Journal: bioRxiv

Article Title: Ribosome Processing Factor-2 Interacts with RPL10A to Regulate Selective Translation during Plant Immunity and Drought Stress

doi: 10.64898/2026.03.12.711238

Figure Lengend Snippet: A. Yeast-two-hybrid assay showing interaction of AtRPF2 and AtRPL10A. AtRPL10A in pDEST32 and AtRPF2 in pDEST22 were expressed and co-transformed in Mav203 yeast cells, then plated on double (-leu, -trp) and triple (-leu, -trp, -his with 20mM 3-AT) dropout media. Krev1-RalGDS and Krev1-RalGDS-m1 used as positive and negative controls, respectively. X-Gal staining was performed to confirm the interactions. B. In-silico protein-protein docking demonstrated the interaction of AtRPF2 with AtRPL10A at different amino acids. The Alpha-fold modelled protein structures were used for docking studies using ClusPro 2.0. C. Molecular dynamic (MD) simulations indicated a stable interaction between AtRPL10A with AtRPF2 protein. The simulations were carried out using full-length proteins on the GrowMacs platform. D. In planta bi-molecular fluorescence complementation (BiFC) assay confirmed the interaction of AtRPF2 with AtRPL10A within the nucleus. The pMDC-cCFP GFP-RPF2 or pMDC-nVENUS GFP-RPL10A constructs in Agrobacterium GV3101 strain were co-expressed transiently in N. benthamiana, and after 48 h, the fluorescence was observed in confocal microscopy. DAPI was used to stain the nucleus. E. Bio-layer interferometry (BLI) assay verified the physical interactions of AtRPF2 with AtRPL10A. The AtRPF2- His in pDEST15 and AtRPL10A- GST in pDEST17 were expressed in E. coli and purified with their respective tags, and the His-tag sensor was used for BLI analysis. F. A Northern blot analysis revealed the accumulation of 5.8S, 18S, and 25S rRNA in AtRPF2 -OE, AtRPL10A -OE, and RNAi Arabidopsis plants. Total RNA was extracted from four-week-old Arabidopsis leaves, immobilized on a nylon membrane, and probed with 5.8S, 18S, and 25S rRNA probes. G. Ribosome profiling from Col-0, AtRPF2 -OE and AtRPF2 -RNAi plants, H. Ribosome profiling from Col-0, AtRPL10A -OE and AtRPL10A -RNAi plants. Ten grams of tissues were used to extract ribosomes and 50-10% sucrose density gradient was used to profile. I. A 35S-methionine [35s-M] incorporation assay in Arabidopsis and J. in N. benthamiana plants demonstrating protein synthesis efficiency in overexpression and RNAi lines. A pulse chase assay with 35s-met incorporation into four-week-old leaf discs for 4 h was conducted. Methionine incorporation was quantified using a liquid scintillation counter. Error bars represent SE, with a two-way ANOVA showing p*<0.05, p**<0.01, p***<0.001, p****<0.0001.

Article Snippet: The proteins were co-incubated with GST resins (Merck), separated by 10% SDS-PAGE, and transferred to a PVDF membrane.

Techniques: Y2H Assay, Transformation Assay, Staining, In Silico, Fluorescence, Bimolecular Fluorescence Complementation Assay, Construct, Confocal Microscopy, Purification, Northern Blot, Membrane, Over Expression, Pulse Chase