Miya et al. 10.1073/pnas.0705147104. |
Fig. 5. Detection of chitin-binding protein in the membrane preparations from Arabidopsis and rice by affinity labeling. Ten micrograms of microsomal membrane preparation was reacted with 0.4 mM GN8-Bio in the presence or absence of 40 mM unlabeled (GlcNAc)8, cross-linked with glutaraldehyde, solubilized, and analyzed by SDS/PAGE. Chitin-binding protein was visualized on the blotted membrane by using a rabbit antibiotin antibody as a primary antibody and a peroxidase-labeled goat anti-rabbit IgG antibody as a secondary antibody. (A) Arabidopsis seedlings and rice cells. (B) Arabidopsis leaves from fully grown plants and cultured cells of both Arabidopsis and rice.
SI Text
Light microscopy.
Two 5-ml drops of a spore suspension containing 5 ´ 105 spores·ml-1 in distilled water were placed on Arabidopsis leaves. Fungal hyphae within the resulting lesions were visualized by staining the inoculated leaves with lactophenol-trypan blue as described (1). Detection of callose deposition (2) and autofluorescence (3) was performed as described.Analysis of reactive oxygen species.
The reactive oxygen species released by Arabidopsis seedlings were quantified by the luminol chemiluminescence method (4). Ten-microliter aliquots of the medium were transferred to 96-well microtiter plates and immediately supplemented with 50 ml of 1.1 mM luminol and 100 ml of 14 mM potassium hexacyanoferrate solution using a programmable injector attached to the luminometer. Chemiluminescence was measured by a microplate luminometer model TR717 (Applied Biosystems).Protein extraction, Western blot analysis, and immunocomplex kinase assay.
Proteins were extracted from seedlings as described (5). The concentration of protein extracts was determined by using protein assay kit (Bio-Rad) with BSA as standard. For Western blot analysis, total proteins (20 mg) were separated by the 4-12% gradient NuPAGE Novex Bis-Tris gel (Invitrogen) and transferred onto PVDF membrane (Millipore). Western blot analysis and the antibodies information were described (5). Immunocomplex kinase assay of MPK3 and MPK6 was performed by using 50 mg of total protein with anti-MPK3 and anti-MPK6 antibodies, respectively, as described (5).RT-PCR.
Whole seedlings were frozen in liquid nitrogen and stored at -80°C. Total RNA was extracted from the Arabidopsis seedlings using an RNeasy Plant Mini kit (QIAGEN). Approximately 12 seedlings per treatment were combined to get appropriate amounts of total RNA. For two-step RT-PCR, first-strand cDNA synthesis was performed using 1 mg of total RNA and QuantiTect Reverse Transcription Kit (QIAGEN). PCR was performed with Takara Ex Taq (Takara) under the following conditions. Denaturing step for 3 min at 94° C; followed by 3-step cycling of denaturizing at 94°C for 0.5 min, annealing at 55°C for 0.5 min, extension at 72°C for 50 seconds; final extension at 72°C for 10 min. The cycle numbers were; 28 for At3g21630 gene and 25 for Actin. The gene-specific primer pairs were designed as follows. At3g21630 forward primer (5'-ggagaagtgtctgcaaaagtag-3') and reverse primer (5'-ctaccggccggacataagactg-3') for the region including the inserted Ds/T-DNA; At3g21630 forward primer (5'-ataagcgtggacaaatctgtt -3') and reverse primer (5'-ctttgctcggaatttctggtc-3') for the region without the inserted Ds/T-DNA; Actin (Accession no.: M20016) forward primer (5'-ggcgatgaagctcaatccaaacg-3') and reverse primer (5'-ggtcacgaccagcaagatcaagacg-3'). RT-PCR for defense gene expression in Fig. 3 was performed with 20 ng of total RNA using OneStep RT-PCR kit (QIAGEN) following the manufacturer's protocol. Amplication was carried out under the following conditions: reverse transcription for 30 min at 50°C: initial PCR activation step for 15 min at 95°C; followed by 3-step cycling of denaturizing at 94°C for 1 min, annealing at 60°C for 1 min, extension at 72°C for 1 min; final extension at 72°C for 10 min. The cycle numbers for each gene were: 22 for PAL; 23 for rbohD and rbohF; 25 for PR-2; 26 for PR-5; 28 for Actin and Nit4. The gene-specific primer pairs were designed based on sequence data obtained from the NCBI website: Nit4 (accession no. U09961) forward primer (5'-ggtaagcaccgcaaactcat-3') and reverse primer (5'-aaacatccaccctcaagtgc-3'); PAL (accession no. X84728) forward primer (5'-attaacggggcacacaagag-3') and reverse primer (5'-agttgagatcgcagccactt-3'); PR-2 (accession no. M90509) forward primer (5'-cgataccttgccaagtccat-3') and reverse primer (5'-tgtaccggaatctgacacca-3'); PR-5 (accession no. M90510) forward primer (5'-cgtacaggctgcaactttga-3') and reverse primer (5'-gcgttgaggtcagagacaca-3'); rbohD (accession no. AF055357) forward primer (5'-ggatgggagacagcaggata-3') and reverse primer (5'-tttggcgacacaaacacaat-3'); rbohF (accession no. AB008111) forward primer (5'-aaggtgatgctcgttctgct-3') and reverse primer (5'-agagggcttttggcttcttc-3'). The amplified products were separated on 1.5% agarose gels and visualized under UV light after staining with ethidium bromide.Microarray analysis.
Microarray analyses were performed using a 60-mer Arabidopsis oligo microarray containing 44,290 features (Agilent Technologies). The RNA extracts from Arabidopsis seedlings were fluorescence labeled according to the manufacture's protocol. Each set of RNAs was labeled again by swapping the dyes (Cy3 and Cy5) to normalize for dye bias. The slides were scanned by an Agilent scanner. Data indicating a lower than 2-fold increase in fluorescence between Cy5 and Cy3 were excluded from further analysis.CERK1-GFP fusion vector
. The CERK1 ORF, except the stop codon, was amplified by PCR using the full-length cDNA (pdz 03321, supplied by RIKEN BioResource Center) as a template and subcloned into pENTRTM/D-TOPO (Invitrogen) and then into pGWB5 expression vector for GFP fusion proteins by using LR clonase reaction. In the expression vector, CERK1 ORF was inserted downstream of 35S promoter and fused to the 5' end of sGFP ORF. All of the constructs were confirmed by sequence analysis.Microsomal membrane preparations
Microsomal membrane fraction was prepared from suspension-cultured rice cells or the leaves/seedlings of A. thaliana as described (6).
Synthesis of biotinylated ligand
GN8-Bio, the conjugate of biocytin hydrazide and N-acetylchitooctaose, was prepared by reductive amination as follows. Five milligrams of N-acetylchitooctaose was dissolved in 5 ml of H2O. Twenty-five milligrams of biocytin hydrazide (PIERCE) and 6.25 mg of NaCNBH3 were added to the mixture. The mixture was heated at 80°C for 1 h on a heating block and stood still overnight at room temperature. The reaction mixture was lyophilized and then washed several times with water by centrifugation (12,000 rpm, 5 min). The supernatant fractions containing GN8-Bio were collected and lyophilized. The dried material was dissolved with water and applied to a Bio-Gel P-2 column (Bio-Rad) to remove remaining reagents. The product was further purified by HPLC using an Inertial ODS-3 column (4.6 ´ 250 mm; GL Sciences) and a gradient elution of methanol (0-50% in water). The eluate was monitored by UV absorption at 220 nm and also by MALDI-TOF/MS to detect GN8-Bio.
SDS/PAGE and Western blotting
SDS/PAGE was carried out on 10% polyacrylamide gel. Western blotting was performed on Immun-Blot PVDF Membrane (Bio-Rad). Detection of biotinylated proteins was performed by using a rabbit antibody against biotin (Rockland, 1:1,000) as a primary antibody and horseradish peroxidase-conjugated goat anti-rabbit IgG (Chemicon, 1:2,000) as a secondary antibody. Biotinylated proteins were detected by the chemiluminescence with Immobilon Western Detection reagents (Millipore).
1. Narusaka Y, Narusaka M, Park P, Kubo Y, Hirayama T, Seki M, Shiraishi T, Ishida J, Nakashima M, Enju A, et al. (2004) Mol Plant-Microbe Interact 17:749-762.
2. Narusaka Y, Narusaka M, Seki M, Ishida J, Shinozaki K, Nan Y, Park P, Shiraishi T, Kobayashi M (2005) Mol Plant Pathol 6:615-627.
3. Dietrich RA, Delaney TP, Uknes SJ, Ward ER, Ryals JA, Dangl JL (1994) Cell 77:565-577.
4. Schwacke R, Hager A (1992) Planta 187:136-141.
5. Ichimura K, Casais C, Peck SC, Shinozaki K, Shirasu K (2006) J Biol Chem 281:36969-36976.
6. Shibuya N, Ebisu N, Kamada Y, Kaku H, Cohn J, Ito Y (1996) Plant Cell Physiol 37:894-898.