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
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
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苹果 S-RNase 与肌动蛋白结合蛋白 MdMVG 相互作用，通过在自花授粉诱导早期抑制其肌动蛋白切断活性来减少花粉管生长
DOI：
10.1111/tpj.13929
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发表时间：
2018
期刊：
影响因子：
7.2
通讯作者：
Li Tianzhong
中科院分区：
文献类型：
--
作者：
Yang Qing;Meng Dong;Gu Zhaoyu;Li Wei;Chen Qiuju;Li Yang;Yuan Hui;Yu Jie;Liu Chunsheng;Li Tianzhong
在S-RNase介导的自交不亲和性中，花柱分泌的S-RNase破坏了自花花粉管的肌动蛋白细胞骨架，最终使其停止生长，但其作用机制尚不清楚。体外生物化学试验表明，S-RNase不结合或切断丝状肌动蛋白（F-actin）。在苹果（Malus domestica）中，我们发现了一种含有肌球蛋白、绒毛蛋白和GRAM的肌动蛋白结合蛋白（MdMVG），它与S-RNase发生物理相互作用，并直接结合和切断F-肌动蛋白。免疫荧光分析和全内反射荧光显微镜观察表明，S-RNase在体外抑制了MdMV G 0的F-肌动蛋白切割活性;在体内，S-RNase在MdMV G 0过表达的情况下，增加了自花授粉诱导过程中肌动蛋白微丝的荧光强度，降低了微丝切割频率和花粉管生长速率。通过诱变在MdMVG的氨基酸基序E-E-K-E-K中产生25个单点、双点和三点突变，并用免疫荧光检测所得突变体，我们鉴定了一个三点突变体MdMVG。（E167 A/E171 A/K185 A），不再具有F-肌动蛋白切断活性或与任何四种S-单体型S-RNA酶相互作用，表明所有三种氨基酸（E167、E171和K185）对于MdMVG的切断活性及其与S-RNA酶的相互作用是必需的。我们得出结论，appleS-RNase与MdMVG相互作用，通过抑制其F-肌动蛋白切断活性来减少自花花粉管生长。
InS‐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 vitrobiochemical assays revealed thatS‐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 withS‐RNase and directly binds and severs F‐actin. Immunofluorescence assays and total internal reflection fluorescence microscopy indicated thatS‐RNase inhibits the F‐actin‐severing activity of MdMVGin vitro.In vivo, the addition ofS‐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 ofMdMVGoverexpression. 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(E167A/E171A/K185A), that no longer has F‐actin‐severing activity or interacts with any of the fourS‐haplotypeS‐RNases, indicating that all three amino acids (E167, E171 and K185) are essential for the severing activity of MdMVG and its interaction withS‐RNases. We conclude that appleS‐RNase interacts with MdMVG to reduce self‐pollen tube growth by inhibiting its F‐actin‐severing activity.