Screening And Identification Of Candidate Proteins Interacting With A RLKs Nip43 In Astragalus Sinicus

In previous work,we have identified a protein Nip43 from the host plant Astragalus sinicus interacted with Nop P of Mesorhizobium huakuii 7653 R by yeast two-hybrid system.The interactions between Nop P and Nip43 were confirmed in vivo and vitro,but little information is known about the downstream of Nip43 signaling pathway.In this work,it was intended to screen and identify any candidate proteins interacting with Nip43 in the host plant A.sinicus.Firstly,the nip43 gene of A.sinicus was cloned to p GBKT7 as a bait plasmid,and then transformed into the yeast strain Y187.After the bait plasmid’s auto-transcriptional activation and toxicity was examined,the AD-c DNA library of A.sinicus was screened.17 positive clones was identified by the reselection on the SD/-Trp-Leu-Ade-His+X-gal plates.After sequencing the c DNA inserts,one positive clone containing an EPN protein out of our interest and importance was chosen for further investigation.The BLAST analysis showed that the EPN contained a superfamily domains,ENTH/ANTH.Secondly,we constructed two deletion mutants of EPN in the AD vector,and transformed them to the yeast strain AH109.The interaction between the Nip43 and the deletion mutants of EPN was tested by the yeast two hybrid respectively.The results indicated that the Nip43 interacted with the N-terminal of EPN,and the crucial interaction domain of EPN was ENTH/ANTH domain(29-198 amino acid residues).Finally,we quantified the expression level of epn by real time PCR,the results showed that nip43 had a high expression level in the infected roots.In the infected roots,the expression level of the epn started to increase from the first day after the inoculation of M.huakuii 7653 R,and reached the peak level on the sixth day,then the expression level of the epn decreased gradually after the sixth day.