Apple S-RNase Combined With MdMVG Reduces The Growth Of The Self-pollen Tube By Inhibiting F-actin Severing

The self-incompatibility is a specific recognition reaction between the style and pollen tube,which runs through the whole style.Normal F-actin cytoskeleton assembly dynamic in pollen tube growth is needed,the assembly including actin depolymerization and polymerization,severing,capping,bundles and so on.Among them,the severing action of F-actins can provide a large number of free and active small fragments of actin in the pollen tube,which provides the basis for the polymerization of actin,capping,and so on.In the process of pollen tube growth,a variety of extracellular stimuli can lead to rapid changes in the cytoskeleton.In gametophytic self-incompatibility S-RNase mediated reaction,S-RNase can be regarded as a kind of extracellular stimuli,after self pollination pollen tube can cause changes in the actin cytoskeleton,resulting in the growth of pollen tube stopped,but how S-RNase influence the severing F-actin action and what kind of proteins involved have not yet been reported.Currently,it was found that S-RNase can enter the pollen tube with the help of MdABCF transporter into self pollen tube.It was unknown whether S-RNase can be directly combined with the actin cytoskeleton depolymerization or severing,polymerization.If S-RNase does not bind directly to the actin filament,what role does S-RNase play in the process?Therefore,we take apple pollen tube as materials,using a variety of technical means to prove that S-RNase through the way of binding 3 severing sites to inhibit MdMVG severing activity,resulting in pollen tube actin cytoskeleton circulation slowing,in the end pollen tube growth is inhibited.The main results are as follows:1.To detect whether S-RNase directly severs F-actin,we first determined whether S-RNase could directly bind to actin:we performed high-speed co-sedimentation experiments for His-S1-RNase,His-S2-RNase,His-S3-RNase,and His-S9-RNase to determine whether each possessed F-actin binding activity.The resulting supernatants and pellets were analyzed separately using SDS-PAGE.The results from this assay showed that none of the four genotypes of S-RNase possessed F-actin binding activity.The four genotypes of S-RNase were each incubated with Alexa-488 phalloidin-stabilized F-actin;no merged fluorescence signal was detected.We also found that none of the four genotypes of S-RNase could bind to F-actin:after hydration,the four genotypes of S-RNase were individually added into the pollen tube culture medium,but no merged fluorescence signal was detected.2.Because S-RNase failed to bind and sever F-actin,we hypothesized that an unknown pollen F-actin severing protein mediated by S-RNase is involved in the process.We used mature peptides of S2-RNase(S2-Mat)as bait to screen a yeast cDNA library prepared from M.domestica cv.'Ralls Janet'pollen(S1 S2).After comparison analysis we found a yet-unknown protein encoding a part of Myosin,Villin and GRAM;therefore,we named it Malus domistica Myosin Villin GRAM(MdMVG).High-speed co-sedimentation and immunofluorescence experiments were performed to verify MdMVG possesses F-actin binding activity.S-RNase,MdMVG and F-actin were incubated in room tempreture for 30min,and it was found that the length of F-actin under four genetypes S-RNase+MdMVG treatment increased as compared to MdMVG treatment alone,indicating that S-RNase weakened the ability of MdMVG to sever F-actin.In vivo,we transiently oerexpressed and RNAi MdMVG in hydrated 'Ralls Janet' pollen tubes by particle bombardment and then added S-RNase to pollen medium.We found that the fluorescence intensity of adding S1-RNase and S2-RNase was increased,the severing frequency of F-actins was reduced,and the pollen tube growth rate was also reduced drasticly.However,no differences were detected between adding S3-RNase and S9-RNase as compared to control,indicating that S-RNase affects the ability of MdMVG to sever self pollen tube F-actin.3.The analysis of the structure and the conserved amino acid ratio of the severing protein showed that the severing site were 167E,171E,185K.In vivo and in vitro experiments confirmed that these three sites are indeed severing sites.In order to determine whether S-RNase inhibits the severing of F-actins by binding to the three severing sites of MdMVG,yeast two hybrid,apple leaf and pollen tube BIFC all showed that S-RNase could not interact with MdMVG E167A,E171A,K 185A.To sum up,the apple style self S-RNase does indeed affect the growth of the pollen tube by binding 3 severing sites to inhibited MdMVG severing F-actins and slow down pollen tube growth.