Plant Physiol. Drug Metab Dispos
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Published on November 12, 2008; 10.1104/pp.108.130260

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Received September 23, 2008
Accepted November 8, 2008

Establishing RNAi as a Reverse Genetic Approach For Gene Functional Analysis in Protoplasts

Zhiyang Zhai , Thanwalee Sooksa-nguan , and Olena K. Vatamaniuk *

Cornell University, Department of Crop and Soil Sciences, Ithaca, NY 14853, USA

* Corresponding author; email: okv2{at}cornell.edu.

Double-stranded (ds)RNA interference (RNAi) is widely used for functional analysis of plant genes and is achieved via generating stable transformants expressing dsRNA in planta. This study demonstrated that RNAi can also be utilized to examine gene functions in protoplasts. Since protoplasts are non-growing cells, effective RNAi-triggered gene-silencing depends not only on a depletion of gene-transcripts, but also on turnover rates of corresponding polypeptides. Herein, we tested whether transient RNAi in protoplasts results in the depletion of a targeted polypeptide and, since protoplasts have limited life span, whether functional assays of RNAi-knock-out genes are feasible in protoplasts. We showed that protoplast transfection with an in vitro-synthesized dsRNA against Arabidopsis thaliana {gamma}-glutamylcysteine synthase (AtECS1), a key enzyme in the synthesis of glutathione (GSH), resulted in a 95% depletion of AtECS1 transcript, a 72% decrease of AtECS1 polypeptide, and a 60% drop in GSH content. These results were comparable to those obtained upon analysis of Arabidopsis seedlings, bearing a cad2-1 mutant allele of AtECS1. We also improved the procedure for RNAi-inactivation of several genes simultaneously. Finally, since we isolated protoplasts from tissues of 14-day-old seedlings instead of one-month-old mature plants, the described procedure is rapid (as it only takes 20 days from seed planting to functional studies), is suitable for analysis of multiple genes in parallel, and is independent of cloning dsRNAs into plant expression vectors. Therefore, RNAi in protoplasts complements existing genetic tools as it allows rapid, cost- and space-efficient initial screening for the selection of genes for further in planta studies.






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