| Self-incompatibility is a kind of specific identification reaction between style and pollen,which is shown in the process of style to pollen.This mechanism is similar to the identification of animals and plants against pathogenic microorganisms and the innate immune response.At present,the results of some researches on cruciferous plants belong to Sporophytic self-incompatibility which had been proved that the similar immune recognition factor determined self-incompatibility reaction.However,S-RNase-mediated Gametophytic self-incompatibility response has not been reported.Currently,the result of study on apple showed that S-RNase could enter pollen tube with the assistance of the ABC transporter protein and then broke the concentration gradient of calcium ions of the pollen tube.In this case,it is not clear that S-RNase how to activate the signaling pathway and which defense responses factors induced to deal with invasive S-RNase in the process of pollen tube absorbing S-RNase.In this study,we used apple and a variety of techniques to prove the process of the S-RNase absorbed into the pollen tube which caused a series of signal response:Ca2+-ROS-JA-MdMYC2 which induced the response of defense protein containing a y-thionine domain in pollen tube.This defensive protein was induced in the process of S-RNase into the pollen tube and inhibited the activity of S-RNase.The main results are as follows:1.We studied the calcium signal at first.We used S-RNase expressed in vitro to incubate apple pollen.The result proved S-RNase could demage calcium ion gradient inside the pollen tube and affect the expression of calcium response protein MdCBL5 again.Generally it was considered that change in calcium gradient affected the expression of Rboh gene.Using fluorescence microscopy we found that ROS signal was weakened at tip but significantly enhanced at pollen tube sub-top;we added S-RNase expressed in vitro to medium to incubate pollen tube,we used qRT-PCR to detect MdRbohH.We found the expression of MdRbohH up-regulated compared with the control.We used HPLC to detect the concentration of JA in pollen.The results showed:the concentration of JA increased compared with the control.Because the concentration of JA in pollen tube was influenced greatly,then we cloned the key transcription factor MdMYC2 in JA signaling pathway.We added S-RNase of apple expressed in vitro to medium to incubate pollen tube,we used qRT-PCR to detect MdMYC2.We found the expression of MdMYC2 up-regulated compared with the control.The result showed that S-RNase could induce Ca2+-ROS-JA-MdMYC2 signal response.2.Using a recombinant protein MdMYC2 and apple genomic DNA banding post Chip-seq analysis,we obtained a high frequency binding sequence.Combining with apple genome comparative analysis,we found that one of these sequences was y-thionin gene(MdDI)promoter which was obtained by S2-RNase screening yeast pollen cDNA library which was done previously.One-hybrid experiments further validated MdMYC2 had conjunction with MdDI promoter sequence.After adding S-RNase to medium to incubate pollen tubes,we found the expression of MdDI was not up-regulated by qRT-PCR.The results indicated that MdD1 was activated by Cat+-ROS-JA-MdMYC2 signal pathway.In order to clarify the relationship between MdDI and S-RNase,we first used yeast two-hybrid,pull-down,bi-molecular fluorescence complementation to further verify the interaction between them.To demonstrate how MdD1 in pollen tube responding to immersion of S-RNase,we added S1-RNase and S9-RNase to the medium respectively to incubate pollen,we detected the trend of MdD1 in pollen tube by fluorescence microscopy.The result showed that MdDl in self-pollen tube and non-self-pollen tube responded to invasion of S-RNase.Using fluorescence microscopy,we found that after blocking MdMYC2,MdD1 in pollen tubes did not move,which revealed that in this process MdD1 did not respond to invasion of S-RNase.3.In order to clarify how MdD1 acting on S-RNase,we purified S-RNase and MdD1 in vitro and then mixed S-RNase and MdD1.We used yeast RNA as a substrate to measure the activity of S-RNase.The results showed that MdD1 inhibit the activity of S-RNase.We segmented S-RNase to clarify the interaction between MdD1 by the way of yeast two-hybrid.The results showed there were exactly interaction between MdD1 and the active site region of S-RNase and no interaction between MdD1 with non-active regions of S-RNase.After site-directed mutagenesis of the active site of S-RNase,no interaction was found in yeast two-hybrid and bimolecular:fluorescence complementation experiments.IP assay in vivo experiments proved in vivo that the non-mutated S-RNase could combine MdD1 in pollen tubes,and could not combined MdD1 after mutation.In summary,the apple pollen MdD1 protein was the defense response factor of pollen tube,which could be activated by Ca2+-ROS-JA-MdMYC2-MdDl signal pathway and was not only in response to the invasion of S-RNase to pollen tube but also inhibited the activity of S-RNase. |
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