Protocol

Centre : Central Potato Research Institute(CPRI)
Name of the Protocol
Development of P. infestans Pol gene RNAi construct
Nature : Novel
Crop / Pathogen : Potato/Phytophthora infestans

Inventors

1. VU Patil
2. BP Singh
3. Sanjeev Sharma
4. Sundaresha S
5. SK Chakrabarti

Objectives

1. To understand pathogen dynamism and to develop late blight resistant varieties through RNAi by reducing pathogen dynamism

Procedure / Summary

The dynamism in P. infestans is mainly due to the movement of transposable elements. Nearly 33% of the genome is covered by transposable Long Terminal Repeats LTR/Gypsy-Ty3 element, which is almost 1/3rd of the genome. Gypsy-Ty3 is well characterized and is independent transposable element and code for its own tranposase enzyme which was targeted for silencing in P. infestans. The strategy adopted was to construct inverted repeat cDNA fragment of Phytophthora Gypsy like element GypsyPi-1a gene intervened by potato granule bound starch synthase (GBSS) intron (iIR-GypsyPi-1a). Transcription of iIR-GypsyPi-1a gene construct in the transgenic potato background would generate hairpin (hp) Gypsy element Pol transcript, which will induce formation of siRNA causing silencing of Pol gene expression. 307bp cDNA sequence of pol was included for construction of sense and antisense fragment of iIR-GypsyPi-1a. The iIR-GypsyPi-1a gene construct under the control of constitutive promoter CaMV 35S on MCS of binary plant transformation vector pRI101. A total of 300 internodal stem cuttings of Kufri Bahar (LB susceptible cultivar) have been transformed for bioefficacy studies.

Applications

1. Understanding pathogen dynamism and development of late blight resistant varieties.