptophan-derived secondary metabolites in disease resistance of Arabidopsis to the oomycete pathogen Phytophthora brassicae. Transcript analysis revealed that genes encoding enzymes involved in tryptophan, camalexin and indole glucosinolate (iGS) biosynthesis are coordinately induced in response to P. brassicae. However, a deficiency in either camalexin or iGS accumulation has only a minor effect on the disease resistance of Arabidopsis mutants. In contrast, the double mutant cyp79B2 cyp79B3, which has a blockage in the production of indole-3-aldoxime (IAOx), the common precursor of tryptophan-derived metabolites including camalexin and iGS, is highly susceptible to P. brassicae. Because cyp79B2 cyp79B3 shows no deficiencies in other tested disease resistance responses, we concluded that the lack of IAOx-derived compounds renders Arabidopsis susceptible despite wild-type-like pathogen-induced hypersensitive cell death, stress hormone signaling and callose deposition. The susceptibility of the double mutant pen2-1 pad3-1, which has a combined defect in camalexin synthesis and PEN2-catalysed hydrolysis of iGS compounds, demonstrates that both camalexin and products of iGS hydrolysis are important for disease resistance to P. brassicae. Products of iGS hydrolysis play an early defensive role, as indicated by enhanced epidermal penetration rates of Arabidopsis mutants affected in iGS synthesis or degradation. Our results show that disease resistance of Arabidopsis to P. brassicae is established by the sequential activity of the phytoanticipin iGS and the phytoalexin camalexin.