Over-expression of NPR1 gene in Brassica juncea Leads to Resistance Against Alternaria brassicae Infection

Author: Rajendra Prasad Meena, Raina Bajpai, Aparna, Rakesh Kumar and Anita Grover

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Abstract

Alternaria blight is one of the serious diseases of Brassica juncea causing 45-58% loss in the yield. In the present days the most common method to control this biotic issue is the chemical method. However, use of these chemicals causes huge damage to the human health, plant health and eventually polluting our ecosystem. Thus, manipulating defense regulatory genes [e.g. NoPR1 (NPR1) gene] is one of the safest strategies which is being followed internationally to manage this disease. Previously in order to develop resistance against Alternaria brassicae, nineteen putative transgenic lines of B.juncea having NPR1 gene under control of 35S promoter were developed. In the present study, these transgenic lines were confirmed for gene integration, expression and its effect on resistance against Alternaria. Gene integration was confirmed by PCR with NPTII primers. In order to reconfirm the NPR1gene integration, PCR was done using 35S forward primer and NPR1 reverse primer. All the nineteen putative lines were found to be positive. Further, RT-PCR with NPR1 primers was done to check levels of gene expression. Eleven lines namely (1, 5, 6, 7, 8, 9, 11, 12, 14, 16, 18) were found to be over expressing NPR1 by about 5 fold over non transgenic control while other eight lines (2, 3, 4, 10, 13, 15, 17, 19) did not show such high expression (only 2 fold). Seven lines encompassing both high NPR1 expressing and not so high NPR1 expressing, were analyzed for disease resistance. Resistance was scored in terms of time of onset of symptoms, lesions number and lesion size. The symptoms of infection were observed on day 3 after inoculation in control plants whereas those in transgenic plants symptoms were observed on day 7 after inoculation. The levels of resistance in-vitro varied from 2.0 to 2.5 fold as compared to the control. In-vivo assays revealed 2 to 3 fold resistance in transgenic plants as compared to control plants. It was found that expression level of NPR1 is directly related to levels of resistance.

Keywords

NPR1, Transgenic, Fungus, Resistance, Brassica, Alternaria

Conclusion

Nineteen putative transgenic Brassica juncea lines having NPR1 gene under the control of 35S promoter were developed in the Plant Pathogen Interacting laboratory of National Institute for Plant Biotechnology. The present study was molecular analysis of putative transgenic lines for gene integration and expression and phenotypic analysis for disease resistance against Alternariabrassicaeinfection.DNA was isolated from control and transgenic plants and PCR was done with NPTII primers (for kanamycin resistance) and with 35S promoter forward primer and NPR1 reverse primers. All the nineteen lines were found to be positive. The study of gene expression was accomplished by isolating RNA from control and transgenic plants and doing RT-PCR with NPR1 primers. Eleven lines were found to over- express NPR1 gene by 5 fold while eight lines were found to over express NPR1gene by 2 fold. Transgenic plants were evaluated after inoculation of one month old plants through in-vitro (by detached leaf method) and in-vivo (on the whole plant) methods. Improved resistance was measured by delay in onset of symptoms and number and size of the lesions. Appearance of symptoms was delayed by 2 days in transgenic plants as compared to the control plants. Number and size of the lesions were reduced from 6 and 7.9 mm in control plants to 3 and 2.7 mm in transgenic plants respectively during in vitro studies. In in-vivo studies also number and size of the lesions were reduced from 5 and 6.2 mm in control to 2 and 2 mm in transgenic plants respectively. It was also seen that transgenic lines showing fewer lesions also displayed small size lesions. From the present study, it was concluded that integrating the NPR1 gene and over-expressing it, leads to improved resistance in B. juncea against A. brassicae.

References

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How to cite this article

Rajendra Prasad Meena, Raina Bajpai, Aparna, Rakesh Kumar and Anita Grover (2023). Over-expression of NPR1 gene in Brassica juncea Leads to Resistance Against Alternaria brassicae Infection. Biological Forum – An International Journal, 15(10): 1322-1329.