LjPP2C, A Protein Phosphatases 2c From Lotus Japonicus, Functions As A Negative Regulator Of MPK6 Pathway

The symbiotic interaction between legumes and rhizobia results in the formation of nitrogen-fixing root nodules is the most efficient and productive way for plant to get nitrogen and play a very important role in the agriculture. Establishion of symbiotic systemsneeds series of signal pathway.Sym RK is a receptor kinase in the pathway and is specificinteract with SIP2. SIP2 is a MAP kinase kinase and play a pivotal role in IT formation and nodule primordium initiation in L.japonicus. In this article, we had idenfified some proteins which was interaction with MPK6 by yeast two-hybrid screening. MPK6 is the downstream target of SIP2. Here, some results are as follows:1. We got MPK6 gene from L.japonicus with PCR.Using report gene Lac Z to test the self-activation of MPK6.Result showed that full length MPK6 had self-activation. Then construct seven deletion mutantsand assay their self-activation using X-Gal in the medium SD/-Trp in vivo plate. Self-activation site of MPK6 locatited between 362 and 368 amino acids.2. Using MPK61-351 as a bait in yeast two-hybrid screening,one c DNA encode a protein phosphatase type 2C, named Lj PP2 C.Lj PP2 C contain a conserved domain PP2 Cc, belonging to PP2 C superfamily.3.Yeast two-hybrid assay, protein pull-down assay showed MPK6 interacted with Lj PP2 C.Using Bi FC to test the interaction between Lj PP2 C and MPK6 in planta.4. Lj PP2 C dephosphorylated MPK6 in Vitro.5. Using Real-time PCR to measure the expression levels of Lj PP2 C m RNA in different tissues. Result showed Lj PP2 C was expressed in all tissues, most in root and leaf.7. Overexpression of Lj PP2 C in L.japonicus with M.loti inoculation. The results showed that the nodule numbers were decrease comparing with control.