Screening And Preliminary Functional Analysis Of Proteins Interact With LRRK1-a Leaf Rolling Regulatory Protein

Rice(Oryza sativa L.)is a major food crops in the world which has important function and significance.Moderating rolling leaves of rice is beneficial for rice to maintain upright posture and enhance the ability of rice to resist lodging,at the same time,it can reduce the mutual occlusion between leaves and improve the utilization efficiency of light in rice.Previous studies found that after LRRK1 was overexpressed in rice,leaves of transgenic lines produced an obvious positive rolling phenotype.The transverse section of the rolling leaves was observed and found that the number and size of bulliform cells in transgenic lines were much less than those in wild type,which was the direct cause of leaf rolling,however,the molecular mechanism of which LRRK1 regulates the growth of bulliform cells remains unclear.In this study,proteins that interact with LRRK1 were screened and identified by immunoprecipitation combined with mass spectrometry(IP-MS)using LRRK1 overexpressed rice materials,then the function of screened proteins was analyzed.This study provided a theoretical basis for analyzing the mechanism of LRRK1 affecting the growth of rice leaf bulliform cells.Specific results of this study are as follows:(1)A total of 91 proteins that may interact with LRRK1 were identified by IP-MS in vivo,it was speculated that Os A7 and OsGF14e were related to the regulation of leaf rolling traits.Os A7 is a plasma membrane H~+-ATPase of rice,which is closely related to the regulation of plant cell p H and cell elongation;OsGF14e is a typical 14-3-3 protein,which plays an important role in signal transduction in plant cells and plant growth and development,and also plays a role in regulating H~+-ATPase in plasma membrane.So we speculated that these two proteins may be related to the development of bulliform cells,the two proteins will be considered as the research object of this study.(2)Os A7 and OsGF14e genes were cloned from total c DNA of rice leaves and bioinformatics analysis was carried out.The CDS of Os A7 was 2 856 bp in length and composed of 951 amino acids.Os A7 belonged toⅡfamily with 81.26%homology as the plasma membrane proton pump AHA2 of Arabidopsis thaliana.The analysis of subcellular localization showed that Os A7 was mainly located in the plasma membrane of rice cells.The CDS of OsGF14e is 789 bp in length and consists of 262 amino acids.OsGF14e and At GF14ωof Arabidopsis thaliana belong to the epsilon group with an homology of 80.23%.OsGF14e is mainly expressed in the cytoplasm of rice cells by subcellular localization.(3)The interactions between LRRK1,OsGF14e and Os A7 were investigated by yeast two-hybridization,Bi FC and Pull down experiments.Yeast two-hybrid vector,bimolecular fluorescence complementary vector and prokaryotic expression vector of OsGF14e,LRRK1and Os A7 were constructed respectively.The interaction analysis experiments were carried out,and the results showed that:OsGF14e can interact with leaf rolling regulatory protein LRRK1,and OsGF14e can also interact with the C-terminal of Os A7,but there is no direct interaction relationship between LRRK1 and Os A7.(4)Overexpression of OsGF14e in rice resulted in irregular leaf rolling phenotype in rice.To verify whether OsGF14e and Os A7 involved in regulating rice leaf rolling or not,overexpression vectors of OsGF14e and Os A7 were constructed,as well as gene editing vectors based on CRISPR/Cas9.All the above plant expression vectors were respectively transferred into Kitaake rice by Agrobacterium-mediated rice transformation method,and positive OsGF14e overexpressed plants were obtained.OsGF14e may be related to the molecular mechanism of LRRK1 regulating rice leaf rolling because rice leaves overexpressed with OsGF14e showed irregular rolling phenotype.In summary,the protein OsGF14e interacting with LRRK1 was identified by mass spectrometry in this study,and the interaction between them was verified through experiments in vivo and in vitro.Meanwhile,this study found that OsGF14e interacts with plasma membrane H~+-ATPase protein Os A7.A large number of studies have shown that this interaction may play a role in regulating the activity of plasma membrane H~+-ATPase.Phenotypic observation of OsGF14e overexpressed plants showed irregular curl phenotype.Therefore,it is speculated that after the overexpression of LRRK1 in rice,a large amount of LRRK1 interacts with OsGF14e,indirectly affecting the interaction between OsGF14e and Os A7,leading to the decrease of enzyme activity of Os A7 as plasma membrane proton pump,inhibiting the growth and development of vesicle cells,and causing rice leaves to curly.These results provide a theoretical basis for revealing the molecular mechanism of LRRK1 regulating rice leaf rolling.