Chitinase Research Articles

English

To evaluate the potential elicitor of homeopathic preparations on bean plants cv Carioca, homeopathics of Corymbia citriodora, Calcarea carbonica, Silicea and Sulphur in dynamisations 12, 24, 30 and 60CH were applied by pulverization throughout the aerial part. Samples of leaf tissue were taken at 6, 12, 24, 48 and 126 h after the treatment (HAT) to analyze the activity of peroxidase (POX), catalase (CAT), chitinase (CHI) and b-1.3-glucanase (GLU), and fifteen days after the application for the total contents of chlorophyll a and b. For the induction of phytoalexin phaseolin, seeds of the same genotype were germinated in the presence of respective treatments, and the production of phytoalexin quantified spectrophotometrically. All treatments increased the activity of POX, CAT, QUI and GLU, in at least one of the schedules evaluated in comparison to the control. The treatments C. citriodora and C. carbonic did not alter the contents of chlorophyll but induced the accumulation of phaseolin. Silicea and Sulphur caused significant reduction in the levels of chlorophyll a and b. The induction values were superior to the trading inductor (harpin), indicating that these homeopathies may come to be utilized as elicitor treatments on bean plants. The results indicate the potential of the treatments applied in the induction of biochemical mechanisms of defense in the bean plants.   Key words: Resistance induction, homeopathy, enzymes, phytoalexins.

Inhibition on brown rot disease and induction of defence response in harvested peach fruit by nitric oxide solution

Nitric oxide (NO) is an important signal molecule involved in numerous plant responses to biotic and abiotic stresses. The effect of nitric oxide (NO) solution on pathogen infection and defence response of peach (Prunus persica (L.) Batsch) fruit against brown rot disease caused by Monilinia fructicola was investigated. The results showed that 15 μmol l−1 NO solution did not significantly inhibit spore germination, germ tube length or pathogenicity of M. fructicola, but significantly reduced disease incidence and lesion areas in the fruit. Although 100 μmol l−1 NO solution effectively inhibited the spore germination, germ tube elongation and pathogenicity of M. fructicola, the high concentration of NO solution caused damage to the fruit. Moreover, 15 μmol l−1 NO enhanced the activities of chitinase (CHI) and β-1,3-glucanase (GNS) in the fruit. RT-PCR analysis showed that the expression of four genes, CHI, GNS, pathogenesis-related protein 1 and 10 genes (PR-1, PR-10) all increased after NO treatment. Conversely, pretreatment with 100 μmol l−1 NO scavenger, 2-4-carboxyphenyl-4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), rendered the fruit relatively susceptible to pathogen infection and inhibited the defence response of the fruit. These results suggest that NO solution treatment can protect peach fruit from pathogen infection by inducing the activities of the defence enzymes and the expression of PR genes.

DL-β-Aminobutyric Acid-Induced Resistance in Soybean against Aphis glycines Matsumura (Hemiptera: Aphididae)

Priming can improve plant innate capability to deal with the stresses caused by both biotic and abiotic factors. In this study, the effect of DL-β-amino-n-butyric acid (BABA) against Aphis glycines Matsumura, the soybean aphid (SA) was evaluated. We found that 25 mM BABA as a root drench had minimal adverse impact on plant growth and also efficiently protected soybean from SA infestation. In both choice and non-choice tests, SA number was significantly decreased to a low level in soybean seedlings drenched with 25 mM BABA compared to the control counterparts. BABA treatment resulted in a significant increase in the activities of several defense enzymes, such as phenylalanine ammonia-lyase (PAL), peroxidase (POX), polyphenol oxidase (PPO), chitinase (CHI), and β-1, 3-glucanase (GLU) in soybean seedlings attacked by aphid. Meanwhile, the induction of 15 defense-related genes by aphid, such as AOS, CHS, MMP2, NPR1-1, NPR1-2, and PR genes, were significantly augmented in BABA-treated soybean seedlings. Our study suggest that BABA application is a promising way to enhance soybean resistance against SA.

Induction of resistance to rice leaf blast by avirulent isolates of Magnaporthe oryzae

Rice blast (Magnaporthe oryzae) disease can cause losses of up to 100% in grain production. A sustainable management should consider alternative control measures. In the present work, biotic resistance induction to blast disease was studied by comparing disease severity and quantifying some pathogenesis-related proteins (PRPs). Rice plants of cultivars Metica-1 and Cica-8 were sprayed with biotic resistance inducers (incompatible isolates of M. oryzae) 48 h before inoculation with challenging isolates (compatible isolates of M. oryzae) under greenhouse conditions. For both cultivars, Metica-1 and Cica-8, the area under the disease progress curve was reduced by more than 80% when plants were induced with avirulent isolates of Magnaporthe oryzae. The biotic inducers also promoted an increase in the activity of peroxidase (POX), β-1.3-glucanase (GLU), chitinase (CHI), and phenylalanine ammonia-lyase (PAL). Systemic expression of resistance was also observed.

Role of Antioxidant Enzymes, Hydrogen Peroxide and PRProteins in the Compatible and Incompatible Interactions of Cowpea (Vigna unguiculata) Genotypes with the Fungus Colletotrichum gloeosporioides

Role of Antioxidant Enzymes, Hydrogen Peroxide and PRProteins in the Compatible and Incompatible Interactions of Cowpea (Vigna unguiculata) Genotypes with the Fungus Colletotrichum gloeosporioides Several diseases, such as anthracnose, which are caused by the fungus species Colletotrichum gloeosporioides, negatively affect the cultivation of the cowpea (Vigna unguiculata). This work was conducted to measured the time-course activities and evaluate the possible roles of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and hydrogen peroxide (H2O2), and the pathogenesis-related proteins (PR-proteins), peroxidase (POX), β-1,3-glucanase (GLU) and chitinase (CHI), in the resistant (TE97) and susceptible (BR3) cowpea genotypes in response to C. gloeosporioides infection.

Influence of water deficit on the molecular responses of Pinus contorta × Pinus banksiana mature trees to infection by the mountain pine beetle fungal associate, Grosmannia clavigera.

Conifers exhibit a number of constitutive and induced mechanisms to defend against attack by pests and pathogens such as mountain pine beetle (Dendroctonus ponderosae Hopkins) and their fungal associates. Ecological studies have demonstrated that stressed trees are more susceptible to attack by mountain pine beetle than their healthy counterparts. In this study, we tested the hypothesis that water deficit affects constitutive and induced responses of mature lodgepole pine × jack pine hybrids (Pinus contorta Dougl. ex Loud. var. latifolia Engelm. ex S. Wats. × Pinus banksiana Lamb.) to inoculation with the mountain pine beetle fungal associate Grosmannia clavigera (Robinson-Jeffrey and Davidson) Zipfel, de Beer and Wingfield. The degree of stress induced by the imposed water-deficit treatment was sufficient to reduce photosynthesis. Grosmannia clavigera-induced lesions exhibited significantly reduced dimensions in water-deficit trees relative to well-watered trees at 5 weeks after inoculation. Treatment-associated cellular-level changes in secondary phloem were also observed. Quantitative RT-PCR was used to analyze transcript abundance profiles of 18 genes belonging to four families classically associated with biotic and abiotic stress responses: aquaporins (AQPs), dehydration-responsive element binding (DREB), terpene synthases (TPSs) and chitinases (CHIs). Transcript abundance profiles of a TIP2 AQP and a TINY-like DREB decreased significantly in fungus-inoculated trees, but not in response to water deficit. One TPS, Pcb(+)-3-carene synthase, and the Class II CHIs PcbCHI2.1 and PcbCHI2.2 showed increased expression under water-deficit conditions in the absence of fungal inoculation, while another TPS, Pcb(E)-β-farnesene synthase-like, and two CHIs, PcbCHI1.1 and PcbCHI4.1, showed attenuated expression under water-deficit conditions in the presence of fungal inoculation. The effects were observed both locally and systemically. These results demonstrate that both constitutive and induced carbon- and nitrogen-based defenses are affected by water deficit, suggesting potential consequences for mountain pine beetle dynamics, particularly in novel environments.

Construction of a cassava PR protein‐interacting network during Xanthomonas axonopodis pv. manihotis infection

Pathogenesis‐related (PR) proteins are inducible antimicrobial defence proteins whose specific function in immunity or in other plant processes have not been deeply studied. Genes coding for PRs have been reported in different plant species with numerous homologous proteins belonging to the same functional protein family. Previously some PR candidate genes were identified in response to cassava bacterial blight (CBB) caused by Xanthomonas axonopodis pv. manihotis (Xam): the genes for hevamine (HEV), chitinase (CHI) and sulphite reductase (SiR) were induced in a resistant cassava cultivar during the response to Xam. To understand their functional roles in defence responses, a protein–protein interaction map was generated based on yeast two‐hybrid (Y2H) assays of these candidate PR proteins. The results showed that the cassava PRs interact with each other and, although hevamine and chitinase belong to the same class of chitinases, they share only four interactors. Co‐regulated expression of PRs and their interactors was observed and similar ontology terms were identified for interactors. Furthermore, an overlap between immune and other metabolic pathways was noticed based on other bioinformatics studies. These results therefore shed more light on the possible function of PR proteins.

The Role of Chitinases in Atopic Dermatitis

Amac: Bu calismada atopik dermatitli hastalardan alinan deri biyopsilerinde asidik memeli kitinaz (AMCase) gen ekspresyonu incelendi. Hastalar ve yontemler: Mayis 2005 Mayis 2007 tarihleri arasinda atopik dermatitli bes yetiskin hasta calismaya dahil edildi. Lezyonlu ve lezyonsuz bolgelerden deri biyopsileri alindi. Gercek zamanli polimeraz zincir reaksiyonu ile AMCase ekspresyonu incelendi. AMCase gen urunleri, beta-aktin ile karsilastirildi. Bulgular: Atopik lezyondan alinan numunelere kiyasla, AMCase yapimi derinin saglikli bolgelerinde daha yuksek oranda idi. Saglikli deri ve atopik deri dokusunda AMCase gen ekspresyonuna rastlandi. Sonuc: AMCase gen ekspresyonu her iki deri bolgesinde izole edildi. Bulgularimiz bu genin sadece mide ve akcigerde sinirli olmadigini ayni zamanda deride de bulundugunu gostermektedir. Anahtar sozcukler: Atopik dermatit; kitin mikropartikulu; kitin; kitinaz. Objectives: In this study, we investigated the acidic mammalian chitinase (AMCase) gene expression in skin biopsy samples taken from patients with atopic dermatitis. Patients and methods: Five adult patients with atopic dermatitis were enrolled in this study between May 2005 and May 2007. Skin biopsy samples were taken from lesional and unaffected areas. AMCase gene expression was tested by real-time polymerase chain reaction. The AMCase gene products were compared with beta-actin. Results: The AMCase production was higher in healthy regions of skin compared to the samples taken from atopic lesion. The AMCase gene expression was isolated in healthy and atopic skin regions. Conclusion: The AMCase gene expression was isolated both skin regions. Our study shown that this gene is not only expressed stomach and lung and also expressed in skin.

Differences in the Induction of Defence Responses in Resistant and Susceptible Muskmelon Plants Infected with Colletotrichum lagenarium

Defence reactions occurring in resistant (cv. Gankezaomi) and susceptible (cv. Ganmibao) muskmelon leaves were investigated after inoculating with Colletotrichum lagenarium. Lesion restriction in resistant cultivars was associated with the accumulation of hydrogen peroxide (H2O2). The activity of antioxidants catalase (CAT) and peroxidase (POD) significantly increased in both cultivars after inoculation, while levels of both CAT and POD activity were significantly higher in the resistant cultivar. Ascorbate peroxidase (APX) increased in both cultivars after inoculation, and level of APX activity was significantly higher in the resistant cultivar. Glutathione reductase (GR) activity significantly increased in both cultivars following inoculation, but was higher in the resistant cultivar, resulting in higher levels of ascorbic acid (AsA) and reduced glutathione (GSH). Phenylalanine ammonia lyase (PAL) significantly increased in inoculated leaves of both cultivars, resulting in higher levels of total phenolic compounds and flavonoids. The pathogenesis-related proteins chitinase (CHT) and β-1, 3-glucanase (GLU) significantly increased following inoculation with higher activity in the resistant cultivar. These findings show that resistance of muskmelon plants against C. lagenarium is associated with the rapid accumulation of H2O2, resulting in altered cellular redox status, accumulation of pathogenesis-related proteins, activation of phenylpropanoid pathway to accumulation of phenolic compounds and flavonoids.

INDUCED RESISTANCE IN TOMATO PLANTS TO CLAVIBACTER MICHIGANENSIS subsp. MICHIGANENSIS BY LENTINULA EDODES AND AGARICUS SUBRUFESCENS (syn. AGARICUS BRASILIENSIS)

Clavibacter michiganensis subsp. michiganensis can cause severe losses to tomato crops and new types of alternative control must be identified. Induced resistance is a potential method. Among resistance inducers, the mushrooms Lentinula edodes (Le) and Agaricus subrufescens (Abl) exhibit the potential to be used in plant disease control. The objectives of this study were: (i) evaluate the potential of these mushrooms and of acibenzolar-S-methyl (aSm) to control bacterial canker; (ii) study the mode of action of these materials by measuring defense-relate enzyme activity; (iii) partially purify biologically active molecules. The aqueous extracts from mushroom isolates, as well as aSm, did not inhibit bacterial growth in vitro. However, 10% (v/v) solutions of extracts from the isolates Le-96/17 and Abl- 26, as well as aSm, reduced wilt in tomato plants under greenhouse conditions. Plants treated with aSm, Abl-26 and Le-96/17 showed an increase in peroxidase and chitinase. Pphenylalanine ammonia-lyase (PAL) and polyphenol oxidase (PPO) activities increased with all treatments. Because isolate Le-96/17 induced the greatest wilt reduction, its aqueous extract was precipitated with ammonium sulfate. The fraction at 40-80% saturation was submitted to anion-exchange chromatography (AEC). The AEC fractions 3 and 4, the inducer aSm and the aqueous extract of Le-96/17 reduced the bacterial disease. The fractions 3 and 4, the 40-80% precipitate and the aqueous extract from Le-96/17 contained more than one protein band. In conclusion, the isolate Le-96/17 induced resistance to C. michiganensis in tomato and partial purification of the inducer molecules was obtained.

Inducers of resistance and silicon on the activity of defense enzymes in the soybean-Phakopsora pachyrhizi interaction

This study aimed to determine the effect of jasmonic acid (JA), Acibenzolar-S-Methyl (ASM) and calcium silicate (a source of soluble silicon, Si), on the potentiation of soybean resistance to Asian soybean rust (ASR). The ASR severity was significantly reduced on plants sprayed with ASM or supplied with Si in comparison to plants sprayed with JA or deionized water. For chitinases (CHI), significant differences in activity between non-inoculated and inoculated plants sprayed with deionized water or with ASM occurred at 72 hours after inoculation (hai), at 24 and 72 hai when sprayed with JA and at 141 hai when supplied with Si. For β-1,3-glucanases (GLU), significant differences in activity between non-inoculated and inoculated plants sprayed with deionized water occurred at 24, 48 and 141 hai, but not until 72 for plants sprayed with ASM. For phenylalanine ammonia-lyases (PAL), significant differences in activity between non-inoculated and inoculated plants occurred only for plants sprayed with ASM at 72 and 141 hai. In conclusion, the ASR symptoms can be mild on plants sprayed with ASM or supplied with Si and that this amelioration likely involved the defense enzymes.

β-Aminobutyric acid induces resistance of mango fruit to postharvest anthracnose caused by Colletotrichum gloeosporioides and enhances activity of fruit defense mechanisms

The effect of β-aminobutyric acid (BABA) on control of anthracnose caused by Colletotrichum gloeosporioides in mango fruit and its possible mechanisms were investigated. The results show that BABA treatments effectively suppressed the expansion of lesion in mango fruit inoculated with C. gloeosporioides during storage at 25°C, with the greatest efficacy being obtained using 100mM BABA. However, BABA at 25–400mM did not exhibit direct antifungal activity against C. gloeosporioides in vitro. Furthermore, BABA treatment at 100mM enhanced the activities of β-1,3-glucanase (GLU), chitinase (CHT) and phenylalanine ammonia lyase (PAL). BABA treatment also contributed to the accumulation of hydrogen peroxide (H2O2), while decreasing the rate of superoxide radical (O2−) production. Concurrently, BABA increased the activity of superoxide dismutase (SOD), while inhibiting catalase (CAT) and ascorbate peroxidase (APX) activities. These results indicate that increased disease resistance of mango fruit after BABA treatment during storage might be attributed to an elicitation of defense response involving in the enhancement of defense-related enzyme activities and modulation of antioxidant system activities.

Impact of carboxymethyl cellulose coating enriched with extract of Impatiens balsamina stems on preservation of ‘Newhall’ navel orange

A new approach to the control of postharvest decay, while maintaining fruit quality, has been implemented by the application of extract of Impatiens balsamina L. stems, a commonly used traditional Chinese medicine, amended coatings to ‘Newhall’ navel orange (Citrus sinensis L., Osbeck). After harvest, navel oranges were dipped in amended coating and then the samples were stored at 5°C and 90–95% RH after being dried naturally. The data suggested that the coating treatment, respectively, reduced the decay rate and weight loss of fruit from 10.2% to 6.1% and from 6.33% to 2.91% after 100 days storage, with none deleterious effects on fruit quality such as soluble solids content (SSC), titratable acidity (TA) and asorbic acid content (AsA). Moreover, the activities of scavenger antioxidant and defense enzymes, including peroxidase (POD), superoxide dismutase (SOD), chitinase (CHI) and β-1,3-glucanase (GLU), were also increased by the coating treatment. It indicates that the film may be an effective and safe alternative preservative for navel orange.

Urinary chitinase 3-like 1, a novel biomarker for acute kidney injury in adult cardiac intensive care patients: a pilot study

in adult cardiac intensive care patients: a pilot study Jorien De Loor, Eric Hoste, Ingrid Herck, Katrien Francois, Luc De Crop, Charlotte Clauwaert, Kristel Demeyere, and Evelyne Meyer 1 Department of Pharmacology, Toxicology and Biochemistry, Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium, Jorien.DeLoor@UGent.be 2 Faculty of Medicine and Health Sciences, Ghent University, 9000 Gent, Belgium

Mecanismos bioquímicos da defesa do algodoeiro à mancha de ramulária mediados pelo silício

Esse estudo investigou o efeito do silício (Si) na resistência do algodoeiro à mancha de ramulária (Ramularia areola). Plantas de algodoeiro (cvs. NuOpal e BRS Buriti) foram cultivadas em solução nutritiva nas concentrações de 0 (-Si) e 2 mM Si L-1 (+Si) e aos 30 dias após emergência foram submetidas à inoculação com uma suspensão de conídios de R. areola. Avaliou-se o período de incubação (PI), o período latente (PL60), a severidade, o número de lesões (NL) por cm² de folha, o tamanho de lesão (TL), a concentração foliar de Si e as atividades das enzimas de defesa peroxidases (POX), polifenoloxidases (PFO), quitinases (QUI), β-1,3-glucanases (GLU) e fenilalanina amônia-liases (FAL). Os dados de severidade foram usados para calcular a área abaixo da curva do progresso da mancha de ramulária (AACPMR). A concentração foliar de Si aumentou em 64% nas plantas supridas com Si em relação às não supridas com esse elemento. Houve aumentos de 10% e 14,7%, respectivamente, para o PI e o PL60 nas plantas supridas com Si. Reduções de 38,6% e 62,4% para o NL e de 17,2% e 26,6% para o TL ocorrem, respectivamente, nas plantas das cvs. NuOpal e BRS Buriti supridas com Si. Houve redução de 35% na AACPMR para as plantas supridas com Si em relação às não supridas. A concentração de compostos fenólicos solúveis totais nas plantas das duas cultivares supridas com Si foi maior aos 21 dias após inoculação (dai) em relação às não supridas com esse elemento. A concentração dos derivados da lignina foi maior dos 15 aos 21 dai apenas para as plantas da cv. BRS Buriti supridas com Si. A atividade da POX foi maior nas plantas das duas cultivares supridas com Si em relação às não supridas com esse elemento dos 15 aos 21 dai. Paras as plantas da cv. NuOpal supridas com Si, as atividades da PFO, QUI, GLU e FAL aumentaram aos 18 dai. As atividades da PFO e da FAL nas plantas da cv. BRS Buriti não foram potencializadas pelo Si. Nas plantas da cv. BRS Buriti supridas com Si, houve aumento nas atividades da QUI e da GLU aos 21 dai em relação às não supridas com Si. Conclui-se que a resistência do algodoeiro à mancha de ramulária foi bioquimicamente potencializada pelo Si, principalmente para as plantas da cv. NuOpal consideradas suscetíveis à mancha de ramulária.

Abstract 111: Analysis of novel insertion polymorphisms of human endogenous retrovirus in lung cancer patients.

Abstract Human endogenous retroviruses (HERVs) are members of the long terminal repeat (LTR) retrotransposon family accounting for about 8% of the human genome. A complete retroviral sequence includes promoter- and enhancer-active LTRs at both ends, but most HERV members appear to lack their mobility because of accumulated mutations within themselves. While the expression of HERV has long been a topic of cancer research, significant association between HERV insertion polymorphisms and cancers has never been reported. Here, we report the analysis of two novel polymorphic insertions of HERV solo LTR (accession no. AB728590 and AB728591) in lung cancer patients. To identify HERV insertion sites in the human genome, we firstly designed primer sequences targeting a conserved LTR region of HML-2, which is the most recently acquired HERV group and has the most intact retroviral sequence. Next, by using inverse PCR, a method amplifying unknown sequences flanking a primer target site, HERV-flanking sequences were amplified and cloned from lung cancer specimens. Finally, thirty two sequences were obtained from 178 clones. While twenty nine sequences were from known HERV loci, three sequences could not be assigned to any HERV loci in databases. Subsequent genomic analyses revealed that two sequences of those three sequences were located in 1p13.2 and 19q12 regions. In fact, we confirmed that HERV sequences were present in those genomic regions by the genotyping PCR and sequencing procedures. These two HERV insertions, HML-2_sLTR(1p13.2) and HML-2_sLTR(19q12), were solo LTRs and were also detected in normal tissues within the same individual, showing that they are polymorphic insertions. Furthermore, a retrospective cohort study indicated a significant prevalence of HML-2_sLTR(1p13.2) homozygosity (LTR/LTR genotype) among female never-smoking patients with lung adenocarcinoma (age => 60; in the west area of Shizuoka Prefecture; odds ratio, 1.97; 95% confidence interval, 1.02-3.81). Our results show that the insertion polymorphism of HML-2_sLTR(1p13.2) is involved in the susceptibility to lung adenocarcinoma in female never-smokers. Interestingly, acidic mammalian chitinase (CHIA), the product of which is known to show anti-apoptotic effects and to be secreted from lung epithelial cells, is located near HML-2_sLTR(1p13.2), suggesting that HML-2_sLTR(1p13.2) may regulate cell viability through the expression of CHIA. This report is the first to indicate an association between an insertion polymorphism of HERV and cancer and suggests that other HERV insertion polymorphisms related to cancer diseases may remain to be found in the human genome. Citation Format: Tomoaki Kahyo, Hiroki Mori, Nobuya Kurabe, Kazuhito Funai, Hiroshi Ogawa, Fumihiko Tanioka, Hong Tao, Suminori Kono, Keitaro Matsuo, Kazuya Shinmura, Haruhiko Sugimura. Analysis of novel insertion polymorphisms of human endogenous retrovirus in lung cancer patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 111. doi:10.1158/1538-7445.AM2013-111

Chitosan and oligochitosan enhance the resistance of peach fruit to brown rot

The effects of chitosan and oligachitosan on resistance induction of peach fruit against brown rot caused by Monilinia fructicola were investigated. Both chitosan and oligochitosan showed significant effect on controlling this disease. Moreover, chitosan and oligochitosan delayed fruit softening and senescence. The two antifungal substances enhanced antioxidant and defense-related enzymes, such as catalase (CAT), peroxidase (POD), β-1,3-glucanase (GLU) and chitinase (CHI), and they also stimulated the transcript expression of POD and GLU. These findings suggest that the effects of chitosan and oligochitosan on disease control and quality maintenance of peach fruit may be associated with their antioxidant property and the elicitation of defense responses in fruit.

Role of Interactions between Autographa californica Multiple Nucleopolyhedrovirus Procathepsin and Chitinase Chitin-Binding or Active-Site Domains in Viral Cathepsin Processing

The binding of Autographa californica multiple nucleopolyhedrovirus chitinase (CHIA) to viral cathepsin protease progenitor (proV-CATH) governs cellular/endoplasmic reticulum (ER) coretention of CHIA and proV-CATH, thus coordinating simultaneous cellular release of both host tissue-degrading enzymes upon host cell death. CHIA is a proposed proV-CATH folding chaperone because insertional inactivation of chiA causes production of proV-CATH aggregates that are incompetent for proteolytic maturation into active V-CATH enzyme. We wanted to determine whether the N-terminal chitin-binding domain (CBD, 149 residues) and C-terminal CHIA active-site domain (ASD, 402 residues) of CHIA bind to proV-CATH independently of one another and whether either domain is dispensable for CHIA's putative proV-CATH folding chaperone activity. We demonstrate that N-terminally green fluorescent protein (GFP)-fused CHIA, ASD, and CBD each colocalize with proV-CATH-RFP in ER-like patterns and that both ASD and CBD independently associate with proV-CATH in vivo using bimolecular fluorescence complementation (BiFC) and in vitro using reciprocal nickel-histidine pulldown assays. Altogether, the data from colocalization, BiFC, and reciprocal copurification analyses suggest specific and independent interactions between proV-CATH and both domains of CHIA. These data also demonstrate that either CHIA domain is dispensable for normal proV-CATH processing. Furthermore, in contrast to prior evidence suggesting that a lack of chiA expression causes proV-CATH to become aggregated, insoluble, and unable to mature into V-CATH, a chiA deletion bacmid virus we engineered to express just v-cath produced soluble proV-CATH that was prematurely secreted from cells and proteolytically matured into active V-CATH enzyme.

Purification and Characterization of Chitinase Produced by Endophytic <i>St</i><i>re</i><i>ptomyceshygroscopicus</i><i> </i>Against Some Phytopathogens

Microorganisms, wh ich secret a co mplex o f mycolytic enzy mes are considered to be possible bio logical control agents of plant d iseases. Since Ch itinases are digestive enzy mes that break down glycosidic bonds in chit in. In this present study, an industrial en zy me chitinase produced by endophyticStreptomyceswas purified and its antifungal act ivity was investigated against phytopathogens i.e. Rhizoctoniasolani,Fusariumoxysporum, Alternaria alternate, Aspergillusniger, Aspergillusflavus, Sclerotiniascleotiorum, Phytophthoraparasiticaand Botrytis cinerea.A chitinasewas produced byendophyticStreptomyceshygroscopicus in cultures containing chitin as the sole substrate that degrade chitin in 0.9 mm zone of clearance. The purification steps included ammoniu m sulfate precip itation, with colu mns of DEA E-Sepharose anion exchange chromatography and Sephacryl S-400 high resolution gel chro matography. The method gave a 5.4 fold increase of the specific activ ity and had a y ield of 18%. The mo lecular weight of the chit inase was found to be around 80.8, 78 and 76 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PA GE) technique. Chit inase was optimally active at pH of 6.0 to7.0 and at 30℃. The ch itanase were found to inhibit the growth of all phytopathogenic fungi.

Physiological and Biochemical Aspects of the Resistance of Banana Plants to Fusarium Wilt Potentiated by Silicon

Silicon amendments to soil have resulted in a decrease of diseases caused by several soilborne pathogens affecting a wide number of crops. This study evaluated the physiological and biochemical mechanisms that may have increased resistance of banana to Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense, after treatment with silicon (Si) amendment. Plants from the Grand Nain (resistant to F. oxysporum f. sp. cubense) and "Maçã" (susceptible to F. oxysporum f. sp. cubense) were grown in plastic pots amended with Si at 0 or 0.39 g/kg of soil (-Si or +Si, respectively) and inoculated with race 1 of F. oxysporum f. sp. cubense. Relative lesion length (RLL) and asymptomatic fungal colonization in tissue (AFCT) were evaluated at 40 days after inoculation. Root samples were collected at different times after inoculation with F. oxysporum f. sp. cubense to determine the level of lipid peroxidation, expressed as equivalents of malondialdehyde (MDA), hydrogen peroxide (H(2)O(2)), pigments (chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids), total soluble phenolics (TSP), and lignin-thioglycolic acid (LTGA) derivatives; the activities of the enzymes phenylalanine ammonia-lyases glucanases (PALs), peroxidases (POXs), polyphenoloxidases (PPOs), β-1,3-glucanases (GLUs), and chitinases (CHIs); and Si concentration in roots. Root Si concentration was significantly increased by 35.3% for the +Si treatment compared with the -Si treatment. For Grand Nain, the root Si concentration was significantly increased by 12.8% compared with "Maçã." Plants from Grand Nain and "Maçã" in the +Si treatment showed significant reductions of 40.0 and 57.2%, respectively, for RLL compared with the -Si treatment. For the AFCT, there was a significant reduction of 18.5% in the +Si treatment compared with the -Si treatment. The concentration of MDA significantly decreased for plants from Grand Nain and "Maçã" supplied with Si compared with the -Si treatment while the concentrations of H(2)O(2) on roots and pigments on leaves significantly increased. The concentrations of TSP and LTGA derivatives as well as the PALs, PPOs, POXs, GLUs, and CHIs activities significantly increased on roots of plants from Grand Nain and "Maçã" from the +Si treatment compared with the -Si treatment. Results of this study suggest that the symptoms of Fusarium wilt on roots of banana plants supplied with Si decreased due to an increase in the concentrations of H(2)O(2), TSP, and LTGA derivatives and greater activities of PALs, PPOs, POXs, GLUs, and CHIs.