Figure 4.

Gene Expression and Subcellular Localization of SIP2.

(A) Expression of SIP2 mRNA in L. japonicus. Roots were harvested 1, 2, 4, 6, 8, 10, and 12 d after inoculation with M. loti. Roots treated with water (uninoculated roots) were also harvested at the same time intervals and served as the mock control. Rhizobium-inoculated roots (IR), stems (S), and leaves (L) were harvested 8 d after inoculation, while nodules (N) were collected 42 d after inoculation. Total RNA was isolated and used for real-time PCR to measure the expression levels of the SIP2 mRNA. The ATPase gene (AW719841) was used as an internal control. Error bars represent sd of the experimental values obtained from three technical replicates.

(B) Subcellular localization of SIP2 in L. japonicus hairy roots. SIP2 was expressed as a GFP fusion protein (GFP:SIP2) under the control of the CaMV35S promoter in L. japonicus hairy roots induced by A. rhizogenes LBA1334. GFP alone served as a control. Bars = 20 μm.

(C) Subcellular localization of GFP-tagged SIP2 in onion cells. SIP2 was expressed as a fusion protein with GFP (GFP:SIP2) under the control of the 35S promoter. Plasmid expressing GFP alone served as a control. The plasmids were delivered to the onion epidermal cells via particle bombardment, and fluorescence images were taken using a confocal laser scanning microscope. Onion epidermal cells expressing GFP:SIP2 were treated with 4% NaCl for 5 min to induce plasmolysis before imaging. Green fluorescence (left) and the corresponding bright-field images (right) were taken using a confocal laser scanning microscope. The overlay images (middle) were produced using Photoshop software. Bars = 50 μm.