Plant Chitinases Research Articles

Genome-Wide Identification of a Maize Chitinase Gene Family and the Induction of Its Expression by Fusarium verticillioides (Sacc.) Nirenberg (1976) Infection.

Maize chitinases are involved in chitin hydrolysis. Chitinases are distributed across various organisms including animals, plants, and fungi and are grouped into different glycosyl hydrolase families and classes, depending on protein structure. However, many chitinase functions and their interactions with other plant proteins remain unknown. The economic importance of maize (Zea mays L.) makes it relevant for studying the function of plant chitinases and their biological roles. This work aims to identify chitinase genes in the maize genome to study their gene structure, family/class classification, cis-related elements, and gene expression under biotic stress, such as Fusarium verticillioides infection. Thirty-nine chitinase genes were identified and found to be distributed in three glycosyl hydrolase (GH) families (18, 19 and 20). Likewise, the conserved domains and motifs were identified in each GH family member. The identified cis-regulatory elements are involved in plant development, hormone response, defense, and abiotic stress response. Chitinase protein-interaction network analysis predicted that they interact mainly with cell wall proteins. qRT-PCR analysis confirmed in silico data showing that ten different maize chitinase genes are induced in the presence of F. verticillioides, and that they could have several roles in pathogen infection depending on chitinase structure and cell wall localization.

Glucanases and Chitinases in Mangifera indica: Identification, Classification, Phylogeny, and Expression Analysis of Defense Genes against Colletotrichum spp.

Plant glucanases and chitinases are defense proteins that participate in pathogenesis; however, very little is known about the glucanase (GLUC) and chitinase (CHIT) gene families in mango. Some mango cultivars are of great economic importance and can be affected by anthracnose, a postharvest disease caused by fungi of the genus Colletotrichum spp. This study identified and characterized 23 putative glucanases and 16 chitinases in the mango genome cv. Tommy Atkins. We used phylogenetic analyses to classify the glucanases into three subclasses (A, B, and C) and the chitinases into four classes (I, II, IV, and V). Information on the salicylic, jasmonic acid, and ethylene pathways was obtained by analyzing the cis-elements of the GLUC and CHIT class I and IV gene promoters. The expression profile of GLUC, CHIT class I, and CHIT class IV genes in mango cv. Ataulfo inoculated with two Colletotrichum spp. revealed different profile expression related to these fungi's level of virulence. In general, this study provides the basis for the functional validation of these target genes with which the regulatory mechanisms used by glucanases and chitinases as defense proteins in mango can be elucidated.

Application of MF3 Microbial Recombinant Protein in Refolding of Plant Chitinase

Application of MF3 Microbial Recombinant Protein in Refolding of Plant Chitinase

APPLICATION OF MICROBIAL RECOMBINANT PROTEIN MF3 IN REFOLDING OF PLANT CHITINASE

Expression of recombinant proteins is important for studying their biological function. Most often, the expression system of the E. coli is used for the primary description of protein properties. However, under overexpression conditions, the rate of aggregation of target proteins often exceeds the rate of proper folding, resulting in the formation of insoluble inclusion bodies. Inclusion bodies are a clear disadvantage of the E. coli expression system because they interfere with the release of target recombinant proteins. One solution to the existing problem is the use of chaperone-like proteins in vitro to refold the target protein. In this work, the recombinant protein MF3 was taken as an example of a chaperone-like protein, which increased the yield of soluble plant chitinase by 92% compared to the yield of this protein using the standard refolding procedure.

Modulation of the Host Defence System by Nematophagous Fungi and Chitosan

Nematophagous fungi (NFs), which are responsible for soil suppression of plant-parasitic nematodes, are multitrophic biocontrol agents. This raises the question of the transition between lifestyles (e.g., endophytism vs. egg parasitism). The NF Pochonia chlamydosporia colonises food crops and promotes their growth and yield. When colonising the plant, P. chlamydosporia induces the plant immunity (PI). However, it also evades the PI. To do this, both endophytic NF and pathogenic fungi (PF) secrete LysM effectors (LysM-effs). LysM effectors have been shown to have diverse functions in different organisms, including the protection of fungal chitin from plant chitinases. P. chlamydosporia is resistant to chitosan, which modulates gene expression in fungi and plants and has antimicrobial properties. P. chlamydosporia chitin deacetylases (CDA) and chitosanases (CSN) also help P. chlamydosporia evade plant immunity, resist exogenous chitosan, and are induced during fungal infection of nematode eggs. NF-chitosan formulations are new biomanagement tools against plant parasitic nematodes, fungal wilt pathogens and insect pests that currently threaten food security crops. Furthermore, omics techniques are useful tools to elucidate the role of CDAs, CSNs, LysM-effs, adhesion proteins and carbohydrate-active enzymes in pathogen–BCA–plant interactions, adhesion and infection to nematode eggs and their modulation by chitosan.

Plant chitinases: Types, structural classification, antifungal potential and transgenic expression in plants for enhanced disease resistance

Plant chitinases: Types, structural classification, antifungal potential and transgenic expression in plants for enhanced disease resistance

Characterization of soybean chitinase genes induced by rhizobacteria involved in the defense against Fusarium oxysporum.

Rhizobacteria are capable of inducing defense responses via the expression of pathogenesis-related proteins (PR-proteins) such as chitinases, and many studies have validated the functions of plant chitinases in defense responses. Soybean (Glycine max) is an economically important crop worldwide, but the functional validation of soybean chitinase in defense responses remains limited. In this study, genome-wide characterization of soybean chitinases was conducted, and the defense contribution of three chitinases (GmChi01, GmChi02, or GmChi16) was validated in Arabidopsis transgenic lines against the soil-borne pathogen Fusarium oxysporum. Compared to the Arabidopsis Col-0 and empty vector controls, the transgenic lines with GmChi02 or GmChi16 exhibited fewer chlorosis symptoms and wilting. While GmChi02 and GmChi16 enhanced defense to F. oxysporum, GmChi02 was the only one significantly induced by Burkholderia ambifaria. The observation indicated that plant chitinases may be induced by different rhizobacteria for defense responses. The survey of 37 soybean chitinase gene expressions in response to six rhizobacteria observed diverse inducibility, where only 10 genes were significantly upregulated by at least one rhizobacterium and 9 genes did not respond to any of the rhizobacteria. Motif analysis on soybean promoters further identified not only consensus but also rhizobacterium-specific transcription factor-binding sites for the inducible chitinase genes. Collectively, these results confirmed the involvement of GmChi02 and GmChi16 in defense enhancement and highlighted the diverse inducibility of 37 soybean chitinases encountering F. oxysporum and six rhizobacteria.

Plant root associated chitinases: structures and functions.

Chitinases degrade chitin, a linear homopolymer of β-1,4-linked N-acetyl-D-glucosamine (GlcNAc) residues found in the cell walls of fungi and the exoskeletons of arthropods. They are secreted by the roots into the rhizosphere, a complex and dynamic environment where intense nutrient exchange occurs between plants and microbes. Here we modeled, expressed, purified, and characterized Zea mays and Oryza sativa root chitinases, and the chitinase of a symbiotic bacterium, Chitinophaga oryzae 1303 for their activities with chitin, di-, tri-, and tetra-saccharides and Aspergillus niger, with the goal of determining their role(s) in the rhizosphere and better understanding the molecular mechanisms underlying plant-microbe interactions. We show that Zea mays basic endochitinase (ZmChi19A) and Oryza sativa chitinase (OsChi19A) are from the GH19 chitinase family. The Chitinophaga oryzae 1303 chitinase (CspCh18A) belongs to the GH18 family. The three enzymes have similar apparent K M values of (20-40 µM) for the substrate 4-MU-GlcNAc3. They vary in their pH and temperature optima with OsChi19A activity optimal between pH 5-7 and 30-40°C while ZmChi19A and CspCh18A activities were optimal at pH 7-9 and 50-60°C. Modeling and site-directed mutation of ZmChi19A identified the catalytic cleft and the active residues E147 and E169 strategically positioned at ~8.6Å from each other in the folded protein. Cleavage of 4-MU-GlcNAc3 was unaffected by the absence of the CBD but diminished in the absence of the flexible C-terminal domain. However, unlike for the soluble substrate, the CBD and the newly identified flexible C-terminal domain were vital for inhibiting Aspergillus niger growth. The results are consistent with the involvement of the plant chitinases in defense against pathogens like fungi that have chitin exoskeletons. In summary, we have characterized the functional features and structural domains necessary for the activity of two plant root chitinases that are believed to be involved in plant defense and a bacterial chitinase that, along with the plant chitinases, may participate in nutrient recycling in the rhizosphere.

The molecular structure, biological roles, and inhibition of plant pathogenic fungal chitin deacetylases.

Chitin/polysaccharide deacetylases belong to the carbohydrate esterases family 4 (CE4 enzymes). They play a crucial role in modifying the physiochemical characteristics of structural polysaccharides and are also involved in a wide range of biological processes such as fungal autolysis, spore formation, cell wall formation and integrity, and germling adhesion. These enzymes are mostly common in fungi, marine bacteria, and a limited number of insects. They facilitate the deacetylation of chitin which is a structural biopolymer that is abundantly found in fungal cell walls and spores and also in the cuticle and peritrophic matrices of insects. The deacetylases exhibit specificity towards a substrate containing a sequence of four GlcNAc units, with one of these units being subjected to deacetylation. Chitin deacetylation results in the formation of chitosan, which is a poor substrate for host plant chitinases, therefore it can suppress the host immune response triggered by fungal pathogens and enhance pathogen virulence and colonization. This review discusses plant pathogenic fungal chitin/polysaccharide deacetylases including their structure, substrate specificity, biological roles and some recently discovered chitin deacetylase inhibitors that can help to mitigate plant fungal diseases. This review provides fundamental knowledge that will undoubtedly lead to the rational design of novel inhibitors that target pathogenic fungal chitin deacetylases, which will also aid in the management of plant diseases, thereby safeguarding global food security.

A Review of the Chitinolytic Enzyme from Actinomycetes from the Western Ghats

Chitinase has a wide distribution of fungi, plants, bacteria, yeast, humans, protozoa, and insects, in the environment. Chitinases have different functions in these organisms. The mineral composition of chitin, nutrients, and bacterial parasitism is often involved in chitinases. Fungal chitinases however play a physiological role in cell division and differentiation. Plant chitinase is part of the plant's immune system against fungal pathogens. In insects, chitinases during their development have been identified as being involved in the molting process. In human gastric juice, chitinases have also been identified. Chitinase chitin degradable enzyme has control over phytopathogens, physiological functions and destruction of chitinous waste.In this review focusing on the various sources of natural chitinase production in Actinomycetes was discussed.

Chitinase of Trichoderma longibrachiatum for control of Aphis gossypii in cotton plants

Chitinase-producing fungi have now engrossed attention as one of the potential agents for the control of insect pests. Entomopathogenic fungi are used in different regions of the world to control economically important insects. However, the role of fungal chitinases are not well studied in their infection mechanism to insects. In this study, Chitinase of entomopathogenic fungi Trichoderma longibrachiatum was evaluated to control Aphis gossypii. For this purpose, fungal chitinase (Chit1) gene from the genomic DNA of T. longibrachiatum were isolated, amplified and characterised. Genomic analysis of the amplified Chit1 showed that this gene has homology to family 18 of glycosyl hydrolyses. Further, Chit1 was expressed in the cotton plant for transient expression through the Geminivirus-mediated gene silencing vector derived from Cotton Leaf Crumple Virus (CLCrV). Transformed cotton plants showed greater chitinase activity than control, and they were resistant against nymphs and adults of A. gossypii. About 38.75% and 21.67% mortality of both nymphs and adults, respectively, were observed by using Chit1 of T. longibrachiatum. It is concluded that T. longibrachiatum showed promising results in controlling aphids by producing fungal chitinase in cotton plants and could be used as an effective method in the future.

The Bursaphelenchus xylophilus candidate effector BxLip-3 targets the class I chitinases to suppress immunity in pine.

Lipase is involved in lipid hydrolysis, which is related to nematodes' energy reserves and stress resistance. However, the role of lipases in Bursaphelenchus xylophilus, a notorious plant-parasitic nematode responsible for severe damage to pine forest ecosystems, remains largely obscure. Here, we characterized a class III lipase as a candidate effector and named it BxLip-3. It was transcriptionally up-regulated in the parasitic stages of B. xylophilus and specifically expressed in the oesophageal gland cells and the intestine. In addition, BxLip-3 suppressed cell death triggered by the pathogen-associated molecular patterns PsXEG1 and BxCDP1 in Nicotiana benthamiana, and its Lipase-3 domain is essential for immunosuppression. Silencing of the BxLip-3 gene resulted in a delay in disease onset and increased the activity of antioxidant enzymes and the expression of pathogenesis-related (PR) genes. Plant chitinases are thought to be PR proteins involved in the defence system against pathogen attack. Using yeast two-hybrid and co-immunoprecipitation assays, we identified two class I chitinases in Pinus thunbergii, PtChia1-3 and PtChia1-4, as targets of BxLip-3. The expression of these two chitinases was up-regulated during B. xylophilus inoculation and inhibited by BxLip-3. Overall, this study illustrated that BxLip-3 is a crucial virulence factor that plays a critical role in the interaction between B. xylophilus and host pine.

Bioinformatics and Expression Analysis of the Chitinase Genes in Strawberry (Fragaria vesca) and Functional Study of FvChi-14.

Plant chitinases (EC 3.2.1.14) are pathogenesis-related (PR) proteins and are well studied in many plant species. However, little is known about the genomic organization and expression of chitinase genes in strawberries (Fragaria vesca). Here, 23 FvChi genes were identified in the genome of strawberry (F. vesca) and divided into GH18 and GH19 subfamilies based on phylogenetic relationships. A detailed bioinformatics analysis of the FvChi genes was performed, including gene physicochemical properties, chromosomal location, exon-intron distribution, domain arrangement, and subcellular localization. Twenty-two FvChi genes showed upregulation after Colletotrichum gloeosporioides infection. Following the exogenous application of SA, FvChi-3, 4, and 5 showed significant changes in expression. The ectopic expression of FvChi-14 in Arabidopsis thaliana increased resistance to C. higginsianum via controlling the SA and JA signaling pathway genes (AtPR1, AtICS1, AtPDF1.2, and AtLOX3). The FvChi-14 protein location was predicted in the cell wall or extracellular matrix. We speculate that FvChi-14 is involved in disease resistance by regulating the SA and JA signaling pathways. The findings of this study provide a theoretical reference for the functional studies of FvChi genes and new candidates for strawberry stress resistance breeding programs.

New Genomic Regions Identified for Resistance to Spot Blotch and Terminal Heat Stress in an Interspecific Population of Triticum aestivum and T. spelta.

Wheat is one of the most widely grown and consumed food crops in the world. Spot blotch and terminal heat stress are the two significant constraints mainly in the Indo-Gangetic plains of South Asia. The study was undertaken using 185 recombinant lines (RILs) derived from the interspecific hybridization of 'Triticum aestivum (HUW234) × T. spelta (H+26)' to reveal genomic regions associated with tolerance to combined stress to spot blotch and terminal heat. Different physiological (NDVI, canopy temperature, leaf chlorophyll) and grain traits (TGW, grain size) were observed under stressed (spot blotch, terminal heat) and non-stressed environments. The mean maturity duration of RILs under combined stress was reduced by 12 days, whereas the normalized difference vegetation index (NDVI) was 46.03%. Similarly, the grain size was depleted under combined stress by 32.23% and thousand kernel weight (TKW) by 27.56% due to spot blotch and terminal heat stress, respectively. The genetic analysis using 6734 SNP markers identified 37 significant loci for the area under the disease progress curve (AUDPC) and NDVI. The genome-wide functional annotation of the SNP markers revealed gene functions such as plant chitinases, NB-ARC and NBS-LRR, and the peroxidase superfamily Cytochrome P450 have a positive role in the resistance through a hypersensitive response. Zinc finger domains, cysteine protease coding gene, F-box protein, ubiquitin, and associated proteins, play a substantial role in the combined stress of spot blotch and terminal heat in bread wheat, according to genomic domains ascribed to them. The study also highlights T. speltoides as a source of resistance to spot blotch and terminal heat tolerance.

Plant Chitinase Mutants as the Catalysts for Chitooligosaccharide Synthesis Using the Sugar Oxazoline Derivatives.

Sugar oxazolines, (GlcNAc)n-oxa (n = 2, 3, 4, and 5), were synthesized from a mixture of chitooligosaccharides, (GlcNAc)n (n = 2, 3, 4, and 5), and utilized for synthesis of (GlcNAc)7 with higher elicitor activity using plant chitinase mutants as the catalysts. From isothermal titration calorimetry, the binding affinity of (GlcNAc)2-oxa toward an inactive mutant obtained from Arabidopsis thaliana GH18 chitinase was found to be higher than those of the other (GlcNAc)n-oxa (n = 3, 4, and 5). To synthesize (GlcNAc)7, the donor/acceptor substrates with different size combinations, (GlcNAc)2-oxa/(GlcNAc)5 (1), (GlcNAc)3-oxa/(GlcNAc)4 (2), (GlcNAc)4-oxa/(GlcNAc)3 (3), and (GlcNAc)5-oxa/(GlcNAc)2 (4), were incubated with hypertransglycosylating mutants of GH18 chitinases from A. thaliana and Cycas revoluta. The synthetic activities of these plant chitinase mutants were lower than that of a mutant of Bacillus circulans chitinase A1. Nevertheless, in the plant chitinase mutants, the synthetic efficiency of combination (1) was higher than those of the other combinations (2), (3), and (4), suggesting that the synthetic reaction is mostly dominated by the binding affinities of (GlcNAc)n-oxa. In contrast, the Bacillus enzyme mutant with a different subsite arrangement synthesized (GlcNAc)7 from combination (1) in the lowest efficiency. Donor/acceptor-size dependency of the enzymatic synthesis appeared to be strongly related to the subsite arrangement of the enzyme used as the catalyst. The A. thaliana chitinase mutant was found to be useful when combination (1) is employed for the substrates.

Plasmodiophora brassicae CBM18 Proteins Bind Chitin and Suppress Chitin-Triggered Immunity

Plants have a sophisticated and multilayered immune system. However, plant pathogens, helped by effector proteins, have found several strategies to evade plant immunity. For instance, the clubroot pathogen Plasmodiophora brassicae is able to turn the roots of susceptible hosts into nutrient-sink galls suppressing pattern-triggered immunity (PTI) and effector-triggered immunity. Chitin, the main component of P. brassicae spore cell walls and a well-known pathogen-associated molecular pattern, can elicit PTI but is also the target of plant chitinases and chitin deacetylases. The fact that P. brassicae does not trigger PTI during infection of susceptible hosts motivated a genome-wide search of genes coding for secreted proteins with domains previously associated with chitin binding. We found that the P. brassicae genome encodes a repertoire of candidate-secreted effectors containing the chitin-binding domain carbohydrate-binding module family 18 (CBM18), chitinase, and chitin deacetylase domains. The role of these proteins in the pathogenicity of the clubroot pathogen is unknown. Here, we characterized two CBM18 proteins, PbChiB2 and PbChiB4, which are transcriptionally activated during infection. Through coprecipitation, we found that recombinant PbChiB2 and PbChiB4 bind to spores and to chitin oligomers. We also showed that both proteins suppress chitin-triggered activation of the map kinase proteins MPK3 and MPK6 in the host Brassica napus. These findings suggest that P. brassicae CBM18 proteins act as effectors for protecting the clubroot pathogen and for suppressing chitin-triggered immunity during infection. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Using arbuscular mycorrhizal fungi (Gigaspora margarita) as a growth promoter and biocontrol of leaf blight disease in eucalyptus seedlings caused by Cylindrocladium quinqueseptatum

The leaf blight disease is a common threat to the growth and production of eucalyptus seedlings in Thailand. This work then aimed to apply a biocontrol approach to help plants survive the disease. Cylindrocladium quinqueseptatum KKU-H4-B12, a pathogen causing leaf blight disease, was isolated from the lesion of eucalyptus leaves. Its pathogenicity was confirmed before use in the field experiments. An AMF Gigaspora margarita KKU-BH-01, previously proved to efficiently promote growth of eucalyptus, was pre-inoculated into eucalyptus seedlings for 30 days prior to pathogen infection. The results showed that an AMF could successfully colonize eucalyptus roots even in the pathogen-infected plants (≥56%). The disease assessment indicated that plants pre-inoculated with AMF and later infected with the pathogen (PM) had a disease reduction of 33.4%. Plant height, SPAD, fresh weights of roots and shoots in PM plants were relatively equal to those of the control plants, suggesting that AMF could relieve detrimental effects of the disease in plants. Moreover, we found that AMF induced plant chitinase and β-1,3-glucanase enzymes resulting in improved disease resistance in plants. This work is the first to report the application of a single AMF species to effectively enhance the growth and help eucalyptus seedlings to survive leaf blight disease.

Expression and Refolding of the Plant Chitinase From Drosera capensis for Applications as a Sustainable and Integrated Pest Management.

Recently, the study of chitinases has become an important target of numerous research projects due to their potential for applications, such as biocontrol pest agents. Plant chitinases from carnivorous plants of the genus Drosera are most aggressive against a wide range of phytopathogens. However, low solubility or insolubility of the target protein hampered application of chitinases as biofungicides. To obtain plant chitinase from carnivorous plants of the genus Drosera in soluble form in E.coli expression strains, three different approaches including dialysis, rapid dilution, and refolding on Ni-NTA agarose to renaturation were tested. The developed « Rapid dilution » protocol with renaturation buffer supplemented by 10% glycerol and 2M arginine in combination with the redox pair of reduced/oxidized glutathione, increased the yield of active soluble protein to 9.5 mg per 1 g of wet biomass. A structure-based removal of free cysteines in the core domain based on homology modeling of the structure was carried out in order to improve the soluble of chitinase. One improved chitinase variant (C191A/C231S/C286T) was identified which shows improved expression and solubility in E. coli expression systems compared to wild type. Computational analyzes of the wild-type and the improved variant revealed overall higher fluctuations of the structure while maintaining a global protein stability. It was shown that free cysteines on the surface of the protein globule which are not involved in the formation of inner disulfide bonds contribute to the insolubility of chitinase from Drosera capensis. The functional characteristics showed that chitinase exhibits high activity against colloidal chitin (360 units/g) and high fungicidal properties of recombinant chitinases against Parastagonospora nodorum. Latter highlights the application of chitinase from D. capensis as a promising enzyme for the control of fungal pathogens in agriculture.

Maize Endochitinase Expression in Response to Fall Armyworm Herbivory.

A large percentage of crop loss is due to insect damage, especially caterpillar damage. Plant chitinases are considered excellent candidates to combat these insects since they can degrade chitin in peritrophic matrix (PM), an important protective structure in caterpillar midgut. Compared to chemical insecticides, chitinases could improve host plant resistance and be both economically and environmentally advantageous. The focus of this research was to find chitinase candidates that could improve plant resistance by effectively limiting caterpillar damage. Five classes of endochitinase (I-V) genes were characterized in the maize genome, and we isolated and cloned four chitinase genes (chitinase A, chitinase B, chitinase I, and PRm3) present in two maize (Zea mays L.) inbred lines Mp708 and Tx601, with different levels of resistance to caterpillar pests. We also investigated the expression of these maize chitinases in response to fall armyworm (Spodoptera frugiperda, FAW) attack. The results indicated that both chitinase transcript abundance and enzymatic activity increased in response to FAW feeding and mechanical wounding. Furthermore, chitinases retained activity inside the caterpillar midgut and enzymatic activity was detected in the food bolus and frass. When examined under scanning electron microscopy, PMs from Tx601-fed caterpillars showed structural damage when compared to diet controls. Analysis of chitinase transcript abundance after caterpillar feeding and proteomic analysis of maize leaf trichomes in the two inbreds implicated chitinase PRm3 found in Tx601 as a potential insecticidal protein.

In silico analysis and overexpression of chitinase class IV gene in Brassica juncea improves resistance against Alternaria brassicae

Indian mustard (Brassica juncea), an elite oilseed crop in the Brassicae family is challenged by various biotic stresses. Alternaria leaf blight is the most devastating disease in B. juncea caused by Alternaria brassicae. Hence engineering B. juncea plants for improved Alternaria disease resistance becomes imperative. Plant chitinases are key players in the plant immune system that offers successful protection to fungal infections over a broad spectrum of diseases. Chitinase class IV gene was isolated and functionally characterized in Indian mustard (Brassica juncea). Based on phylogenetic analysis, Bj chitinase class IV gene was closely related to Brassica napus followed by other Brassica species. To understand the defensive role of this gene, it was cloned in (Pet28a+) vector and expressed in the prokaryotic expression host Escherichia coli BL21. The purified protein showed antifungal activity against A. brassicae. To further discern its role in disease resistance, B. juncea transgenic lines were raised by overexpressing Bj chitinase IV gene and further confirmed by PCR and southern blotting. In addition, independent B. juncea transgenic lines with high transcript levels of chitinase class IV gene were selected for disease assays against A. brassicae infection. Pathological studies showed that the rate of A. brassicae lesions development and size was significantly reduced in the Bj chitinase transgenic lines compared with non transgenic plants. Interestingly, Bj chitinase transgenic lines showed increased activity of ROS scavenging enzymes when compared to control plants. Collectively our results suggest that overexpression of Bj chitinase IV gene in B. juncea improves resistance against Alternaria leaf blight.