The Functional Analysis Of BnaRPLS34 In The Self-incompatibility Response Of Brassica NA-PUS. L

Ribosomal proteins have long been known for their role in m RNA directed protein synthesis but their function in other cellular activities like,signalling pathways,inflorescence,and in self-incompatibility response are yet to be explored.In bisexual angiosperms,the stamen and stigma are in close proximity to each other which enhances the chance of selfcrossing.To avoid self-crossing flowering plants,have independently evolved Selfincompatibility system which prevents self-crossing and promotes outcrossing.The out crossing in turn increases hybrid vigour and genetic variation.In Brassica napus L selfincompatibility system involves alleles specific interaction of pollen secreted small cysteinerich(SCR/SP11)protein and stigmatic S-receptor kinase(SRK).This interaction leads to the activation of E3 ubiquitin ligase Armadillo repeat-containing 1(ARC1),resulting in proteasomal degradation of compatibility factors necessary for successful fertilization.However,except for the degradation of compatibility factors,exocyst complex protein(Exo70A1),glyoxlase 1(GLO1)and Phospholipase D1(PLD1)the targets of ARC1 and the intracellular signalling networks regulated by these genes are still elusive.In this study,we identified a group of Ribosomal larger subunit protein genes(RPLSs),which collectively showcased a unique expression pattern in self-compatible response relative to self-incompatible and unpollinated condition.By coupling time course transcriptome analysis with tissue expression pattern and quantitative real time PCR(q RT-PCR)of pollination bioassay we validated their unique expression.However,the physico-chemical nature,genome wide distribution and evolutionary analysis of these RPLs are also reported.CRISPR/Cas9 mediated functional characterization of one RPL protein Bna RPLS34 was also performed which suggested it to be involved in the self-compatibility response.Overall,our findings suggested the important role of RPLLs as potential targets in self-incompatible response and explored a novel compatibility factor in the self-incompatibility system.The stepwise findings of our study are as follows 1?Genome-wide analysis of RPLSs in B.napusThis bioinformatic study was initiated in Arabidopsis thaliana TAIR database.From the gene family search ribosomal proteins were investigated which all belonged to a single super family named as the ‘‘Cytoplasmic ribosomal protein gene family''(CRPG)according to the database.The CRPG family contain both the Ribosomal larger subunit proteins(RPLs)and the ribosomal smaller subunit proteins(RPSs).Since we were interested only in RPLs all the corresponding genes were extracted while the rest were ignored.This yielded a total of 123 RPLs which were used as query sequences for the identification of RPLs in B.napus,B.rapa and B.oleraceae.A total of 522 Bna RPSLSs,269 Bra RPLSs and 257 Bol RPLSs were extracted.The increased number of RPLSs in B.napus might be resulted due to Whole genome triplication(WGT).Since the analysis of too much genes would be too complex we first phylogenetically analysed A.thaliana and B.napus RPLSs.All duplicates and poorly aligned sequences were removed.All the genes of the identified clade were retrieved and used as queries for homologues identification in B.rapa and B.oleraceae.This phylogenetic analysis resulted in a total of 69 genes which were divided into four clades,name as L13,L29,L34 and L36.The nomenclature of the clade is consistent with the domain shared by the members of each clade.This analysis also led us to identify 27 Bna RPLSs which aroused from a same clade.Intriguingly,transcriptomic analysis,validated via q RT-PCR exposed them to share the same high expression pattern in the self-compatible response while showing minimal expression in the self-incompatible response of B.napus.2?Expression analysis of identified Bna RPLSsThe phylogenetic analysis led us to identify 27 Bna RPLSs which shared the same expression pattern in self-(in)compatible response of B.napus.The same trend was found when the genes were subjected q RT-PCR quantification analysis.All of these genes showed high expression in self-compatible pollination while showed minimal expression in selfincompatible pollination.3?Functional Analysis of Bna RPLS34(GSBRNA2T00045497001)At present,the report ingress of plant ribosome protein function,mainly focuses on protein synthesis,cell growth,division and development,and there is no research on whether they regulate self-incompatibility response and downstream signalling pathway.Through CRISPR/Cas9 technology,we constructed a mutant of the Bna RPLS34 gene,which significantly breaks the self-compatibility of Brassica napus.Through q PCR analysis we found that the Bna RPLS34 mutants significantly inhibited the self-compatibility and its positive regulatory genes SRK and ARC1 compared to wild types,while THL1/2,EXO70A1 and GLO1 were significantly increased.Based on the above studies,we speculate that Bna RPLS34 is a potential candidate responsible for positive regulation of the self-compatibility response of B.napus,and it is expounded that its molecular mechanism is a signal transduction pathway that enriches the self-compatibility of B.napus.