Inoculation of Paenibacillus illinoisensis alleviates root mortality, activates of lignification-related enzymes, and induction of the isozymes in pepper plants infected by Phytophthora capsici

To investigate the variations of the enzymes responsible for lignification, after inoculation with Phytophthora capsici and/or Paenibacillus illinoisensis KJA-424, in relation to biocontrol of Phytophthora blight in pepper, roots of two-month-old plants were inoculated with P. capsici inoculation (P), and co-inoculation of P. capsici and P. illinoisensis cell cultures (P + A). Root mortality of pepper plants induced by inoculation of P. capsici was completely recovered by co-inoculation with antagonistic KJA-424. At day 7, peroxidase (POD) activity increased by 36.7% in P-treated roots but by 7.1% only in P + A-treated, compared with control. Polyphenol oxidase (PPO) activity increased for 3 days and then drastically decreased in P-treated roots but maintained a constant level in control and P + A-treated. At day 7, PPO activity in P-treated leaves decreased but recovered to the level of control in the P + A-treated. Three major POD isozymes (45, 53, and 114 kDa) were shown in P-treated roots, while two major (53 and 114 kDa) in control and P + A-treated, suggesting that the 45 kDa of POD was actively induced in P-treated roots but not induced in P + A-treated roots. A PPO isozyme of 80 kDa was induced in P-treated roots but not induced by co-treated with KJA-424. In leaves, the POD isozyme of 45 kDa appears to be systemically induced in P-treated only. The PPO isozyme of 80 kDa in leaves was not induced by pathogen challenge but recovered by co-inoculated with P. illinoisensis. All these results suggest that the inoculation of an antagonist, P. illinoisensis alleviates root mortality, activates of lignification-related enzymes and induction of the isozymes in pepper plants infected by P. capsici.     

Treatment of Paenibacillus illinoisensis suppresses the activities of antioxidative enzymes in pepper roots caused by Phytophthora capsici infection

Summary The activity of antioxidative enzymes after inoculation of pepper (Capsicum annuum L. Chungok) with a pathogen, Phytophthora capsici (P), the causal agent of Phytophtora blight and dual inoculation of pathogen and an antagonist, Paenibacillus illinoisensis KJA-424 (P+A), were measured and compared with that of non-inoculated (C) roots. Root mortality was significantly reduced by about 84% in P+A treatment compared with P treatment alone. When compared to the non-inoculated (C) roots, malondialdehyde (MDA) concentration gradually decreased by 52.4% in 7 days only in P-treated roots and hydrogen peroxide (H₂O₂) was not significantly affected by the treatment for 5 days but significantly decreased in the P+A-treated roots at day 7. P-treatment continuously induced peroxidase (POD) and superoxide dismutase (SOD), resulting in significant increases of 36.7% and 27.7% at day 7, respectively, compared to the control. In P+A-treated roots, the activities of POD and SOD also increased for 5 days but returned to the control level at day 7. Catalase activity fluctuated but again increased over the 7-day period following P+A inoculation. These results indicate that an antagonist P. illinoisensis KJA-424 alleviated root mortality and suppressed the elevated activities of POD and SOD in the root of pepper plant root caused by P.␣capsici infection.     

The Effect of Pseudomonas fluorescens and Fusarium oxysporum f.sp. cubense on Induction of Defense Enzymes and Phenolics in Banana

The effect of Pseudomonas fluorescens treatment and Fusarium oxysporum f. sp. cubense inoculation on induction of phenylalanine ammonia-lyase (PAL), peroxidase (POX), chitinase, -1,3-glucanase and accumulation of phenolics in banana (Musa sp.) was studied. When banana roots were treated with P. fluorescens strain Pf10, a two-fold increase in phenolic content in leaf tissues was recorded 3 – 6 d after treatment. Challenge inoculation with F. oxysporum, the wilt pathogen, steeply increased the phenolic content in P. fluorescens-treated banana plants. Significant increase in POX activity was detected 6 – 9 d after P. fluorescens treatment. PAL, chitinase and -1,3-glucanase activities increased significantly from 3 d after P. fluorescens treatment and reached the maximum 6 d after treatment. Challenge inoculation with F. oxysporum further increased the enzyme activities. These results suggest that the enhanced activities of defense enzymes and elevated content of phenolics may contribute to bioprotection of banana plants against F. oxysporum.     

Induction of pathogenesis-related proteins during biocontrol of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> with <Emphasis Type="Italic">Pseudomonas aureofaciens</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L. Merr.) plants

To investigate the biocontrol effectiveness of the antibiotic producing bacterium, Pseudomonas aureofaciens 63–28 against the phytopathogen Rhizoctonia solani AG-4 on Petri plates and in soybean roots, growth response and induction of PR-proteins were estimated after inoculation with P. aureofaciens 63–28 (P), with R. solani AG-4 (R), or with P. aureofaciens 63–28 + R. solani AG-4 (P + R). P. aureofaciens 63–28 showed strong antifungal activity against R. solani AG-4 pathogens in Petri plates. Treatment with P. aureofaciens 63–28 alone increased the emergence rate, shoot fresh weight, shoot dry weight and root fresh weight at 7 days after inoculation, when compared to R. solani AG-4; P + R treatment showed similar effects. Peroxidase (POD) and β-1,3-glucanase activity of P. aureofaciens 63–28 treated roots increased by 41.1 and 49.9%, respectively, compared to control roots. POD was 26% greater in P + R treated roots than R. solani treated roots. Two POD isozymes (59 and 27 kDa) were strongly induced in P + R treated roots. The apparent molecular weight of chitinase from treated roots, as determined through SDS-PAGE separation and comparison with standards, was about 29 kDa. Five β-1,3-glucanase isozymes (80, 70, 50, 46 and 19 kDa) were observed in all treatments. These results suggest that inoculation of soybean plants with P. aureofaciens 63–28 elevates plant growth inhibition by R. solani AG-4 and activates PR-proteins, potentially through induction of systemic resistance mechanisms.     

Acibenzolar-S-methyl induced resistance to <Emphasis Type="Italic">Phytophthora capsici</Emphasis> in pepper leaves

The leaves of pepper (Capsicum anuum L.) were inoculated with Phytophthora capsici Leonian 3 d after treatment with acibenzolar-S-methylbenzo [1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester (ASM) and resistance to Phytophthora blight disease was investigated. Results showed that P. capsici was significantly inhibited by ASM treatment by up to 45 % in planta. The pepper plants responded to ASM treatments by rapid and transient induction of L-phenylalanine ammonia-lyase (PAL), increase in total phenol content and activities of chitinase and β-1,3-glucanase. No significant increases in enzyme activities were observed in water-treated control plants compared with the ASM-treated plants. Therefore it may be suggested that ASM induces defense-related enzymes, PAL activity, PR proteins and phenol accumulation in ASM-treated plants and contribute to enhance resistance against P. capsici.     

Biocontrol Ability of <Emphasis Type="Italic">Lysobacter antibioticus</Emphasis> HS124 Against <Emphasis Type="Italic">Phytophthora</Emphasis> Blight Is Mediated by the Production of 4-Hydroxyphenylacetic Acid and Several Lytic Enzymes

Several rhizobacteria play a vital role in plant protection, plant growth promotion and the improvement of soil health. In this study, we have isolated a strain of Lysobacter antibioticus HS124 from rhizosphere and demonstrate its antifungal activity against various pathogens including Phytophthora capsici, a destructive pathogen of pepper plants. L. antibioticus HS124 produced lytic enzymes such as chitinase, β-1,3-glucanase, lipase, protease, and an antibiotic compound. This antibiotic compound was purified by diaion HP-20, silica gel, sephadex LH-20 column chromatography and high performance liquid chromatography. The purified compound was identified as 4-hydroxyphenylacetic acid by gas chromatography-electron ionization (GC-EI) and gas chromatography-chemical ionization (GC-CI) mass spectrometry. This antibiotic exhibited destructive activity toward P. capsici hyphae. In vivo experiments utilizing green house grown pepper plants demonstrated the protective effect of L. antibioticus HS124 against P. capsici. The growth of pepper plants treated with L. antibioticus culture was enhanced, resulting in greater protection from fungal disease. Optimum growth and protection was found when cultures were grown in presence of Fe(III). Additionally, the activities of pathogenesis-related proteins such as chitinase and β-1,3-glucanase decreased in roots, but increased in leaves with time after treatment compared to controls. Our results demonstrate L. antibioticus HS124 as a promising candidate for biocontrol of P. capsici in pepper plants.     

Purification,characterization and differential hormonal regulation of a β-1,3-glucanase and two chitinases from chickpea (Cicer arietinum L.)

Chickpea (Cicer arietinum L.) cell-suspension cultures were used to isolate one -1,3-glucanase (EC 3.2.1.29) and two chitinases (EC 3.2.1.14). The -1,3-glucanase (M_r = 36 kDa) and one of the chitinases (M_r = 32 kDa) belong to class I hydrolases with basic isoelectric points (10.5 and 8.5, respectively) and were located intracellularly. The basic chitinase (BC) was also found in the culture medium. The second chitinase (M_r = 28 kDa), with an acidic isoelectric point of 5.7, showed homology to N-terminal sequences of class III chitinases and represented the main protein accumulating in the culture medium. Polyclonal antibodies raised against the basic -1,3-glucanase (BG) and the acidic chitinase (AC) were shown to be monospecific. The anti-AC antiserum failed to recognize the BC on immune blots, confirming the structural diversity between class I and class III chitinases. Neither chitinase exhibitied lysozyme activity. All hydrolases were endo in action on appropriate substrates. The BC inhibited the hyphal growth of several test fungi, whereas the AC failed to show any inhibitory activity. Expression of BG activity appeared to be regulated by auxin in the cell culture and in the intact plant. In contrast, the expression of neither chitinase was apparently influenced by auxin, indicating a differential hormonal regulation of -1,3-glucanase and chitinase activities in chickpea. After elicitation of cell cultures or infection of chickpea plants with Ascochyta rabiei, both system were found to have hydrolase patterns which were qualitatively and quantitatively comparable. Finally, resitant (ILC 3279) and susceptible (ILC 1929) cultivars of chickpea showed no appreciable differences with regard to the time and amount of hydrolase accumulation after inoculation with spores of A. rabiei.Abbreviations AC acidic chitinase - BC basic chitinase - BG = basic -1,3-glucanase - CM-Chitin-RBV carboxymethylated-chitin-remazol brilliant violet - 2,4-D 2,4-dichlorophenoxyacetic acid - ILC international legume chickpea - M_r relative molecular mass - pI isoelectric point - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis We thank the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie for financial support and ICARDA, Aleppo, Syria, for the provision of seed material. We also thank Dr. B. Fritig (Institut de Biologie Moléculaire des Plantes, CNRS, Straßbourg, France) and Dr. F. Meins, Jr. (Friedrich-Miescher-Institut, Basel, Switzerland) for their kind gifts of antibodies.     

Primary structure and expression of mRNAs encoding basic chitinase and 1,3-β-glucanase in potato

Infection of potato leaves (Solanum tuberosum L. cv. Datura) by the late blight fungus Phytophthora infestans, or treatment with fungal elicitor leads to a strong increase in chitinase and 1,3--glucanase activities. Both enzymes have been implicated in the plant's defence against potential pathogens. In an effort to characterize the corresponding genes, we isolated complementary DNAs encoding the basic forms (class I) of both chitinase and 1,3--glucanase, which are the most abundant isoforms in infected leaves. Sequence analysis revealed that at least four genes each are expressed in elicitor-treated leaves. The structural features of the potato chitinases include a hydrophobic signal peptide at the N-terminus, a hevein domain which is characteristic of class I chitinases, a proline- and glycine-rich linker region which varies among all potato chitinases, a catalytic domain, and a C-terminal extension. The potato 1,3--glucanases also contain a N-terminal hydrophobic signal peptide and a C-terminal extension, the latter comprising a potential glycosylation site. RNA blot hybridization experiments showed that basic chitinase and 1,3--glucanase are strongly and coordinately induced in leaves in response to infection, elicitor treatment, ethylene treatment, or wounding. In addition to their activation by stress, both types of genes are regulated by endogenous factors in a developmental and organ-specific manner. Appreciable amounts of chitinase and 1,3--glucanase mRNAs were found in old leaves, stems, and roots, as well as in sepals of healthy, untreated plants, whereas tubers, root tips, and all other flower organs (petals, stamen, carpels) contained very low levels of both mRNAs. In young leaves and stems, chitinase and 1,3--glucanase were differentially expressed. While chitinase mRNA was abundant in these parts of the plant, 1,3--glucanase mRNA was absent. DNA blot analysis indicated that in potato, chitinase and 1,3--glucanase are encoded by gene families of considerable complexity.     

Oligogalacturonide signal transduction,induction of defense-related responses and protection of grapevine against<Emphasis Type="Italic"> Botrytis cinerea</Emphasis>

Grapevine (Vitis vinifera L.) is vulnerable to a variety of pathogenic fungi, among them Botrytis cinerea, the causal agent of grey mould, is responsible for worldwide yield losses that would be even more important without a successful control that relies mainly on fungicides. In the present work we investigated an alternative way of using oligogalacturonides (OGA) to induce defense responses in grapevine and protection against B. cinerea. Kinetic experiments with grapevine cells showed that OGA induced a rapid and transient generation of H₂O₂, followed by differential expression of nine defense-related genes and stimulation of chitinase and -1,3-glucanase activities. Inhibition of OGA-induced oxidative burst by diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, lowered induction levels of six genes and chitinase activity. Interestingly, the induction of three other genes and -1,3-glucanase activity were inhibited by K252a, a protein kinase inhibitor, but not by DPI. Treatment of grapevine leaves with OGA also reduced infection by B. cinerea by about 55–65%. Accordingly, DPI or K252a with or without OGA increased the susceptibility of grapevine leaves to B. cinerea. We suggest that treatment of grapevine with OGA elicits different signalling pathways, which might act in tandem with the oxidative burst to increase grapevine defense responses required for protection against B. cinerea.Abbreviations AOS Active oxygen species - Chit Chitinase - DPI Diphenylene iodonium - -Glu -1,3-Glucanase - GST Glutathione-S-transferase - MAP Mitogen-activated protein - OGA Oligogalacturonides - PAL Phenylalanine ammonia-lyase - PR Pathogenesis-related - PGIP Polygalacturonase inhibiting protein - PIN Serine-proteinase inhibitor - STS Stilbene synthase     

Differential induction of chitinase in Piper colubrinum in response to inoculation with Phytophthora capsici,the cause of foot rot in black pepper

Plant chitinases have been of particular interest since they are known to be induced upon pathogen invasion. Inoculation of Piper colubrinum leaves with the foot rot fungus, Phytophthora capsici leads to increase in chitinase activity. A marked increase in chitinase activity in the inoculated leaves was observed, with the maximum activity after 60 h of inoculation and gradually decreased thereafter. Older leaves showed more chitinase activity than young leaves. The level of chitinase in black pepper (Piper nigrum L.) upon inoculation was found to be substantially high when compared to P. colubrinum. RT–PCR using chitinase specific primers revealed differential accumulation of mRNA in P. colubrinum leaves inoculated with P. capsici. However, hyphal extension assays revealed no obvious differences in the ability of the protein extracts to inhibit growth of P. capsici in vitro.     

Diverse mechanisms adopted by fluorescent <Emphasis Type="Italic">Pseudomonas</Emphasis> PGC2 during the inhibition of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> and <Emphasis Type="Italic">Phytophthora capsici</Emphasis>

Rhizoctonia solani and Phytophthora capsici are two of the most destructive phytopathogens occurring worldwide and are only partly being managed by traditional control strategies. Fluorescent Pseudomonas isolates PGC1 and PGC2 were checked for the antifungal potential against R. solani and P. capsici. Both the isolates were screened for the ability to produce a range of antifungal compounds. The results of this study indicated the role of chitinase and β-1,3-glucanase in the inhibition of R. solani, however, antifungal metabolites of a non-enzymatic nature were responsible for inhibition of P. capsici. The study confirmed that multiple and diverse mechanisms are adopted by the same antagonist to suppress different phytopathogens, as evidenced in case of R. solani and P. capsici.     

Transgenic grapefruit plants expressing the PAPETALA3-IPT gp gene exhibit altered expression of PR genes

Transgenic grapefruit plants (Citrus paradisi cv. ‘Duncan’) with the isopentenyltransferase (ipt) gene under the control of APETALA3 promoter have been produced using Agrobacterium-mediated transformation. The relative expression level of the ipt gene was between 2.3 and 7 times higher in transformed plants than in the wild-type but despite the presence of a tissue-specific promoter, the expression was not limited only to flower tissue. Increased levels of trans-zeatin riboside between 9.4 and 32-fold found in transgenic grapefruit were considered the consequence of ectopic expression of the ipt gene. Chlorophyll levels in fully expanded uppermost leaves were also about 30% higher in transgenic than in wild-type plants. Involvement of cytokinins in control of expression of three pathogenesis-related protein genes: β-1,3-glucanase, a stress related PR gene 24P220, and an acidic chitinase, 24P262 was examined. Expression of β-1,3-glucanase, and 24P220 gene were significantly enhanced in transgenic plants while the expression of chitinase was reduced to low levels. Our results confirm the effect of cytokinins on expression of genes implicated in the response of grapefruit plants to pathogen attack and suggest a possible role of cytokinins in pathogen resistance.     

An agglutinating chitinase with two chitin-binding domains confers fungal protection in transgenic potato

Brassica juncea BjCHI1 is a unique chitinase with two chitin-binding domains. Here, we show that, unlike other chitinases, potato-expressed BjCHI1 shows hemagglutination ability. BjCHI1 expression in B. juncea seedlings is induced by Rhizoctonia solani infection, suggesting its protective role against this fungus. To verify this, transgenic potato (Solanum tuberosum L. cv. Desiree) plants expressing BjCHI1 generated by Agrobacterium-mediated transformation were challenged with R. solani. We also transformed potato with a cDNA encoding Hevea brasiliensis -1,3-glucanase, designated HbGLU, and a pBI121-derivative that contains cDNAs encoding both BjCHI1 and HbGLU. In vitro fungal bioassays using Trichoderma viride showed that extracts from transgenic potato lines co-expressing BjCHI1 and HbGLU inhibited fungal growth better than extracts from transgenic potato expressing either BjCHI1 or HbGLU, suggesting a synergistic effect. Consistently, in vivo fungal bioassays with soil-borne R. solani on young transgenic potato plants indicated that the co-expressing plants showed healthier root development than untransformed plants or those that expressed either BjCHI1 or HbGLU. Light microscopy and transmission electron microscopy revealed abundant intact R. solani hyphae and monilioid cells in untransformed roots and disintegrated fungus in the BjCHI1-expressing and the BjCHI1 and HbGLU co-expressing plants. Observations of collapsed epidermal cells in the co-expressing potato roots suggest that these proteins effectively degrade the fungal cell wall, producing elicitors that initiate other defense responses causing epidermal cell collapse that ultimately restricts further fungal penetration.     

Ethylene-induced chitinase and β-1,3-glucanase accumulate specifically in the lower epidermis and along vascular strands of bean leaves

We have studied the spatial pattern of accumulation of chitinase (EC 3.2.1.14) and -1,3-glucanase (EC 3.2.1.39) in ethylene-treated leaves of bean (Phaseolus vulgaris L.). Electron-microscopical examination of chemically fixed tissue demonstrated the presence of large electron-dense aggregates in the vacuoles of ethylene-treated leaf cells. No such vacuolar structures were observed in untreated control cells. Immunogold labelling with antisera directed against the basic forms of chitinase and -1,3-glucanase indicated that the vacuolar aggregates were the major site of accumulation of chitinase and -1,3-glucanase. The chitinase- and -1,3-glucanase-containing vacuolar aggregates were not randomly distributed within the leaf tissue but were restricted to the lower epidermal cells and to parenchyma cells adjacent to vascular strands. In addition, heavy -1,3-glucanase labelling was observed over spongy plugs of expanded middle-lamella material that appear to occlude the transition regions between the airspaces underlying the stomata and those throughout the rest of the leaf. Some labelling was also seen to extend along the surface layer of the cell walls lining all of the airspaces. Protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting as well as enzyme-activity measurements showed that the peeled lower epidermis of the ethylene-treated leaves contained on a protein and on a per-weight basis several times more chitinase and -1,3-glucanase than the remainder of the leaf.Abbreviation in Text SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Abbreviations in Micrographs AS air space - C chloroplast - EP (epidermal) cell - G guard cell - P parenchyma cell - S stoma - V vacuole - VE] vein - VP vascular parenchyma cell - W cell wall - X xylem We thank Dr. L.A. Hadwiger, Pullman, Wash., and Dr. U. Vögeli, Lexington, Ky., for their kind gifts of antibodies. This work was supported by the National Science Foundation grant DCB-8615763 to L.A.S.     

Exogenous systemin has a contrasting effect on disease resistance in mycorrhizal tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis>) plants infected with necrotrophic or hemibiotrophic pathogens

A study was performed to determine the effect of the systemin polypeptide on the bio-protective effect of arbuscular mycorrhizal fungi (AMF) in tomato plants infected with Alternaria solani, Phytophthora infestans or P. parasitica. Before infection, tomato plants were colonized with two different AMF, Glomus fasciculatum or G. clarum. In addition, a group of inoculated plants was treated with systemin, just after emergence. The exogenous application of systemin marginally suppressed the resistance against A. solani leaf blight observed in G. fasciculatum mycorrhizal plants but significantly enhanced it in plants colonized with G. clarum. Systemin induced resistance to P. parasitica in leaves of G. fasciculatum mycorrhizal plants, in which AMF colonization alone was shown to have no protective effect. Conversely, none of the treatments led to resistance to root or stem rots caused by P. infestans or P. parasitica. The above effects did not correlate with changes in the activity levels of β-1,3-glucanase (BG), chitinase (CHI), peroxidase (PRX), and phenylalanine ammonium lyase (PAL) in leaves of infected plants. However, they corroborated previous reports showing that colonization by AMF can lead to a systemic resistance response against A. solani. Systemic resistance to A. solani was similarly observed in non-mycorrhizal systemin-treated plants, which, in contrast, showed increased susceptibility to P. infestans and P. parasitica. The results indicated that the pattern of systemic disease resistance conferred by mycorrhizal colonization was dependent on the AMF employed and could be altered by the exogenous application of systemin, by means of a still undefined mechanism.     

Glutamate activates cation currents in the plasma membrane of<Emphasis Type="Italic"> Arabidopsis</Emphasis> root cells

The effect of glutamate on plant plasma membrane cation transport was studied in roots of Arabidopsis thaliana (L.) Heynh. Patch-clamp experiments using root protoplasts, ²²Na⁺ unidirectional fluxes into intact roots and measurements of cytosolic Ca²⁺ activity using plants expressing cytosolically-targeted aequorin in specific cell types were carried out. It was demonstrated that low-millimolar concentrations of glutamate activate within seconds both Na⁺ and Ca²⁺ currents in patch-clamped protoplasts derived from roots. The probability of observing glutamate-activated currents increased with increasing glutamate concentration (up to 29% at 3 mM); half-maximal activation was seen at 0.2–0.5 mM glutamate. Glutamate-activated currents were voltage-insensitive, instantaneous (completely activated within 2–3 ms of a change in voltage) and non-selective for monovalent cations (Na⁺, Cs⁺ and K⁺). They also allowed the permeation of Ca²⁺. Half-maximal Na⁺ currents occurred at 20–30 mM Na⁺. Glutamate-activated currents were sensitive to non-specific blockers of cation channels (quinine, La³⁺, Gd³⁺). Although low-millimolar concentrations of glutamate did not usually stimulate unidirectional influx of ²²Na⁺ into intact roots, they reliably caused an increase in cytosolic Ca²⁺ activity in protoplasts isolated from the roots of aequorin-transformed Arabidopsis plants. The response of cytosolic Ca²⁺ activity revealed a two-phase development, with a rapid large transient increase (lasting minutes) and a prolonged subsequent stage (lasting hours). Use of plants expressing aequorin in specific cell types within the root suggested that the cell types most sensitive to glutamate were in the mature epidermis and cortex. The functional significance of these glutamate-activated currents for both cation uptake into plants and cell signaling remains the subject of speculation, requiring more knowledge about the dynamics of apoplastic glutamate in plants.Abbreviations GLR Gene in plants encoding glutamate receptor-like protein - iGluRs Ionotropic glutamate receptors     

Effect of chitinase antisense RNA expression on disease susceptibility of Arabidopsis plants

Chitinases accumulate in higher plants upon pathogen attack are capable of hydrolyzing chitin-containing fungal cell walls and are thus implicated as part of the plant defense response to fungal pathogens. To evaluate the relative role of the predominate chitinase (class I, basic enzyme) of Arabidopsis thaliana in disease resistance, transgenic Arabidopsis plants were generated that expressed antisense RNA to the class I chitinase. Young plants or young leaves of some plants expressing antisense RNA had <10% of the chitinase levels of control plants. In the oldest leaves of these antisense plants, chitinase levels rose to 37–90% of the chitinase levels relative to vector control plants, most likely because of accumulation and storage of the enzyme in vacuoles. The rate of infection by the fungal pathogen Botrytis cinerea was measured in detached leaves containing 7–15% of the chitinase levels of control plants prior to inoculation. Antisense RNA was not effective in suppressing induced chitinase expression upon infection as chitinase levels increased in antisense leaves to 47% of levels in control leaves within 24 hours after inoculation. Leaves from antisense plants became diseased at a slightly faster rate than leaves from control plants, but differences were not significant due to high variability. Although the tendency to increased susceptibility in antisense plants suggests that chitinases may slow the growth of invading fungal pathogens, the overall contribution of chitinase to the inducible defense reponses in Arabidopsis remains unclear.     

Pest and disease resistance enhanced by heterologous suppression of a <Emphasis Type="Italic">Nicotiana plumbaginifolia</Emphasis> cytochrome P450 gene

The functional role of theNicotiana plumbaginifolia cytochrome P450 gene CYP72A2 was investigated in transgenic plants. N. tabacum plants transformed with a sense or antisense CYP72A2 construct exhibited diminished heights, branched stems, smaller leaves and deformed flowers. Western blot analysis revealed reduced levels of a 58kDa protein corresponding to CYP72A2, suggesting that the CYP72A2 homolog was suppressed in the sense and antisense plants. Transgenic plants had increased resistance to Manduca sexta larvae that consumed about 35 to 90 less of transgenic versus control leaves. A virulent strain of Pseudomonas syringae pv. tabaci induced a disease-limiting response followed by a delayed and decreased development of disease symptoms in the transgenics. CYP72A2 gene mediated resistance suggests that the plant-pest or -pathogen interactions may have been modified by changes in bioactive metabolite pools.     

Activation of Glycosidases as a Consequence of Infection Stress in Fusarium Wilt of Tomato

Changes of β-1,3-glucanase, chitinase, β-1,4-glucosidase and N-acetylglucosaminidase activity have been investigated in relation to the development of symptoms and colonization by the pathogen in roots, stems and leaves of susceptible (‘Improved, Pearson’) and resistant (‘Improved Pearson VF11’) tomato plants infected by Fusarium oxysporum f. sp. lycopersici. Glycosidase activities increased after inoculation to different extents depending on the plant part and cultivar. Increases were always higher in susceptible than in resistant plants. Changes in the β-1,3-glucanase activity after inoculation were particularly large in stems of infected plants. In contrast, chitinase activity increased more in roots than in stems. The β-1,3-glucosidase and chitinase activity decreased slightly from the basal to the apical third of stems. The trend of changes of the glycosidase activity generally were well related with the severity of disease symptoms and the fungal colonization of basal stem segments. There was no evidence that the increase of glycosidase activity after the infection was directly related with the resistance to Fusarium wilt in tomato.