Mutant materials and growth conditions
Wild type rice (Oryza sativa cv. 9522) and mutant plants were grown in the paddy fields of Shanghai Jiao Tong University (30 °N 121 °E) from June to September (the natural growing season) according to standard local practice. Stem lengths were measured, and grains were harvested, at maturity. Differences in mature plant height, flag leaf width (7-day old seedlings) and seed length/width were analyzed using ruler and Excel. The osfh5 mutant in the 9522 background was available from previous work (Zhang et al., 2011). The rip1, rip2 rip3 and rips mutants were generated in 9522 via CRISPR-Cas9 as previously described (Xie et al, 2015). CRISPR-Cas9 guide RNAs and screening primers are shown in Table S1.
Yeast two hybrid (Y2H) assays
Yeast two hybrid experiments were performed according to the manufacturer’s instructions (Clontech). The coding sequences of OsFH5, P1, P2 and P3 were fused in-frame with the sequence encoding the GAL4 DNA-binding domain of the bait vector pGBKT7 (BK). The coding sequences of RIP1 and SIAH1 were cloned in the prey vector pGADT7 (AD). Primers for cloning are given in Table S1. Each bait–prey pair was co-transformed into Saccharomyces cerevisiae strain AH109 as previously described (Lecrenier et al, 1998). Yeast was grown for 2–3 days in liquid YPDA medium (Yeast Extract Peptone Dextrose Medium) at 28°C, then dilutions were spotted onto selective SD media containing 20 mg/ml X-gal, and grown for a further 2–3 days. Three amino acid–-deficient medium (–his –leu –trp) and four amino acid–deficient medium (–his –leu –trp –ade) was used to test the binding ability of protein interaction.
To analyze formation of homo- and hetero-oligomers between RIP proteins, RIP1 was cloned into pGBKT7, and paired with prey vectors (pGADT7) encoding SIAH1–6 domains from RIP1 to RIP6, respectively. Transfection and selective growth as above.
Bimolecular fluorescence complementation (BiFC)
DNA encoding RIP1–RIP6, SIAH1, and OsFH5 were amplified and fused with nYFP, cYFP, and/or eGFP, cloned into pXY104 and pXY106 vectors (From Prof. Hongquan Yang’s Lab of SJTU), using primers shown in Table S1. Constructs were individually or co-transformed into 2-day Nicotiana benthamiana leaves using Agrobacterium-mediated transformation as previously described (Li et al, 2017). After 24–36 h in the dark, tobacco leaf cells were observed under 50 % glycerol with a Leica sp5 confocal laser scanning microscope.
Phylogenetic analyses
The RIP1 protein sequence was used for BLASTp screening of other proteins from National Center for Biotechnology Information (NCBI) and https://www.arabidopsis.org/. Matching protein sequences were aligned with the software Molecular Evolutionary Genetics Analysis (MEGA) and used to create an evolutionary tree. Branch support was assessed with 1000 bootstrap replicates (Felsenstein, 1985, Sanderson and Wojciechowski, 2000).
Transient gene expression in rice protoplasts
Rice protoplasts were prepared according to Zhang et al (2011) with the following modifications. Wild type 9522 seeds were germinated in dark for 2 weeks in the dark. 50–60 seedlings (no seeds) were harvested, cut into 0.5 mm lengths, and incubated in 0.6 M mannitol in the dark for 10 min. The mannitol solution was discarded and replaced with enzymatic solution (1.5 % (w/w) cellulase RS, 0.75 % (w/w) macerozyme R-10, 0.6 M mannitol, 10 mM MES (pH 5.7), 10mM CaCl2 and 0.1 % BSA), and vacuum infiltrated for 1 h, before shaking at 60–80 rpm in the dark for 4–5 h in room temperature. The enzymatic hydrolysate was removed by 1ml syringe. Cells were gently resuspended in W5 solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl and 2 mM MES (pH 5.7)), and the protoplasts were released after shaking at room temperature for 1 h in dark. Protoplasts were collected after filtrated into small bottles through 40µm nylon mesh (Millipore) and washed 3 times with fresh W5 solution at 1500 RPM for 3 min. Protoplasts were collected and precipitated with an appropriate amount of MMG solution (0.4 M mannitol, 15 mM MgCl2 and 4 mM MES (pH 5.7)), and resuspended to a concentration of 2×106 cells/ml in room temperature.
For transient protoplast transformation, 10 µg of plasmid DNA and 200 µl of protoplast solution were combined in a 2 ml Eppendorf tube, to which 220 µl (w/v) PEG was gentled added, and mixed gently at room temperature for 20 min. 1 ml of W5 solution was added, and protoplasts gently pelleted at 200 g for 1 min. The supernatant was discarded, and 1 ml W5 solution was used to resuspend the protoplasts and transfer them to a 12-well plate (moistened in advance with 1 mL 5% BSA). Plates were incubated in the dark at 22°C overnight, and again pelleted gently at 200 g for 1 min. The supernatant was discarded, and the fluorescence signal was observed by confocal fluorescence microscopy, eGFP excitation is 488 nm, YFP excitation is 514 nm, chloroplasts can fluoresce spontaneously.
In vitro protein stability assays
Protein expression expressing OsFH5, RIP1-eGFP, RIP5-eGFP, and RIP6-eGFP were introduced separately into N. benthamiana leaves via Agrobacterium-mediated transformation, as described above. After 36–48 h in the dark, tobacco leaves were harvested, ground under liquid nitrogen, and extracted with a non-denaturing extraction buffer (50 mM Tris-MES (pH 8.0), 0.5 M sucrose, 1 mM MgCl2, 10 mM EDTA, 5 mM DTT, protease inhibitor cocktail (Sigma, 1 complete Mini tablet per 10 ml of extraction buffer)).
OsFH5 extract was mixed with different ratios (0, 1, 2, 4, or 8× vol) of RIP1 extract, and incubated to 0 min, or for 10 min at room temperature in the presence and absence of 20 µM MG132 (Sigma), a proteosome inhibitor. A OsFH5 negative control was also used (a 9× vol of RIP1 extract). Mixtures were immediately run on a protein gel, transferred to a western blot, and probed with anti-OsFH5 antibody (Zhang et al) or anti-eGFP antibody to detect RIP protein, as previously described.
Root and microfilament observation
Roots from 3 d seedlings grown in light incubator were used for picture with Asana mirror (Leica). Microfilaments from cv. 9522 and rips roots were stained using the glycerol method (Olysla egers and Verbelen, 1998). Roots from 3 d seedlings grown in light incubator were incubated in PEM buffer (100 mM PIPES, 10 mM EGTA, 5 mM MgSO4, and 0.3 M mannitol, pH 6.9) that contains 1% (w/v) glycerol (Sigma Aldrich) and 6.6 mM Alexa Fluor 488-phalloidin staining (Invitrogen). After a 30 min incubation, root tips were observed in 50% glycerol with a Leica TCS SP5 confocal laser scanning microscope equipped with a 363 1.46–numerical aperture HC PLANs objective to determine microfilament lengths in lateral root cells. Actin filament abundance and bundling (skewness) were measured using image J.
Accession numbers
RIP1, Os02g0293400; RIP2, Os05g0238200; RIP3, Os01g0234900; RIP4, Os02g0128800; RIP5, Os03g0356414; RIP6, l Os07g0659800. Arabidopsis RIP homologs: At2g41980, At3g13672, At3g58040, At5g53360, At4g27880, At3g61790.