• Spin column
• Cellulose powder
• Purification method
• E.coli expression
• RNAi
• dsRNA production
• Plant protection
• Botrytis cinerea

Botrytis cinerea is a plant pathogen that causes production losses worth billions of dollars every year and due to its genomic plasticity, it tends to develop resistance to fungicides.
Recently, new approaches based on RNA interference (RNAi) by exogenous application of dsRNA are emerging in the crop protection scenario. The aim of this work is to develop a protocol for the production and purification of dsRNA of B. cinerea genes using transformed E. coli cells as biofactories.
E. coli HT115(DE3) cells were transformed with L4440 plasmids into which the B. cinerea virulence genes BcBmp1, BcBmp3, and BcPls1 and the GFP gene as a control were individually inserted. The growth kinetics of cells were studied in three media (LB, TB and YT2x). The induction of dsRNA was performed in TB using IPTG 2 hours after inoculation, and the bacterial pellet was collected at 2 and 4 hours after induction. dsRNA purification was carried using two types of cellulose (CF11 and CE).
Growth kinetics revealed that TB is more suitable for the growth of BcBmp1, BcBmp3, BcPls1 E. coli transformed cells except GFP. Evaluation of dsRNA purification using two types of cellulose in micro-spin column revealed improved dsRNA yield by CF11. Among the genes the highest dsRNA production was achieved by BcPls1 while among the post-induction times the best yields were obtained by 4 hours post-induction. We concluded that medium composition and biomass production are related to the amount of dsRNA produced. Furthermore, fragment size and its sequence composition are other crucial parameters to consider for large-scale dsRNA production.