Use of the rhizobial type III effector gene nopP to improve Agrobacterium rhizogenes-mediated transformation of Lotus japonicus

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.

Keywords: Agrobacterium rhizogenes; Effector; Hairy roots; Lotus japonicus; Plant transformation.

Fig. 1

Pictures illustrating the described transformation procedure. a Roots from 5-day-old L. japonicus MG20 seedlings were removed. The arrow indicates the cutting site at the bottom of the hypocotyl. Seedlings were co-cultivated with A. rhizogenes for 30 min. c Seedlings were transferred to agar plates containing ½ strength Gamborg’s B5 Salts and Vitamins medium. d The agar with seedlings was covered with a sterile filter paper and the plate sealed with parafilm. e Plants were weekly transferred to a fresh agar plate and covered by a new filter paper. f Analysis by fluorescence microscopy (28 dpi): Selected plants showing at least one red fluorescent root were placed into plastic jar units and inoculated with M. loti MAFF303099. g Plants with formed nodules (arrow) were harvested 4 weeks later. h Analysis of roots and nodules by fluorescence microscopy. The picture shows a red fluorescent root with two nodules. Bars = 1 mm in a, 1 cm in b, 2 cm in c–e, 500 μm in f, 2 cm in g, and 500 μm in h

Fig. 2

Formation of hairy roots on L. japonicus seedlings induced by different A. rhizogenes strains carrying pISV-DsRed1. The binary vector pISV-DsRed1 was introduced into the A. rhizogenes strains LBA1334, K599 and LBA9402. Each strain was inoculated on 50 plants. Plants were analyzed at 28 dpi. Data indicate means ± SE. Different letters above columns indicate statistically significant differences (Duncan’s Multiple Range test, P < 0.05). a Total number of formed hairy roots per plant (n = 50). b Microscopic analysis of formed hairy roots under bright field conditions (top) and for red fluorescence (RF) emission (bottom). Bar = 500 μm. c Transformation efficiency as determined by the percentage of red fluorescent hairy roots per plant. Data were obtained from plants showing at least one red fluorescent root (n = 8 for LBA1334; n = 20 for K599; n = 23 for LBA9402)

Fig. 3

Transformation of L. japonicus with pISV-DsRed1 containing effector genes. The effector genes nopL, nopM, nopP and nopT of Sinorhizobium sp. NGR234 were cloned into pISV-DsRed1. A. rhizogenes LBA9402 bacteria carrying the constructed binary vectors were used for transformation. Microscopic analysis of formed hairy roots was performed under bright field conditions (top) and for red fluorescence (RF) emission (bottom) at 28 dpi. Bar = 500 μm. RNA from selected red fluorescent roots was isolated for qRT-PCR analysis to detect effector gene expression (5 plants per RNA extraction). LjUbiquitin was used as a reference gene to normalize the transcript abundance value of a given effector gene. Control plants transformed with pISV-DsRed1 (without effector gene) showed weak background signals in the qRT-PCR analysis. Data indicate means ± SE (n = 3; 3 RNA extractions)

Fig. 4

Expression of nopP in hairy roots results in an increased transformation efficiency. L. japonicus seedlings were transformed with A. rhizogenes LBA9402 bacteria carrying pISV-DsRed1 containing nopL, nopM, nopP and nopT, respectively. Plants transformed with pISV-DsRed1 (without effector gene) served as a control (C). Hairy roots were analyzed for red fluorescence emission at the time of harvest. Histograms indicate means ± SE. Different letters above columns indicate statistically significant differences (Duncan’s Multiple Range test, P < 0.05). a Transformation efficiency as determined by the percentage of red fluorescent roots per plant (28 dpi). Each binary vector was examined on 40 plants. Data were obtained from plants showing at least one red fluorescent root (n = 21 for the control (C); n = 18 for nopL; n = 15 for nopM; n = 22 for nopP; n = 16 for nopT). b Transformation efficiency of control plants (C) and nopP expressing plants as determined by the gridline intersection method (45 dpi). Plants showing red fluorescence in at least one hairy root were analyzed (n = 8 for both test groups)

Fig. 5

Expression of nopP in hairy roots of L. japonicus does not affect nodule formation. A. rhizogenes LBA9402 carrying the pISV-DsRed1 control vector or pISV-DsRed1-nopP was used to induce hairy roots. Data indicate means ± SE. Different letters above columns indicate statistically significant differences between control (C) and nopP expressing plants (Duncan’s Multiple Range test, P < 0.05). a Transformation efficiency (percentage of red fluorescent roots) for plants showing at least one red fluorescent root (28 dpi). The selected plants (n = 15 for the control (C); n = 18 for nopP) were then transferred to jars and inoculated with M. loti MAFF303099. b Microscopic analysis of formed nodules was performed under bright field conditions (top) and for red fluorescence (RF) emission (bottom) 28 days later. Bar = 500 μm. c Total number of nodules per plant and number of nodules showing red fluorescence at the time of harvest