Apple S‐RNase interacts with an actin‐binding protein, MdMVG, to reduce pollen tube growth by inhibiting its actin‐severing activity at the early stage of self‐pollination induction

Abstract

In S-RNase-mediated self-incompatibility, S-RNase secreted from the style destroys the actin cytoskeleton of the self-pollen tubes, eventually halting their growth, but the mechanism of this process remains unclear. In vitro biochemical assays revealed that S-RNase does not bind or sever filamentous actin (F-actin). In apple (Malus domestica), we identified an actin-binding protein containing myosin, villin and GRAM (MdMVG), that physically interacts with S-RNase and directly binds and severs F-actin. Immunofluorescence assays and total internal reflection fluorescence microscopy indicated that S-RNase inhibits the F-actin-severing activity of MdMVG in vitro. In vivo, the addition of S-RNase to self-pollen tubes increased the fluorescence intensity of actin microfilaments and reduced the severing frequency of microfilaments and the rate of pollen tube growth in self-pollination induction in the presence of MdMVG overexpression. By generating 25 single-, double- and triple-point mutations in the amino acid motif E-E-K-E-K of MdMVG via mutagenesis and testing the resulting mutants with immunofluorescence, we identified a triple-point mutant, MdMVG , that no longer has F-actin-severing activity or interacts with any of the four S-haplotype S-RNases, indicating that all three amino acids (E167, E171 and K185) are essential for the severing activity of MdMVG and its interaction with S-RNases. We conclude that apple S-RNase interacts with MdMVG to reduce self-pollen tube growth by inhibiting its F-actin-severing activity.

Genome-Wide Identification and Characterization of CIPK Family and Analysis Responses to Various Stresses in Apple (Malus domestica)

“…As one of the woody perennial species, the apple tree often suffers from a variety of environmental stresses making it urgent to comprehend the function of the CIPK genes in stress responses in order to improve crop tolerances against adverse environments [ 27 ]. In this study, all the CIPK genes were identified by apple genome-wide analysis, their evolutionary relationship phylogenetically was evaluated, and their structure and chromosomal distribution of the CIPK genes were analyzed.…”

Cytoskeleton, Transglutaminase and Gametophytic Self-Incompatibility in the Malinae (Rosaceae)

“…The authors speculated that S-RNase simultaneously decrease Ca 2+ current and inhibit ROS formation; however, as Ca 2+ currents and ROS production are mutually interconnected, it is difficult to distinguish the order in time between tip-localized ROS disruption and Ca 2+ current decrease [28]. Cytoplasmic Ca 2+ changes drastically affected the dynamics of AFs and MTs [13,28]. If AFs are not arranged correctly, this causes improper secretion and thus results in deeply altered cell wall deposition.…”

“…In both apple and pear, the SI response certainly involves the cytoskeletal apparatus in a way that is not yet fully clarified. Yang and coworkers recently studied the timing of the events affecting the cytoskeleton during the SI response in apple; they demonstrated that AF depolymerization takes place within 6 minutes after SI induction, while pollen tube MTs are disrupted and form foci only 60 min after SI induction [13,51]. Therefore, MTs are likely to be a secondary target of the SI process but could also play more roles that are important during the SI response.…”

“…Specifically, S-RNase inhibits the F-actin-severing activity of MdMVG. thereby modifying the dynamics of AFs and reducing pollen tube growth [13]. The above data suggested that the alteration of AFs can occur at least in two ways: on the one hand S-RNase could act directly on proteins that regulate actin dynamics, on the other hand the effect of S-RNase could be achieved by altering factors such as Ca 2+ , which in turn affect the activity of actin-binding proteins or activate various enzymes, such as TGase, for which actin is one target.…”

“…Other processes, including changes in cytoplasmic Ca 2+ concentration and actin filaments (AFs) alteration, are likely to be involved [12]. This Ca 2+ -signaling cascade leads downstream to pollen tube tip growth arrest, depolymerization of AFs and consequently programmed-cell death (PCD) [13]. Both pollen-style interaction and SI response occur with different mechanisms in different plant families; however, since cytoplasmic Ca 2+ concentration and the proper organization of the cytoskeleton are crucial for pollen tubes elongation, they might be common targets in mediating the SI process.…”