File:Figure 1.LysM-Containing LYP4 and LYP6 Are Rice Plasma Membrane Proteins.png

Figure 1.LysM-Containing LYP4 and LYP6 Are Rice Plasma Membrane Proteins. (A) Phylogenetic tree and domain structure diagram of LYPs in rice andArabidopsis. The phylogenetic tree was generated using MEGA4. Full-length amino acids sequences of plant LYPs were selected for generating a bootstrap neighbor-joining phylogenetic tree. Creye2 fromChlamydomonas reinhardtii was used as an outgroup. Bootstrap probabilities were obtained from 1000 replicates. A scale bar is indicated. A textfile of the alignment used to generate this tree is available as Supplemental Data Set 1 online. The N-terminal signal peptide, LysM, and the C-terminal GPI anchor signal(v-site) of each LYP are colored in orange, green, and yellow, respectively. (B)LYP4 and LYP6 localize at rice plasma membrane. Os-LYP4 and Os-LYP6 with GFP inserted behind the N-terminal signal peptide were individually expressed in rice protoplasts and visualized by confocal microscopy. FM4-64 dye was used to stain the plasma membrane. (C) LYP4 and LYP6 localize in the microsomal fraction. LYP4-GFP and LYP6-GFP were individually coexpressed with CEBiP-HA and GFP in rice protoplasts. Microsomal and soluble fractions of protoplast lysates were separated through Suc gradient centrifugation. Distribution of individual proteins and the endogenous tubulin was analyzed through immunoblotting with anti-GFP, anti-HA, and antitubulin antibodies. (D) Relative expression levels ofLYP4andLYP6in different rice tissues and developmental stages. The expression levels of these genes were determined by qPCR, and the expression level of each gene in rice calli is set as 100%. (E)Upregulation ofLYP4andLYP6transcripts in rice seedlings byX. oryzae. Five-day-old rice seedlings were incubated withX. oryzaesuspension (10^5cells/mL) or mock treated (sterile water) for the indicated period. The expression levels of these genes were examined by qPCR, and the induction fold of each gene was calculated by the gene expression level inX. oryzae–treated seedlings relative to that in mock-treated seedlings at the same time point. hpi, h postinoculation. (F)Induction of LYP4andLYP6expression in mature rice leaf and root by bacterial pathogenX. oryzae. The mature leaf (the fourth leaf) and the primary root from the indicatedPromoter:GUStransgenic rice at thefive-leaf stage were immersed intoX. oryzaesuspension (10^5 cells/mL) for 2 h before GUS staining. Bars = 1 mm. (G) Induction of LYP4andLYP6expression by diverse MAMPs. Five-day-old rice seedlings were treated with 100mg/mL of insoluble PGNXoo , soluble PGNXoo muropeptides, insoluble crab shell chitin, soluble chitin fragmentN-acetylchitohexaose, soluble LPS, or 100 nMflg22 for 1 h, and the induction of each gene was examined by qPCR. The experiments in(B) to (G) were repeated three times with similar results. The data in(D), (E),and(G) represent the mean 6SDof nine samples from three independent tests.