Chitinase Gene Expression Research Articles

Dataset on recombinant expression of an ancient chitinase gene from different species of Leishmania parasites in bacteria and in Spodoptera frugiperda cells using baculovirus.

The data presented here is related to negative results obtained with the recombinant expression of chitinase from four species of Leishmania parasites in two expression systems, performed in order to investigate the molecular characteristics of the Leishmania chitinase and its possible application in leishmaniasis diagnosis. Thus, heterologous Leishmania sp chitinase proteins were expressed in bacteria using the prokaryotic expression vector pET28a and Escherichia coli Mach-T1, and in Spodoptera frugiperda (Sf9) insect cells, using the eukaryotic bac-to-bac expression system (Thermo Fisher Scientific) to produce recombinant baculoviruses to infect Sf9. Biochemical and cellular analysis of the various recombinant forms of the Leishmania sp chitinase produced in prokaryotic and eukaryotic expression systems were performed through SDS-PAGE and Western blotting. Chitinase produced and purified from bacteria presented low yield and formed inactive aggregates. Heterologous chitinase obtained after infection of Sf9 insect cells with all the four Leishmania species recombinant baculoviruses presented high yield of insoluble proteins. Dot-blot serological tests presented inconclusive results against the recombinant Leishmania sp chitinases produced in both expression systems. The experiments described in this paper can help researchers to avoid errors when choosing a recombinant expression systems to produce Leishmania parasites proteins for biotechnological purposes.

Thermophilic Chitinases: Structural, Functional and Engineering Attributes for Industrial Applications.

Chitin is the second most widely found natural polymer next to cellulose. Chitinases degrade the insoluble chitin to bioactive chitooligomers and monomers for various industrial applications. Based on their function, these enzymes act as biocontrol agents against pathogenic fungi and invasive pests compared with conventional chemical fungicides and insecticides. They have other functional roles in shellfish waste management, fungal protoplast generation, and Single-Cell Protein production. Among the chitinases, thermophilic and thermostable chitinases are gaining popularity in recent years, as they can withstand high temperatures and maintain the enzyme stability for longer periods. Not all chitinases are thermostable; hence, tailor-made thermophilic chitinases are designed to enhance their thermostability by direct evolution, genetic engineering involving mutagenesis, and proteomics approach. Although research has been done extensively on cloning and expression of thermophilic chitinase genes, there are only few papers discussing on the mechanism of chitin degradation using thermophiles. The current review discusses the sources of thermophilic chitinases, improvement of protein stability by gene manipulation, metagenomics approaches, chitin degradation mechanism in thermophiles, and their prospective applications for industrial, agricultural, and pharmaceutical purposes.

Grainy head signaling regulates epithelium development and ecdysis in Blattella germanica.

The transcription factor grainy head (Grh) functions in the protection of the epithelium against the external environment by generating strongly adhesive layers, and this function is conserved in vertebrates and invertebrates. In Drosophila, the top model for holometabolous insects, Grh is necessary during embryonic development, epidermal differentiation, central nervous system specification and epithelial repair. However, the function of this gene in hemimetabolous insect epithelia remains unknown. To examine the function of Grh signaling in regulating epithelium development in Hemimetabola, we focused on the Blattella germanica epidermal layer using a gene knockdown strategy. The spatiotemporal expression pattern of BgGrh was detected, and knockdown of BgGrh and BgCad96ca, which provide positive feedback to BgGrh, caused severe defects in new epithelium development and impeded the molting process required to discard the old integument. Knockdown of the expression of BgGrh and BgCad96ca caused increased expression of chitin synthase gene (BgCHS1) and chitinase gene (BgCht5), the upregulations of which should be mediated by the higher level of hormone receptor 3 (BgHr3) gene. In conclusion, epithelium development is regulated by Grh signaling, which might represent a potential target for the control of urban pest cockroaches.

EFFECT OF CHITOSAN FORMULATIONS OF DIFFERENT BIOLOGICAL ORIGIN ON TOBACCO (NICOTIANA TABACUM L.) PR-GENES EXPRESSION

During the life cycle, plants sustain a significant impact of external abiotic and biotic stress factors. Their reactions to the factors are complex and multi-level; they include changes in the transcriptomic, cellular, and physiological levels. The effect of chitosan formulations of different biological origin and molecular weight on the relative expression pattern of five pathogenesis-related (PR-)genes of tobacco (PR1a, PR2a, PR2b, PR3a and PR3b) was studied. Based on the analysis of nucleotide sequences deposited in the GenBank, specific primers for the amplification of exon regions of the five PR-genes were designed and the optimal conditions for real-time PCR, using SYBR Green dye determined. The relative expression pattern of the genes under study was influenced by the molecular weight of the chitosan used, the time passed after treatment and the very gene; The expression was generally higher when low-molecular chitosan fractions were used. Regardless of the biological origin of the elicitor, the formulations had the smallest effect on the expression of gene PR1a and the highest effect on the expression of the main chitinase gene (PR3b). Nicotiana tabacum L. leaves possess sensory systems triggering response reactions on the transcriptional level. The nature of the interaction between chitosan and cell sensor may differ, however, depending on the biological origin of chitosan, its molecular weight and the degree of deacetylation. The obtained results prove high sensitivity of defensive plant systems to the tested chitosan formulations and the ability of these formulations to penetrate tissues and interact with the relevant cellular sensors.

Proteome Responses to Individual Pathogens and Abiotic Conditions in Rice Seedlings.

Rice plants under field conditions experience various biotic and abiotic stresses and are adapted to survive using a molecular cross-talk of genes and their protein products based on the severity of a given stress. Seedlings of cultivated variety ASD16 (resistant to fungal disease, blast; tolerant to abiotic stress, salinity) were subjected to salt, drought, high temperature and low temperature stress as well as infection by Rhizoctonia solani and Xanthomonas oryzae pv. oryzae (causing reemerging diseases such as sheath blight and leaf blight), respectively, the sheath blight and bacterial leaf blight pathogens. Leaf proteome was analyzed using two-dimensional electrophoresis and differentially expressed proteins were identified using mass spectrometry. In addition to many other differentially expressed proteins, acidic endochitinase was found to be upregulated during fungal infection and drought treatment, and a germin-like protein upregulated during fungal infection and high temperature stress. These two proteins were further validated at the gene expression level using reverse transcription-PCR in dual stress experiments. Pot culture plants were subjected to fungal infection followed by drought and drought followed by fungal infection to validate chitinase gene expression. Similarly, plants subjected to fungal infections followed by high temperature stress and vice versa were used to validate the expression of germin-like protein-coding gene. The results of the present study indicate that chitinase and germin-like protein are potential targets for further exploration to develop rice plants resistant or tolerant to biotic and abiotic stresses.

Investigation of the quorum-sensing regulon of the biocontrol bacterium Pseudomonas chlororaphis strain PA23.

Pseudomonas chlororaphis strain PA23 is a biocontrol agent capable of protecting canola from stem rot disease caused by the fungal pathogen Sclerotinia sclerotiorum. PA23 produces several inhibitory compounds that are under control of a complex regulatory network. Included in this cascade is the PhzRI quorum sensing (QS) system, which plays an essential role in PA23 biocontrol, as well as CsaRI and AurRI, which have not yet been characterized in PA23. The focus of the current study was to employ RNA sequencing to explore the spectrum of PA23 genes under QS control. In this work, we investigated genes under the control of the main QS transcriptional regulator, PhzR, as well as those differentially expressed in an AHL-deficient strain, PA23-6863, which constitutively expresses an AiiA lactonase, rendering the strain QS defective. Transcriptomic profiling revealed 545 differentially expressed genes (365 downregulated; 180 upregulated) in the phzR mutant and 534 genes (382 downregulated; 152 upregulated) in the AHL-deficient PA23-6863. In both strains, decreased expression of phenazine, pyrrolnitrin, and exoprotease biosynthetic genes was observed. We have previously reported that QS activates expression of these genes and their encoded products. In addition, elevated siderophore and decreased chitinase gene expression was observed in the QS-deficient stains, which was confirmed by phenotypic analysis. Inspection of the promoter regions revealed the presence of “phz-box” sequences in only 58 of the 807 differentially expressed genes, suggesting that much of the QS regulon is indirectly regulated. Consistent with this notion, 41 transcriptional regulators displayed altered expression in one or both of the QS-deficient strains. Collectively, our findings indicate that QS governs expression of approximately 13% of the PA23 genome affecting diverse functions ranging from secondary metabolite production to general metabolism.

Recombinants Rhizobacteria Enhanced Biological Control of Rhizoctonia solani infected Faba Bean

This study aimed to test the effect of Rhizobium recombinants on the vegetative growth and yield components of faba bean under biotic stress of Rhizoctonia solani. Tweleve transconjugants resulted from six crosses were evaluated for their antigonism against Rhizoctonia solani. The results showed variations in chitin hydrolysis after genome shortening. At the molecular level, the recipient strains of rhizobia showed more similarity with each other. However, the donar strains of Serratia and pseudomonas showed less similarity. Meanwhile , Rhizobium transconjugants genomes have a similar distribution of bands. Both transconjugants Tr 4 and Tr15 showed more genome size than the other ones due to high level of plasmid transfer. The same transconjugants recorded higher values of chitin hydrolysis zone due to higher expression of chitinase genes. Most transconjugants showed significant increase in nodulation above the uninoculated plants grown in the infected soil. Rhizobium inoculants improved the yield , chlorophyll concentration and NPK content of the plants grown in the soil infected with Rhizoctonia solani compared with uninoculated plants. The results indicated that Rhizobium inoculants improved the growth and yield of plants grown under biotic stress condition. In addition, improved rhizobia with chitinolytic enzymes producing genes could inhibit significantly the growth of pathogenic fungi.

Peach-gum: A promising alternative for retarding the ripening and senescence in postharvest peach fruit

Abstract To evaluate the potential of peach-gum coating in retarding postharvest fruit ripening and softening, the effects of peach-gum treatment on storage performance and transcriptomes of peach fruit were studied during cold storage. Compared to the controls (CK), treatment with all tested concentrations (1 %, 5 %, and 10 %, v/v) of peach-gum repressed ethylene production and fruit softening, and to some extent, prevented weight loss. Peach-gum treatment did not alter malic acid, citric acid, quinic acid, glucose, fructose, or sucrose content, but it repressed the reduction in sorbitol. Transcriptomic analysis revealed that the expression of numerous genes related to fruit softening and cell wall degradation were repressed by peach-gum treatment, in accordance with the delayed softening observed. Meanwhile, the expression of senescence-associated genes, chitinase genes, and pathogenesis-related genes that were up-regulated during cold storage, were also inhibited by peach-gum treatment. Among the genes differentially expressed between peach-gum-treated and control fruit, genes involved in indole-3-acetic acid (IAA) transport and auxin response were relatively statistically overrepresented. A total of 90 transcription factors belonging to 26 families were differentially expressed. 21 of 23 zinc finger proteins from the four TF families, C2H2, C3H, CO, and Dof, were up-regulated in peach-gum-treated fruit. Additionally, abscisic acid and IAA content were markedly lower in peach-gum-treated fruit than in control fruit. Taken together, our study demonstrated that peach-gum can potentially serve as a new edible coating to preserve peach fruit. These results establish the basis for the future development of improved peach-gum-based edible coatings, by incorporating other effective compounds, and provide valuable information for further investigation of the regulatory mechanisms underlying fruit ripening and senescence in peaches.

Identification and expression analysis of chitinase genes in Zizania latifolia in response to abiotic stress

Zizania latifolia is the second-most cultivated aquatic vegetable in China, because of its nutritional and health-giving properties, and its cultivation is of considerable economic importance. Plant chitinases, as members of the glycoside hydrolase family, are considered primarily to be proteins involved in plant response to biotic and abiotic stresses, although they are also involved in many other physiological and morphological processes. In order to analyze the expression of chitinase genes under abiotic stress conditions in Z. latifolia and to better understand the functionality of these genes in this plant, we carried out a BLAST search of the Z. latifolia genome database and phylogenetic analysis based on the neighbor-joining method. Eleven chitinase genes were identified (ZlChi1-11) and classified into the chitinase classes I, II and IV. Seedlings were exposed to various abiotic stresses including salt, heat, cold, drought and the stress hormone abscisic acid (ABA) and expression patterns of all 11 chitinase genes in Z. latifolia were determined by quantitative reverse-transcription PCR. The results showed that most ZlChis were differentially expressed under abiotic stress conditions. The expression patterns indicated that most ZlChis responded to abiotic stresses within 6 h, although some ZlChis reached peak expression at 12 h or 18 h of abiotic stress treatment. Expression of ZlChi2 and ZlChi5 was upregulated under each of the stress treatments, while that of ZlChi10 or ZlChi11 was not detected under any conditions. This is the first report of expression analysis of all members of the glycoside hydrolase family 19 genes in Z. latifolia under abiotic stress, which provides basic information and insights into the putative roles of these genes in response to abiotic stress. The results of this study should serve as the basis for further functional characterization of these gene families in Zizania latifolia and related members of the Poaceae.

The combined use of the antagonistic yeast Hanseniaspora uvarum with β-aminobutyric acid for the management of postharvest diseases of kiwifruit

Significant losses in harvested kiwifruit can be directly attributed to decay fungi. In the present study, the use of an antagonistic yeast, Hanseniasporauvarum, combined with β-aminobutyric acid (BABA), a non-proteinogenic amino acid, was evaluated as a treatment for controlling postharvest infections of kiwifruit, artificially-inoculated with either Botrytis cinerea or Alternaria alternata. Natural infection of treated kiwifruits was also assessed. H.uvarum or BABA as stand-alone treatment, significantly reduced gray mold (B. cinerea) and black rot (A. alternata) on kiwifruit, relative to untreated control fruit, and also reduced the level of natural infection. The combination of H.uvarum and BABA, however, provided a superior level of postharvest disease control compared to either treatment alone. The growth of H.uvarum growth in kiwifruit wounds was not affected by BABA. Treatment of kiwifruit with either H.uvarum or BABA individually or in combination induced the gene expression and enzyme activity of chitinase and β-1,3-glucanase in kiwifruit. The ability of BABA to enhance the biocontrol efficacy of H.uvarum may be partially attributed to the elicitation of defense response in kiwifruit. The combined use of H.uvarum and BABA represents a promising alternative approach to the use of synthetic fungicides for the control of postharvest diseases in kiwifruit.

Genome-wide identification and characterization of Chitinase gene family in Brassica juncea and Camelina sativa in response to Alternaria brassicae

Chitinases belong to the group of Pathogenesis-related (PR) proteins that provides protection against fungal pathogens. This study presents the, genome-wide identification and characterization of chitinase gene family in two important oilseed crops B. juncea and C. sativa belonging to family Brassicaceae. We have identified 47 and 79 chitinase genes in the genomes of B. juncea and C. sativa, respectively. Phylogenetic analysis of chitinases in both the species revealed four distinct sub-groups, representing different classes of chitinases (I-V). Microscopic and biochemical study reveals the role of reactive oxygen species (ROS) scavenging enzymes in disease resistance of B. juncea and C. sativa. Furthermore, qRT-PCR analysis showed that expression of chitinases in both B. juncea and C. sativa was significantly induced after Alternaria brassicae infection. However, the fold change in chitinase gene expression was considerably higher in C. sativa compared to B. juncea, which further proves their role in C. sativa disease resistance to A. brassicae. This study provides comprehensive analysis on chitinase gene family in B. juncea and C. sativa and in future may serve as a potential candidate for improving disease resistance in B. juncea through transgenic approach.

Bioinformatics analysis and expression of chitinase genes in Armillaria gallica

Armillaria gallica is a facultative parasitic fungus which is the only nutrient source of Gastrodia elata during its cultivation.Chitinase,as a glycosidic hydrolytic enzyme,plays an important role in the growth,development,stress tolerance and symbiotic signal transduction of A. gallica. There were 22 chitinase genes in A. gallica. Bioinformatics analysis of amino acid sequence of these chitinase genes revealed that 12 chitinase genes contained glycosidase 18 family( GH18) domain. Chitinase amino acid sequences of A. gallica,A. ostoyae,G. elata,Saccharomyces cerevisiae and Trichoderma harzianum were analyzed byclustering trees,so as to further predict the gene function of chitinase in A. gallica. Induction of A. gallica branching with strigolactone analogue GR24,high-throughput sequencing technology based on the induction of branch group( MHJ1),uninduced branch group( MHJ2) and blank control group( MHJ3) is used to detect the expression quantity,the transcription level data of 22 chitinase genes were obtained and the heat map was generated for expression pattern analysis. It was found that 8 genes may be involved in physiological processes such as A. gallica branching,cell wall degradation and remodeling. In this paper,the function of chitinase gene in A. gallica was just preliminarily analyzed and predicted.

The deletion of the AcMNPV ac124 gene resulted in a decrease in chitinase transcription

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac124 gene has been previously characterized as a viral pathogenicity factor. In this study, an ac124-knockout virus (vAc124KO) was generated to examine the role of the ac124 gene in the context of the AcMNPV genome during infection. Our results showed that the absence of ac124 does not affect the production of budded virus (BV) and occlusion bodies (OBs) in infected Sf9 cells. Western blotting analysis showed that the deletion of ac124 does not affect the temporal expression and the relative levels of GP64, VP39, P6.9, and polyhedrin. qRT-PCR analysis showed that the transcription level of chitinase but not the adjacent cathepsin in vAc124KO infected cells was significantly lower than that of the vAcWT infected cells from 24 to 96 h p.i. Luciferase assays showed that the overexpression of Ac124 could significantly improve the ability of chitinase promoter to initiate reporter genes. Based on the above data, we hypothesize that Ac124 binds to the promoter of chitinase to regulate the expression of chitinase gene.

Expression of ChiA74∆sp and its truncated versions in Bacillus thuringiensis HD1 using a vegetative promoter maintains the integrity and toxicity of native Cry1A toxins

Our objective was to determine whether a recombinant chitinase ChiA74∆sp of Bacillus thuringiensis and its truncated versions (ChiA74∆sp-60, ChiA74∆sp-50) could be produced in B. thuringiensis HD1 with no detrimental effect on the size and insecticidal activity of the native bipyramidal Cry crystal. chiA-p, the promoter used to drive chitinase gene expression, was active during vegetative growth of Cry-B. HD1 recombinants showed increases from ~7- to 12-fold in chitinase activity when compared with parental HD1 and negligible or no effect on the volume of bipyramidal crystals was observed. HD1/ChiA74∆sp-60 showed increases from 20% to 40% in the yield of Cry1A per unit of culture medium when compared with parental HD1 and HD1/ChiA74∆sp-50, HD1/ChiA74∆sp. Inclusion bodies presumably composed of the enzyme attached to native Cry1A crystals of recombinant strains were observed; these inclusions were likely responsible for the enhancements in chitinase activity. Western blot analysis using polyclonal anti-ChiA74∆sp showed a weak signal with proteins of ~50 kDa in sporulated and lysed cells of recombinant strains. Bioassays against Spodoptera frugiperda using sporulated/lysed samples of the recombinant strains did not show statistically significant differences in LC50s when compared with HD1.

Changes in exoskeleton surface roughness and expression of chitinase genes in mud crab Macrophthalmus japonicus following heavy metal differences of estuary

Risk assessment of heavy metals is important for the health evaluation of inhabiting species in aquatic ecosystem. This study investigated whether chitin exoskeleton of mud crab Macrophthalmus japonicus is affected by heavy metals in estuary sediments in Korea. We compared heavy metal concentrations and analyzed the expression of M. japonicus chitinase genes, which play the crucial role in the formation of chitin exoskeleton. Concentrations of heavy metals were highly observed in crab body inhabiting Hampyeong among estuarine sites. High expressions of chitinase 1 were observed in crab gill and hepatopancreas from Myodo, which is the site with the lowest concentration of heavy metal in crab body. The surface roughness of the exoskeleton decreased with the increased concentration of heavy metals accumulated in the crab body. These results suggest that the total bioconcentration of heavy metals in crabs affected the expression of chitinase genes and changes in the exoskeleton surface roughness.

Overexpression of MdATG18a in apple improves resistance to Diplocarpon mali infection by enhancing antioxidant activity and salicylic acid levels

Marssonina apple blotch, caused by Diplocarpon mali, is one of the most serious diseases of apple. Autophagy plays a key role in pathogen resistance. We previously showed that MdATG18a has a positive influence on drought tolerance. Herein, we describe how overexpression (OE) of MdATG18a enhances resistance to D. mali infection, probably because less H2O2 but more salicylic acid (SA) is accumulated in the leaves of OE apple plants. Expression of chitinase, β-1,3-glucanase, and SA-related marker genes was induced more strongly by D. mali in OE lines. Transcript levels of other important MdATG genes were also drastically increased by D. mali in OE plants, which indicated increased autophagy activities. Taken together, these results demonstrate that OE of MdATG18a enhances resistance to infection by D. mali and plays positive roles in H2O2-scavenging and SA accumulations. Our findings provide important information for designing strategies which could induce autophagy to minimize the impact of this disease on apple production.

Effects of chitosan treatment on avocado postharvest diseases and expression of phenylalanine ammonia-lyase, chitinase and lipoxygenase genes

Stem-end rot (Lasiodiplodia theobromae) and anthracnose (Colletotrichum gloeosporioides) are major postharvest diseases in avocado that cause severe postharvest losses throughout the supply chain. One of the strategies to reduce development of such decay agents resides in the application of resistance inducers (e.g. chitosan), capable of reducing fungal growth and inducing resistance in fruit tissues.The influence of chitosan treatment (1% or 1.5% w/v) was investigated on decay incidence, gene expression of phenylalanine ammonia-lyase (PAL), chitinase (CHI) and lipoxygenase (LOX) and antioxidant enzyme activity [superoxide dismutase (SOD) and catalase (CAT)] in i) drop-inoculated (without wounding) or ii) artificially infected (with wounding) avocado (cv. ‘Hass’) with L. theobromae or C. gloeosporioides pathogens and also in iii) naturally infected (natural inoculum) avocado. Fruit were dipped in 1% or 1.5% w/v chitosan dissolved in water for 3 min, followed by storage for 14 d and 28 d respectively at 7.5 °C, and thereafter for 5 d at 18 °C to simulate market shelf conditions.Chitosan at 1.5% significantly reduced the incidence of stem-end rot and anthracnose in both inoculated and naturally infected avocados. The up-regulation of PAL and down-regulation of LOX genes moderately allowed higher epicatechin contents (90 mg kg−1 FW) in the exocarp, which could have contributed to improved anthracnose control. The up-regulation of CHI genes and higher SOD activity could have contributed to control of stem-end rot. Chitosan solution (1.5%) retained moderate levels of C7 sugars and firmness up to 5 d shelf life. The control of stem-end rot and anthracnose of avocados obtained with 1.5% chitosan can be ascribed to a combination of its antifungal and eliciting properties.

Acidic mammalian chitinase tuning after enteric helminths eradication in inflammatory respiratory disease patients.

This study aimed at investigating the presence of intestinal parasitic infections in inflammatory respiratory diseases patients during the disease attack, and measuring the acidic mammalian chitinase (AMCase) gene expression in blood before and after infection eradication. This case-control study included 123 inflammatory respiratory diseases patients and 120 apparently healthy individuals. Repeated stool examination was done, while total and specific IgE were measured. AMCase gene expression was analysed by real time-polymerase chain reaction (RT-PCR). Infection was detected in 32.5% of the diseased and 23.25% of the healthy individuals. Higher rate of the helminthic infection was detected (23.57) in comparison to the protozoal (12.19%) in the patients. A significantly higher rate of infection with the chitin-rich helminths "Enterobius vermicularis & Hymenolepis nana" and level of anti-Dermatophagoide-IgE were reported in the patients (14.63%, 6.5% and 23.57%, respectively). AMCase expression was significantly higher in helminths-infected patients than the noninfected, or protozoa infected. After infection eradication, AMCase expression significantly declined in the previously helminth-infected patients (mean ± SD=13.9±3.918 before and 4.515±1.93 after), but insignificantly affected in the protozoa infected (mean ± SD=2.095±.285 before and 2.675±1.181 after). Chitin-rich intestinal helminths are suspected to precipitate Th2-immune response in remote tissues by enhancing systemic AMCase expression through intestinal mucosa and macrophages irritation.

Expression of disease resistance in genetically modified grapevines correlates with the contents of viral sequences in the T-DNA and global genome methylation.

Increased tolerance to pathogens is an important goal in conventional and biotechnology-assisted grapevine breeding programs worldwide. Fungal and viral pathogens cause direct losses in berry production, but also affect the quality of the final products. Precision breeding strategies allow the introduction of resistance characters in elite cultivars, although the factors determining the plant's overall performance are not fully characterized. Grapevine plants expressing defense proteins, from fungal or plant origins, or of the coat protein gene of grapevine leafroll-associated virus 3 (GLRaV-3) were generated by Agrobacterium-mediated transformation of somatic embryos and shoot apical meristems. The responses of the transformed lines to pathogen challenges were investigated by biochemical, phytopathological and molecular methods. The expression of a Metarhizium anisopliae chitinase gene delayed pathogenesis and disease progression against the necrotrophic pathogen Botrytis cinerea. Modified lines expressing a Solanum nigrum osmotin-like protein also exhibited slower disease progression, but to a smaller extent. Grapevine lines carrying two hairpin-inducing constructs had lower GLRaV-3 titers when challenged by grafting, although disease symptoms and viral multiplication were detected. The levels of global genome methylation were determined for the genetically engineered lines, and correlation analyses demonstrated the association between higher levels of methylated DNA and larger portions of virus-derived sequences. Resistance expression was also negatively correlated with the contents of introduced viral sequences and genome methylation, indicating that the effectiveness of resistance strategies employing sequences of viral origin is subject to epigenetic regulation in grapevine.

Enhancement of nematicidal potential through cloning and expression of chitinase gene from Bacillus subtilis subsp. Subtilis BTN7A strain.

A gene encoding chitinase from B. subtilis has been isolated after optimization of PCR conditions. It was cloned with two different prometers, T7 promoter of the pJET1.2/blunt cloning vector and the SP6 promoter of pGEM®-T Easy vector. After transforming E. coli DH5α, two transformants were selected, CHI-NRC-4 from the first vector and T-CHI-NRC-6 from the second vector, and used for further studies. The complete CDS sequence of chitinase gene was determined and submitted to GenBank with the accession number KX268692.1. Culture supernatants of E. coli (CHI-NRC-4) and E. coli (T-CHI-NRC-6) were investigated for their inhibitory effect on M. javanica egg hatch under laboratory conditions. Result showed up to 96% inhibition in egg hatching due to both E. coli transformants as compared to control which reflect the same expression efficiency of both used prometers. A greenhouse experiment was carried out to evaluate the nematicidal effect of culture supernatants of the two transformts E. coli (CHI-NRC-4) and E. coli (T-CHI-NRC-6) against M. javanica infected eggplant. Obtained results showed a significant reduction in nematode population in soil and roots and enhancement in eggplant growth parameters as compared to control.