Identification And Functional Analysis Of Incompatible Receptor Kinase Srk Interacting Protein In Brassica Napus

Self-incompatibility(SI)system is a well-known strategy employed by plants that enables the pistil to accept compatible pollen or reject self-incompatible pollen to avoid inbreeding and promote outcrossing.The transduction of self-incompatibility signals in Brassica napus L.is mediated by SRK receptor kinase.Until now,only an ubiquitination degradation pathway mediated by ARC1 has been found,and other downstream components in SRK signaling pathway remain largely unknown.So,searching for the downstream signaling substrates of SRK is very important for improving the selfincompatibility signaling pathway.In this study,self-incompatibility Brassica napus‘W-3',S70 and self-compatibility line Westar were used as experimental materials.Yeast two-hybrid,gene cloning,expression analysis,genetic transformation,phosphoproteomics and other techniques were employed.The main results of the self-incompatibility and downstream signal molecules results are shown as follows: 1.Quantitative phosphoproteomics analysis of compatible/incompatible pollinations in Brassica napus L.Phosphoproteomic analysis was performed to identify the phosphoproteins in selfcompatible line Westar and self-incompatible(SC)line ‘W-3' in the stigma of Brassica napus.Phosphoproteome analysis led to the identification of 4,109 phosphopeptides,representing 1978 unique protein groups.The plant-pathogen interactions,cell wall modification,m RNA surveillance,RNA degradation was enriched.The plant hormone signal transduction,especially,the Brassinolide,Abscisic acid,and Ethephon signal pathway proteins were highly induced in pistils pollinated with incompatible pollen.2.Yeast two-hybrid identification of BnA5.ZML1 interacting with SRKThe kinase domain of SRK was used as a bait to screen a yeast two-hybrid c DNA library of self-incompatible line‘W-3''s stigma,and a GATA-type transcription factor BnA5.ZML1 was identified.Their interactions were verified in vitro and vivo.Furthermore,it was found that the CCT domain of BnA5.ZML1 is the main region interacting with SRK kinase.3.BnA5.ZML1 was a compatible factor induced by compatible pollenTissue-specific expression pattern of BnA5.ZML1 was performed by reversetranscription PCR(RT-PCR).BnA5.ZML1 was expressed in root,stem,leaf,flower,silique and stigma,but the expression level in stigma is much high than other tissues.Real-time PCR showed that BnA5.ZML1 was induced when pollinated with compatible pollen.Using CRISPR-Cas9 technology to knock out BnA5.ZML1,we found that pollen grains adhered and pollen tubes penetrated the stigmatic surface were significantly decreased in bna5.zml1 mutant.Furthermore,the number of seeds per pod were significantly reduced in bna5.zml1 mutant than wildtype.Overexpression of BnA5.ZML1 can partially breakdown the selfincompatibility.This also indicates that BnA5.ZML1 is a compatible factor in the selfincompatibility response.4.BnA5.ZML1 affects self-compatibility/incompatibility by inhibiting the expression of SRKTo further explore the molecular mechanisms of BnA5.ZML1 mutants and overexpressed phenotypes,we examined the maker genes involved in the selfincompatibility signaling pathway.The expression level of SRK was down regulated in BnA5.ZML1 overexpression line and up regulated in bna5.zml1 homozygous mutant by quantitative RT-PCR.In addition,BnA5.ZML1 inhibited the expression of SRK by DualLuciferase Reporter(DLR)assay,indicating that BnA5.ZML1 regulates selfincompatibility by inhibiting the expression of SRK.Yeast one hybrid and EMSA assays confirmed that BnA5.ZML1 can not interact with SRK promoter,indicating that BnA5.ZML1 may interact with other proteins and bind to the SRK promoter to inhibit the expression of SRK gene.5.SRK influence the subcellular localization of BnA5.ZML1BnA5.ZML1 and SRK were co-transformed into N.benthamiana,the BnA5.ZML1 is mainly expressed in the nucleus in the absence of SRK,when the presence of SRK,BnA5.ZML1 can be localized on the cytoplasm and partially colocalized with SRK on the plasma membrane.6.The importance of the SRK kinase domain in transmitting self-incompatibility signalThe class I SRK overexpression transgenic plant were obtained by fusion of the GFP tag and FLAG tag to the C-terminus and N-terminus of the class I SRK gene,respectively,and driven by the SRK self-promoter and transformed into the class II self-incompatible material S70.Subsequently,analysis of these transgenic plants showed that the GFP tag affected the activity of the C-terminal kinase region of SRK to transmit self-incompatibility signal.The N-terminal fusion protein FLAG-SRK was successfully expressed in class II S70 by pollination experiment and expression analysis.It is verified that the signal pathways of class I and class II self-incompatibility are conservative.The creation of this material provides favorable conditions for subsequent validation of in vivo protein interactions and IP-MS to find self-incompatible pathway related proteins.