Chimeric Chitinase Research Articles

Enhanced antifungal activity of sugarcane cv. NCo310 expressing chimeric chitinase 42

The development of sustainable and environmentally safe strategies for crop protection against fungal pathogens is a significant challenge in agriculture. This paper discusses the successful transfer of a chimeric chitinase 42 gene into sugarcane using the biolistic method, resulting in an enhanced antifungal activity. Chitinases possess the enzymatic capability to degrade chitin, a key component of the cell walls in various fungal phytopathogens. In this research, we employed a chimeric chitinase 42 that combines the 42 kDa chitinase from Trichoderma atroviridae with a chitin binding domain from Serratia marcescens chitinase B. This chimeric chitinase exhibits higher antifungal activity against phytopathogenic fungi. PCR analysis confirmed the integration of the transgene into the genome of transformants, while the expression of the transgene was assessed using reverse transcription-PCR. Transgenic lines showed to have different chitinase activity and a high increase in chitinase activity was detected in lines T3, T5 and T6 with about 18 U/mg chitinase activity compared to the control plants (2.8 U/mg). Antifungal activity of transgenic lines T3, T5 and T6 on sugarcane fungal pathogens was shown in radial diffusion and detached leaf assays. The enhanced antifungal activity observed in the transformed sugarcane lines indicates the potential of chimeric chitinase 42 as an effective antifungal agent.

Structural modeling, expression and purification of Chimeric chitinase 42 containing His-tag in Nicotiana tabacum hairy root system

Chimeric chitinase42 (Chit42 containing ChBD) has great potential as a candidate for digesting and recycling chitin as a beneficial nutrient, which can be produced in bioreactors. The plant is one of the most efficient bioreactors that can produce the eukaryotic proteins in active forms. With the plant hairy root system, it is possible to express a variety of recombinant proteins cost-effectively, easily, and quickly. Due to the huge amount of proteins in plants, protein purification can be facilitated by the use of the His-tag. In this research, different computer programs were used for the three-dimensional structural analysis of Chimeric chitinase42 containing His-tag. The results showed that these comparative modeling approaches had a remarkable degree of accuracy in predicting the fused protein structure. The Z-score of -9.38 and -3.64 predicted for Chit42 and ChBD by ProSA represents the good quality of the model. In addition, bioinformatic observations showed that the His-tag was exposed and can be used to purify the Chimeric chitinase42. The Chimeric chitinase42 containing a His-tag was expressed in Nicotiana tabacum hairy roots, and the role of the His-tag in the detection by Western blot and purification using a Ni-NTA column was investigated. The presence of the Chimeric chitinase42 was confirmed by analyzing root extracts using SDS-PAGE and Western blot. The purification step was achieved using the His-tag and the Ni-NTA column. The plant-derived Chimeric chitinase42 was confirmed to be biologically active by measuring the chitinase activity of the purified protein on a media plate containing colloidal chitin.

Effect of the type of inoculation medium on chimeric chitinase gene transfer to lisianthus (Eustoma grandiflorum [Raf.] Shinn) for resistance to the fungal disease Fusarium solani

Effect of the type of inoculation medium on chimeric chitinase gene transfer to lisianthus (Eustoma grandiflorum [Raf.] Shinn) for resistance to the fungal disease Fusarium solani

The profile change of defense pathways in phaseouls vulgaris L. by biochemical and molecular interactions of Trichoderma harzianum transformants overexpressing a chimeric chitinase

Trichoderma species as biocontrol agents protect plants against several pathogens via multiple mechanisms. This study was aimed to evaluate the in vitro biocontrol activities and, as well as in vivo induction of systemic resistance of Phaseolus vulgaris L. by two Trichoderma harzianum transformants overexpressing a chimeric chitinase42 with chitin binding domain (T13 and T15) and wild-type (Tw) strain with and without Rhizoctonia solani. In order to achieve this goal, T. harzianum strains were compared in vitro for their potential to antagonize activity against R. solani. Also, the expression of biocontrol-associated genes (chit42 and qid74) and the volatile compounds (VOCs) profile with biocontrol activity was analyzed from T. harzianum strains. The real-time PCR was used to measure the expression of defense-related genes involved in systemic acquired resistance (SAR) and induced systemic resistance (ISR) pathways. The results showed that recombinant strains T15 (92%) and T13 (85%) had the higher inhibitory effects against R. solani compared to the Tw strain. Also, gene expression analysis of T. harzianum revealed the up-regulation of biocontrol-associated genes in recombinant strains. Analysis of VOCs from three T. harzianum strains identified a total of twenty-two compounds, including five that were produced by all of them .VOCs analysis of recombinant strains revealed the production of compounds, like caryophyllene oxide, 2-methyl-1-butanol, α-farnesene, and β-bisabolene. These compounds had been reported to induce plant defensive reactions against stress. Inoculation of plants with T13 and specially T15 strains, restricted the R. solani growth. Treatment of bean plants with recombinant strains led to increase in protection mechanisms including enhanced defense enzymes activity and higher expression of defense-related genes, as compared with Tw. The ability of recombinant strains to have better colonization within the soil and the root surface, describes its positive effects on defense mechanisms against R. solani.

Increased antifungal activity of Chit42 from Trichoderma atroviride by addition of a chitin binding domain

Chitin is the main component of the cell wall of plant pathogenic fungi. Chitinase 42 (Chit42) from Trichoderma atroviride (PTCC5220) plays a significant role in the biocontrol activity of this fungus against fungal pathogens. This enzyme lacks a chitin binding domain (ChBD) which is involved in its binding to crystalline chitin. In this research, a chimeric chitinase (Chit42+ ChBD) containing a strong chitin binding capacity was constructed by fusing a ChBD from chitinase 18-10 to Chit42 both from isolate PTCC5220. The construct was cloned and overexpressed in Escherichia coli BL21 (DE3). The fusion of ChBD improved the affinity to crystalline and colloidal chitin and also the thermal and chemical stability of the chimeric chitinase, when compared with the native Chit42. In vitro assays indicated that the chimeric chitinase showed higher antifungal activity toward plant pathogenic fungi.

Co-transformation of canola by chimeric chitinase and tlp genes towards improving resistance to Sclerotinia sclerotiorum.

Canola (Brassica napus) plants were co-transformed with two pathogenesis-related protein genes expressing a Trichoderma atroviride chitinase with a chitin-binding domain (chimeric chitinase) and a thaumatin-like protein (tlp) from Oryza sativa conferring resistance to phytopatogenic fungi by Agrobacterium-mediated transformation. The putative transgenic plants were confirmed by PCR. After measuring the specific activity of the chimeric chitinase and glucanase activity for tlp genes, transgenic plants with high specific activity were selected for southern blot analysis to confirm the copy number of the genes. In vitro assays, the antifungal activity of crude extracted protein against Sclerotinia sclerotiorum showed that the inhibition percentage in double transgenic plants was between 55 and 62, whereas the inhibition percentage in single-gene transformants (chimeric chitinase) ranged from 35 to 45 percent. Importantly, in greenhouse conditions, the double transgenic plants showed significant resistance than the single-gene transformant and wild type plants. The results in T2 generation using the intact leaf inoculation method showed that the average lesion diameters were 10, 14.7 and 29mm for the double transformant, single-gene transformant and non-transgenic plants, respectively. Combined expression of chimeric chitinase and tlp in transgenic plants showed significantly enhanced resistance against S. sclerotiorum than the one that express single-gene transformant plants. These results suggest that the co-expression of chimeric chitinase and tlp can confer enhanced disease resistance in canola plant.

Pathogen-induced expression of chimeric chitinase gene containing synthetic promoter confers antifungal resistance in transgenic canola

Controlling the expression of genes related to plant defense mechanisms is crucial when engineering plants with increased resistance to pathogens. In this study, synthetic promoters were placed upstream of a chimeric chitinase defense gene to produce transformation vectors. Canola plants were transformed with three constructs, pGDEC, pGMPC, pBISM2, containing synthetic promoters, synthetic pathogen-inducible promoter (SP-DDEE; parsley D and E17 elements + minimal promoter), SP-MP (minimal promoter), and the CaMV35S constitutive promoter, respectively. The results of reverse transcriptase PCR (RT-PCR) and enzyme activity assays show that the synthetic pathogen-inducible promoter (SP-DDEE) was responsive to the chitin and fungal elicitors, whereas negative control pGMPC did not respond. Furthermore promoters were assessed in the transgenic lines for antifungal activity against two phytopathogenic fungi. Results indicated that total proteins from transgenic lines carrying the SP-DDEE promoter when treated with elicitors strongly inhibited fungal growth, particularly Sclerotinia sclerotiorum, the major pathogen of canola. Overall, results indicate that not only was the SP-DDEE synthetic promoter highly responsive to the pathogen elicitors, but the inducible expression of the chimeric chitinase gene, when controlled by the SP-DDEE promoter, was also adequate to inhibit the fungal growth and development.

Co-expression of chimeric chitinase and a polygalacturonase-inhibiting protein in transgenic canola (Brassica napus) confers enhanced resistance to Sclerotinia sclerotiorum.

Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum is one of the major fungal diseases of canola. To develop resistance against this fungal disease, the chit42 from Trichoderma atroviride with chitin-binding domain and polygalacturonase-inhibiting protein 2 (PG1P2) of Phaseolus vulgaris were co-expressed in canola via Agrobacterium-mediated transformation. Stable integration and expression of transgenes in T0 and T2 plants was confirmed by PCR, Southern blot and RT-PCR analyses. Chitinase activity and PGIP2 inhibition were detected by colorimetric and agarose diffusion assay in transgenic lines but not in untransformed plants. The crude proteins from single copy transformant leaves having high chitinase and PGIP2 activity (T16, T8 and T3), showed up to 44% inhibition of S. sclerotiorum hyphal growth. The homozygous T2 plants, showing inheritance in Mendelian fashion (3:1), were further evaluated under greenhouse conditions for resistance to S. sclerotiorum. Intact plants contaminated with mycelia showed resistance through delayed onset of the disease and restricted size and expansion of lesions as compared to wild type plants. Combined expression of chimeric chit42 and pgip2 in Brassica napus L. provide subsequent protection against SSR disease and can be helpful in increasing the canola production in Iran.

Enhanced resistance to Sclerotinia sclerotiorum in Brassica napus by co-expression of defensin and chimeric chitinase genes.

Sclerotinia stem rot caused by Sclerotinia sclerotiorum is one of the major fungal diseases of Brassica napus L. To develop resistance against this fungal disease, the defensin gene from Raphanus sativus and chimeric chit42 from Trichoderma atroviride with a C-terminal fused chitin-binding domain from Serratia marcescens were co-expressed in canola via Agrobacterium-mediated transformation. Twenty transformants were confirmed to carry the two transgenes as detected by polymerase chain reaction (PCR), with 4.8% transformation efficiency. The chitinase activity of PCR-positive transgenic plants were measured in the presence of colloidal chitin, and five transgenic lines showing the highest chitinase activity were selected for checking the copy number of the transgenes through Southern blot hybridisation. Two plants carried a single copy of the transgenes, while the remainder carried either two or three copies of the transgenes. The antifungal activity of two transgenic lines that carried a single copy of the transgenes (T4 and T10) was studied by a radial diffusion assay. It was observed that the constitutive expression of these transgenes in the T4 and T10 transgenic lines suppressed the growth of S. sclerotiorum by 49% and 47%, respectively. The two transgenic lines were then let to self-pollinate to produce the T2 generation. Greenhouse bioassays were performed on the transgenic T2 young leaves by challenging with S. sclerotiorum and the results revealed that the expression of defensin and chimeric chitinase from a heterologous source in canola demonstrated enhanced resistance against sclerotinia stem rot disease.

A comparative study of transgenic canola (Brassica napus L.) harboring either chimeric or native Chit42 genes against phytopathogenic fungi

Canola (Brassica napus L.), an agro-economically important crop in the world, is sensitive to many fungal pathogens. One strategy to combat fungal diseases is genetic engineering through transferring genes encoding the pathogenesis-related (PR) proteins such as chitinase which cause the chitin degradation of fungal cell wall. Chitinase Chit42 from Trichoderma atroviride (PTCC5220) plays an important role in biocontrol and has high antifungal activity against a wide range of phytopathogenic fungi. This enzyme lacks a chitin binding domain (ChBD) which is involved in binding activity to insoluble chitin. In the present study, we investigated the effect of chitin binding domain fused to Chit42 when compared with native Chit42. These genes were over-expressed under the CaMV35S promoter in B. napus, R line Hyola 308. Transformation of cotyledonary petioles was achieved by pBISM2 and pBIKE1 constructs containing chimeric and native Chit42 genes respectively, via Agrobacterium method. The insertion of transgenes in T0 generation was verified through polymerase chain reaction (PCR) and Southern blot analysis. Antifungal activity of expressed chitinase in transgenic plants was also investigated by bioassays. The transgenic canola expressing chimeric chitinase showed stronger inhibition against phytopathogenic fungi that indicates the role of chitin binding domain.

Protein Engineering of Chit42 Towards Improvement of Chitinase and Antifungal Activities

The antagonism of Trichoderma strains usually correlates with the secretion of fungal cell wall degrading enzymes such as chitinases. Chitinase Chit42 is believed to play an important role in the biocontrol activity of Trichoderma strains as a biocontrol agent against phytopathogenic fungi. Chit42 lacks a chitin-binding domain (ChBD) which is involved in its binding activity to insoluble chitin. In this study, a chimeric chitinase with improved enzyme activity was produced by fusing a ChBD from T. atroviride chitinase 18-10 to Chit42. The improved chitinase containing a ChBD displayed a 1.7-fold higher specific activity than chit42. This increase suggests that the ChBD provides a strong binding capacity to insoluble chitin. Moreover, Chit42-ChBD transformants showed higher antifungal activity towards seven phytopathogenic fungal species.

Physicochemical study of a novel chimeric chitinase with enhanced binding ability

Chitinases are slow-reacting but important enzymes as they are anticipated to have diverse applications. The role of a chitin-binding domain (ChBD) in enhancing the quality of binding is essential information for purposeful engineering of chitinases. The idea of making hybrid chitinases by fusing a known ChBD to a chitinase, which naturally lacks ChBD is of interest especially for bio-controlling purposes. Therefore, in the present study, the ChBD of Serratia marcescens chitinase B was selected and fused to the fungal chitinase, Trichoderma atroviride Chit42. Both Chit42 and chemric Chit42 (ChC) showed similar activity towards colloidal chitin with specificity constants of 0.83 and 1.07 min(-1), respectively, same optimum temperatures (40°C), and similar optimum pH (4 and 4.5, respectively). In the presence of insoluble chitin, ChC showed higher activity (70%) and obtained a remarkably higher binding constant (700 times). Spectroscopic studies indicated that chimerization of Chit42 caused some structural changes, which resulted in a reduction of α-helix in ChC structure. Chemical and thermal stability studies suggested that ChC had a more stable structure than Chit42. Hill analysis of the binding data revealed mixed-cooperativity with positive cooperativity governing at ChC concentrations below 0.5 and above 2 µM in the presence of insoluble chitin. It is suggested that the addition of the ChBD to Chit42 affords structural changes which enhance the binding ability of ChC to insoluble chitin, improving its catalytic efficiency and increasing its thermal and chemical stability.

Designing a new chitinase with more chitin binding and antifungal activity

Chitinases have the ability of chitin digestion that constitutes a main compound of the cell wall in many of the phytopathogens such as fungi. Chitinase Chit42 from Trichoderma atroviride PTCC5220 is considered to play an important role in the biocontrol activity of this fungus against plant pathogens. Chit42 lacks a chitin binding domain (ChBD). We have produced a chimeric chitinase with stronger chitin-binding capacity by fusing to Chit42 a ChBD from Serratia marcescens Chitinase B. The fusion of ChBD improved the affinity to crystalline and colloidal chitin and also the enzyme activity of the chimeric chitinase when compared with the native Chit42. The chimeric chitinase showed higher antifungal activity toward phytopathogenic fungi.

Swapping the chitin-binding domain in Bacillus chitinases improves the substrate binding affinity and conformational stability

Chitinase from Bacillus thuringiensis and Bacillus licheniformis consisting of an N-terminal catalytic domain (GH18) and a C-terminal chitin-binding domain (ChBD), were cloned and characterised. In order to study the importance of individual domains, chimeric chitinases (BtGH-BliChBD and BliGH-BtChBD) were constructed using domain swapping as a strategy to exchange the CBD of BtGH-ChBD with that of BliGH-ChBD and vice versa. Both chimeric chitinases showed increased affinity to colloidal chitin. BtGH-BliChBD was different from the three other chitinases studied concerning optimum temperature and pH. Additionally, BtGH-BliChBD and BliGH-BtChBD showed significant improvement in functional stability, conformational stability, and binding ability towards insoluble chitinous substrates compared to those of the native chitinases.

Analysis of the involvement of chitin-binding domain of ChiCW in antifungal activity, and engineering a novel chimeric chitinase with high enzyme and antifungal activities.

An antifungal chitinase, ChiCW, produced by Bacillus cereus 28-9 is effective against conidial germination of Botrytis elliptica, the causal agent of lily leaf blight. ChiCW as a modular enzyme consists of a signal peptide, a catalytic domain, a fibronectin type-III-like domain, and a chitin-binding domain. When two C-terminal domains of ChiCW were truncated, ChiCWdeltaFC (lacking the chitin-binding domain and fibronectin type III-like domain) lost its antifungal activity. Since ChiCWdeltaAC (lacking the chitin-binding domain) could not be expressed in Escherichia coli as ChiCWdeltaFC did, a different strategy based on protein engineering technology was designed to investigate the involvement of the chitin-binding domain of ChiCW (ChBD(ChiCW)) in antifungal activity in this study. Because ChiA1 of Bacillus circulans WL-12 is a modular enzyme with a higher hydrolytic activity than ChiCW but not inhibitory to conidial germination of Bo. elliptica and the similar domain composition of ChiA1 and ChiCW, the C-terminal truncated derivatives of ChiA1 were generated and used to construct chimeric chitinases with ChBD(ChiCW). When the chitin-binding domain of ChiA1 was replaced with ChBD(ChiCW), the chimeric chitinase named ChiAAAW exhibited both high enzyme activity and antifungal activity. The results indicate that ChBD(ChiCW) may play an important role in the antifungual activity of ChiCW.

A family 19 chitinase ( Chit30) from Streptomyces olivaceoviridis ATCC 11238 expressed in transgenic pea affects the development of T. harzianum in vitro

Embryo axes excised from mature seeds of pea ( Pisum sativum L.) cv. ‘Sponsor’ were used as explants for Agrobacterium-mediated transformation using pGreenII 0229 binary vectors. The vectors harbored a chimeric chitinase gene ( chit30), driven by the constitutive 35S promoter or the elicitor inducible stilbene synthase ( vst) promoter from grape ( Vitis vinifera L.). The secretion signal of the bacterial chitinase gene from Streptomyces olivaceoviridis ATCC 11238 (DSM 41433) was replaced by the A. thaliana basic chitinase leader sequence. Functional properties of the recombinant gene were tested in tobacco as a model system before the long process of pea transformation was undertaken. Several transgenic pea clones were obtained and the transgenic nature confirmed by different molecular methods. The accumulation and activity of chitinase in stably transformed plants were examined by Western blot analysis and in-gel assays, which showed the presence of an additional 3 isoform bands. Using in vitro bioassays with Trichoderma harzanium as a model, we found an inhibition or delay of hyphal extension, which might indicate enhanced antifungal activity compared with non-transformed pea plants. Up to the 4th generation, the transgenic plants did not show any phenotypic alterations compared with non-transgenic control plants.

Improving the insecticidal activity of Bacillus thuringiensis subsp. aizawai against Spodoptera exigua by chromosomal expression of a chitinase gene.

A transcriptionally fused gene comprising the P19 gene from Bacillus thuringiensis subsp. israelensis fused with a chitinase gene (chiBlA) from B. licheniformis was integrated into the B. thuringiensis subsp. aizawai BTA1 genome by homologous recombination. The resulting B. thuringiensis subsp. aizawai strain (INT1) showed growth and sporulation comparable with that of the wild-type strain. INT1 produced four chitinases of different molecular masses (i.e., 66, 55, 39, 36 kDa). Three of these (66, 55, 36 kDa) were derived from the cloned chiBlA gene, whereas the 39-kDa chitinase originated from BTA1. Using surface contamination bioassays, the 50% lethal concentration of lyophilized whole culture broth of INT1 against Spodoptera exigua neonate larvae was 12.2 microg/cm2, compared with 30.8 microg/cm2 for BTA1. Bioassays using filtered culture supernatant of INT1 (110 microg/cm2) together with trypsin-activated purified Cry1C protein of B. thuringiensis (1,280 ng/cm2) showed 75.0% mortality, compared with 56.7% mortality for Cry1C combined with BTA1 at the same concentration. Using scanning electron microscopy, clear perforations were observed in S. exigua fifth instar peritrophic membranes incubated with either crude or purified chitinase, or isolated from fifth instar S. exigua fed purified chitinase since the first instar. These results show that chitinase can increase the activity of B. thuringiensis subsp. aizawai against S. exigua. This is the first documentation of expressing a chimeric chitinase gene on the chromosome of B. thuringiensis; and chromosomal integration might be used as a potential technique for strain improvement.

Increased antifungal and chitinase specific activities of Trichoderma harzianum CECT 2413 by addition of a cellulose binding domain.

Trichoderma harzianum is a widely distributed soil fungus that antagonizes numerous fungal phytopathogens. The antagonism of T. harzianum usually correlates with the production of antifungal activities including the secretion of fungal cell walls that degrade enzymes such as chitinases. Chitinases Chit42 and Chit33 from T. harzianum CECT 2413, which lack a chitin-binding domain, are considered to play an important role in the biocontrol activity of this strain against plant pathogens. By adding a cellulose-binding domain (CBD) from cellobiohydrolase II of Trichoderma reesei to these enzymes, hybrid chitinases Chit33-CBD and Chit42-CBD with stronger chitin-binding capacity than the native chitinases have been engineered. Transformants that overexpressed the native chitinases displayed higher levels of chitinase specific activity and were more effective at inhibiting the growth of Rhizoctonia solani, Botrytis cinerea and Phytophthora citrophthora than the wild type. Transformants that overexpressed the chimeric chitinases possessed the highest specific chitinase and antifungal activities. The results confirm the importance of these endochitinases in the antagonistic activity of T. harzianum strains, and demonstrate the effectiveness of adding a CBD to increase hydrolytic activity towards insoluble substrates such as chitin-rich fungal cell walls.

A new approach to directed gene evolution by recombined extension on truncated templates (RETT)

We describe a new approach to in vitro DNA recombination technique termed recombined extension on truncated templates (RETT). RETT generates random recombinant gene library by template-switching of unidirectionally growing polynucleotides from primers in the presence of unidirectional single-stranded DNA fragments used as templates. RETT was applied to the recombination of two homologous chitinase genes from S. marcescens ATCC 21074 and S. liquefaciens GM1403. When the shuffled genes were examined by restriction mapping and sequence analysis, it was found that chimeric genes were produced at a high frequency (more than 70%) between two chitinase genes with 83% of sequence identity. The number of crossovers within each chimeric gene ranged from one to four, and the recombination points were randomly distributed along entire DNA sequence. We also applied RETT to directed evolution of a chitinase variant for enhancing thermostability. Chimeric chitinases that were more thermostable than the parental enzyme were successfully obtained by RETT-based recombination.

Addition of substrate-binding domains increases substrate-binding capacity and specific activity of a chitinase from Trichoderma harzianum

Chitinase Chit42 from Trichoderma harzianum CECT 2413 is considered to play an important role in the biocontrol activity of this fungus against plant pathogens. Chit42 lacks a chitin-binding domain (ChBD). We have produced hybrid chitinases with stronger chitin-binding capacity by fusing to Chit42 a ChBD from Nicotiana tabacum ChiA chitinase and the cellulose-binding domain from cellobiohydrolase II of Trichoderma reesei. The chimeric chitinases had similar activities towards soluble substrate but higher hydrolytic activity than the native chitinase on high molecular mass insoluble substrates such as ground chitin or chitin-rich fungal cell walls.