Single or Multiple Gene Silencing Directed by U6 Promoter-Based shRNA Vectors Facilitates Efficient Functional Genome Analysis in Medicago truncatula

Short hairpin RNA (shRNA) expression vectors are an effective means to deliver double-stranded RNA that mediates sequence-specific cleavage of target transcripts in RNA interference (RNAi). Previously, we constructed shRNA expression vectors based on U6 small nuclear RNA (snRNA) gene promoters of Medicago truncatula. To facilitate screening of shRNA candidates, a protoplast-based dual luciferase assay system was developed, which consists of a shRNA-expressing effector plasmid and a target gene-carrying reporter plasmid. RNAi efficiency of a given shRNA could be measured by comparing two luciferase activities derived from transcripts with an intact or interrupted 3′ untranslated region as a result of RNAi. Using a multiple U6-shRNA cassette plasmid that enables targeting of multiple genes simultaneously, the expression of two Δ¹-pyrroline-5-carboxylate synthetase genes (MtP5CS1 and MtP5CS2) was silenced with a U6 plasmid carrying two 27-nucleotide-stem shRNA cassettes targeting each gene. The decreased transcript levels were accompanied by decreased protein levels and decreased free proline contents. To examine possible U6-shRNA-mediated RNAi in nodules, two remorin-encoding MtREM genes were targeted by single and double U6-shRNA plasmids. Silencing of MtREM2.2, an essential nodulation gene, MtREM1.1, an uncharacterized gene, or both inhibited nodule formation in transgenic roots. Moreover, M. truncatula roots transformed with a suppressor-overexpresser construct wherein MtREM2.2-targeting U6-shRNA and GmREM2.3-overexpressing cassettes were placed tandemly showed decreased MtREM2.2 transcripts yet survived nitrogen-free medium, indicating complementation of MtREM2.2 with the overexpressed soybean gene. These results demonstrate that U6 promoter-based shRNA-expressing plasmids are valuable for multiple gene functional analyses in protoplasts and nodules of M. truncatula.