Isolation of Pollen-expressed Actin as a Candidate Protein Interacting with S-RNase in Prunus avium L.

Abstract

Many species of the Rosaceae family exhibit an S-ribonuclease (S-RNase)-based self-incompatibility (SI) system. In this system, pistil and pollen specificities are conferred by S-RNase and S locus F-box protein, respectively. In addition to these specificity determinants, other SI general factors have been observed to be essential for an SI reaction. To isolate SI general factors in Prunus, yeast two-hybrid (Y2H) screening was performed against the sweet cherry (Prunus avium L.) pollen cDNA library using the N-terminal (NT) and C-terminal (CT) regions of P. avium (Pav) S⁶-RNase as bait. Among 31 genes isolated by Y2H screening, the interaction between actin homolog (PavAct1) and S-RNase was further examined by in vitro assays because some T2/S-type RNase family proteins, such as RNASET2 and ACTIBIND, are assumed to exert their cytotoxicity through its actin-binding ability. Although the GST pull-down assay did not detect any interaction between GST-tagged recombinant PavAct1 and non-reduced S-RNase, interaction between the recombinant PavAct1 and reduced S-RNase was observed. Furthermore, filamentous actin (F-Act) cosedimentation assay and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) crosslinking assay using rabbit actin demonstrated that reduced S-RNases interacted with both F-Act and globular actin (G-Act). These results collectively suggested that S-RNase, which may be fully or partially reduced in the pollen tube cytoplasm, could bind actin to disrupt the coordinated actin dynamics.