THE EFFECTIVENESS OF RECOMBINANT CHITINASE OBTAINED FROM BARLEY (Hordeum vulgare L.) AGAINST POTATO PATHOGENS

Abstract

Globally, fungi and fungi-like organisms, with over 8,000 species, cause more plant diseases than any other group of plant pests. Chitinases possess an anti-fungal role in disease resistance. They represent the pathogen-related protein group that cleaves β-1,4-glycoside bond of chitin present in the cell wall of filamentous fungi by catalysing the hydrolytic cleavage. In this study, we have amplified 672 base pair (bp) coding regions of the plant chitinase gene of Hordeum vulgare, over-expressed in Eschercia coli (E. coli) host BL21 DE3 strain by 0.5mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. In western blotting, the recombinant chitinase protein transferred on nitrocellulose membrane was detected at 26 kDa with histidine-tagged antibody reactions. By applying the nickel charged sepharose column affinity chromatography method, a single purified band of recombinant chitinase protein was found at 26 kDa in SDS-PAGE. The purified recombinant chitinase protein during the agar well diffusion method inhibited the growth of three phytopathogenic fungi in a significant way; Alternaria alternate, Rhizoctonia solani and Fusarium oxysporum in qualitative in-vitro antifungal assays. The purified recombinant chitinase protein showed up to 82.6% reduction in A. alternate hyphal growth; 77% reduction of hyphal growth in Rhizoctonia solani and 78% reduction of Fusarium's hyphal growth in invitro quantitative antifungal assays, thus depicting a strong antifungal potential. Conclusively, plant derived chitinase genes are superior in protecting the crops from phytopathogenic fungi if cloned in a suitable plant vector and expressed.

Proteome Phenotypes Discriminate the Growing Location and Malting Traits in Field-Grown Barley

“… 53 Plant endochitinases have antifungal properties, and a potential inhibitory effect against fungal pathogens was previously reported in barley. 54 In Arabidopsis, abiotic stresses, particularly heat stress, brought about downregulation of most chitinase genes. 55 Germin-like proteins are also involved in responses to pathogen and abiotic stresses in plants; in a study on the multigene family-encoding germin-like proteins of barley, it has been found that a pathogen attack or hydrogen peroxide are strong signals for germin-like protein subfamilies.…”

“…It has been suggested that this difference might be related to the rain and humidity of the growing environment . Plant endochitinases have antifungal properties, and a potential inhibitory effect against fungal pathogens was previously reported in barley . In Arabidopsis, abiotic stresses, particularly heat stress, brought about downregulation of most chitinase genes .…”

Quantitative Label-Free Comparison of the Metabolic Protein Fraction in Old and Modern Italian Wheat Genotypes by a Shotgun Approach

“…Instead, an antifungal R protein and the 26 kDa endochitinase 2 appear down-regulated in comparison with Simeto and Russello, respectively. These proteins are usually up-regulated under fungal (e.g., Aspergillus parasiticus or A. flavus ) attacks accompanied by drought stress and might contribute to increase the resistance of the plant to adverse biotic and abiotic stimuli [ 42 , 43 ]. Altogether, the differences observed in the abundance of both the inhibitors and stress-related proteins suggest that the old genotypes, and in particular Timilia, in comparison with the modern Simeto might present different mechanisms of defense.…”

Liposomal Formulation of Botanical Extracts may Enhance Yield Triggering PR Genes and Phenylpropanoid Pathway in Barley (Hordeum vulgare)

“…The data showed that in the resistant genotypes, PR transcripts accumulated higher and earlier than in the susceptible genotypes; the most prominent differences were observed in PR1 and PR5 genes, which were strongly activated [ 31 ]. Recombinant chitinase protein (PR3-4) from seeds of H. vulgare significantly inhibited the growth of the hyphae of three phytopathogenic fungi ( Alternaria alternate, Rhizoctonia solani and Fusarium oxysporum ) in in vitro qualitative antifungal assays [ 32 ]. Furthermore, an increase in the abundance of peroxidases and PR-3 and PR-5 was observed after infection of barley ears with macroconidia suspension of Fusarium graminearum [ 33 ].…”