Fig. 2: BID1 encodes a nodule-specific, ER membrane-localized SPP.
From: A signal peptide peptidase is required for ER-symbiosome proximal association and protein secretion
a A diagram showing BID1 gene structure and the position of the target mutation in rough mapping. BID1 was mapped to the upper arm of Chromosome 1, in the interval between markers 001e_01 and h2_116k17a. A 8,857-bp fragment from Chromosome 2, 44,572,929-44,581,777 bp, is inserted into the 5’ UTR of MtrunA17_Chr1g0147151, 102 bp ahead of the start codon. b Expressing GFP-fused candidate gene genomic sequence driven by its own promoter could complement the bid1 phenotype. Please note that GFP tag need be inserted at N-terminus of BID1. Bar=2 mm. c BID1 is a multi-transmembrane domain-containing SPP protein. The transmembrane domains were shown, and two conserved aspartate residues critical for peptidase activity were labeled red. PAL, a conserved motif important for peptidase activity; KKXX, the C-terminal ER membrane localization signal. Numbers indicated positions of the amino acids. d BID1 is expressed exclusively in nodules in promoter-GUS reporter assay. Zone I, nodule meristem, Zone II, infection zone, the zone rhizobia infect nodule cells, Zone III, differentiation zone, the zone where symbiosomes undergo differentiation, Zone IV, fixation zone, the zone symbiosomes fix nitrogen. Bar=500 μm. e BID1 localized specifically at ER membrane when expressed in the leaves of Nicotiana benthamiana. N. benthamiana leaves co-transformed with 35 S::GFP-BID1 and mCherry-HDEL ER reporter were analyzed under confocal microscopy at 2 days post-transformation. Bar=10 μm. f, BID1 localized to the ER membrane in nodule cells. A construct expressing pBID1::GFP-BID1 together with mCherry-HDEL reporter was transformed into bid1 mutants. Bar=10 μm. g and h Fluorescent intensity measurement of GFP-BID1 and mCherry-HDEL in indicated regions of tobacco leaf and nodule cells respectively. Fluorescent peaks of GFP-BID1 overlapped with mCherry-HDEL signals in both cell types. Fluorescent intensity was measured as grey value of pixels using ImageJ software. Roi, region of interest. Experiments were repeated more than 3 times with similar results.
