Expression of a fungal endochitinase gene in transgenic tomato and tobacco results in enhanced tolerance to fungal pathogens

Abstract

Development of transgenic Nicotiana tabacum and Lycopersicon esculentum expressing an endochitinase (ech42) gene from biocontrol fungus Trichoderma virens using Agrobacterium-mediated genetic transformation is reported in this paper. Integration of transgene in the genome of transgenic plants was demonstrated using polymerase chain reaction and Southern-blot hybridization, while expression was ascertained by reverse transcription polymerase chain reaction. Histochemical analysis confirmed the expression of GUS enzyme in transformed shoots. Levels of endochitinase enzyme in transgenic plants were found to be up to 10 fold higher compared to control plants. Endochitinase enzyme of 42 kDa was also visualized on SDS-PAGE gel using fluorimetric zymogram in transgenic plants. Endochitinase activity was found to be higher in leaf and stem than the root tissue in transgenic tomato plants. Transgenic lines of both plants showed enhanced resistance to fungal pathogens and a strong negative correlation was found between expression level of endochitinase enzyme and size of disease lesions. Inheritance of transgene, expression and resistance to fungal pathogens of T 1 transgenic tobacco lines was also analysed. The results of the present studies show that ech42 is a promising candidate gene for developing fungal disease resistance in tomato plants.

Expression of an endochitinase gene from Trichoderma virens confers enhanced tolerance to Alternaria blight in transgenic Brassica juncea (L.) czern and coss lines

“…PCR was carried out according to Shah et al (2010). PCR analysis was also carried out with 5 T 1 plants each from 5 different T 1 lines to confirm the stable integration of transgene.…”

“…One μg of DNase-treated and purified RNA was taken for cDNA synthesis using affinity script multi temperature cDNA synthesis kit (Stratagene, USA). Two μl of this reaction mix was used for PCR amplification of 'ech42'and a housekeeping gene 'actin' (Shah et al 2010).…”

“…Endochitinase activity in transgenic plants (each from different T 0 and T 1 lines) was measured by fluorimetric assay and fluorimetric zymogram was performed for visualization of chitinase activity as described earlier by Shah et al (2010). All the reagents used were from Sigma and assay was repeated at least twice with three replicates.…”

“…1707) were obtained from the Indian Type Culture Collection, Division of Plant Pathology, IARI, New Delhi. Bioassay for sensitivity of transgenic lines against the fungal pathogens were done as described earlier (Shah et al 2010). Three independent experiments with three replicates were carried out and the data was analyzed using Analysis of Variance (ANOVA) and Pearson's correlation using Microcal™ Origin pro 6.1.…”

“…Chitinase and glucanase gene from tomato have been introduced into B. juncea and the transgenic plants showed resistance to A. brassicae (Mondal et al 2003(Mondal et al , 2007. We have earlier cloned and transferred an endochitinase gene from T. virens to tobacco and tomato and the transgenic lines exhibited enhanced tolerance to some fungal pathogens (Shah et al 2010).…”