Yan Wang1, Feng Yang1, Peng-Fei Zhu1, Asaf Khan1, Zhi-Ping Xie2, Christian Staehelin3. 1. State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-Sen University, East Campus, Guangzhou, 510006, China. 2. State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-Sen University, East Campus, Guangzhou, 510006, China. xiezping@mail.sysu.edu.cn. 3. State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-Sen University, East Campus, Guangzhou, 510006, China. cst@mail.sysu.edu.cn.
Abstract
BACKGROUND: Protocols for Agrobacterium rhizogenes-mediated hairy root transformation of the model legume Lotus japonicus have been established previously. However, little efforts were made in the past to quantify and improve the transformation efficiency. Here, we asked whether effectors (nodulation outer proteins) of the nodule bacterium Sinorhizobium sp. NGR234 can promote hairy root transformation of L. japonicus. The co-expressed red fluorescent protein DsRed1 was used for visualization of transformed roots and for estimation of the transformation efficiency. RESULTS: Strong induction of hairy root formation was observed when A. rhizogenes strain LBA9402 was used for L. japonicus transformation. Expression of the effector gene nopP in L. japonicus roots resulted in a significantly increased transformation efficiency while nopL, nopM, and nopT did not show such an effect. In nopP expressing plants, more than 65% of the formed hairy roots were transgenic as analyzed by red fluorescence emitted by co-transformed DsRed1. A nodulation experiment indicated that nopP expression did not obviously affect the symbiosis between L. japonicus and Mesorhizobium loti. CONCLUSION: We have established a novel protocol for hairy root transformation of L. japonicus. The use of A. rhizogenes LBA9402 carrying a binary vector containing DsRed1 and nopP allowed efficient formation and identification of transgenic roots.
BACKGROUND: Protocols for Agrobacterium rhizogenes-mediated hairy root transformation of the model legume Lotus japonicus have been established previously. However, little efforts were made in the past to quantify and improve the transformation efficiency. Here, we asked whether effectors (nodulation outer proteins) of the nodule bacterium Sinorhizobium sp. NGR234 can promote hairy root transformation of L. japonicus. The co-expressed red fluorescent protein DsRed1 was used for visualization of transformed roots and for estimation of the transformation efficiency. RESULTS: Strong induction of hairy root formation was observed when A. rhizogenes strain LBA9402 was used for L. japonicus transformation. Expression of the effector gene nopP in L. japonicus roots resulted in a significantly increased transformation efficiency while nopL, nopM, and nopT did not show such an effect. In nopP expressing plants, more than 65% of the formed hairy roots were transgenic as analyzed by red fluorescence emitted by co-transformed DsRed1. A nodulation experiment indicated that nopPexpression did not obviously affect the symbiosis between L. japonicus and Mesorhizobium loti. CONCLUSION: We have established a novel protocol for hairy root transformation of L. japonicus. The use of A. rhizogenes LBA9402 carrying a binary vector containing DsRed1 and nopP allowed efficient formation and identification of transgenic roots.