Functional Analysis Of Spinach SoCPR5 And SoSIM Genes In Plant Immunity

Spinach(Spinacia oleracea L.),belongs to Spinacia of Amaranth family,is a green vegetable with high nutritional value and a variety of vitamins.Both its yield and quality are influenced by diseases.Therefore,improving the disease resistance has a great significance in spinach breeding.The cell cycle signaling pathway plays an important role in balancing development and immunity of plants.It is known that CONSTITUTIVE EXPRESSOR OF PATHOGENSIS-RELATED GENES5(CPR5)is an important immune protein and the cyclin-dependent kinase inhibitors(CKIs)are the core cell cycle regulators.The Arabidopsis CPR5(AtCPR5)could not only negatively regulate plant immunity and cell death,but also promote the development of trichomes.As the Arabidopsis cpr5 mutant exhibits early senescence,warf and enhanced immunity.Besides,the trichomes of cpr5 mutant are branchless while the wild type trichomes are often three-branched.The SIM(SIMESE)and SMR1(SIMESE-1),which belong to the CKIs in Arabidopsis,function redundantly not only to positively regulate plant immunity,but also inhibit the cell proliferation of trichomes.The sim smr1 mutant trichomes are multicellular and clustered whereas Arabidopsis wild-type trichomes are unicellular and single,meanwhile,they are susceptiable to the pathogen with less immunity.In the spinach genome,we identified the homologs of Arabidopsis CPR5 and SIM referred to as Spinacia oleracea CPR5(SoCPR5)and Spinacia oleracea SIM(SoSIM),respectively.In order to study their function,we transferred SoCPR5 into cpr5 mutants of Arabidopsis,and the SoSIM into Arabidopsis sim smr1 double mutants,respectively.Our experimental results show that in the cpr5/35S:SoCPR5 transgenic plants,the spontaneous cell death and enhanced immunity are suppressed,and the trichomes are also fully restored to the wild type phenotype by SoCPR5.It shows that SoCPR5 function as its counterpart AtCPR5 in regulating plant immunity,PCD and trichomes development.In the 35S:SIM transgenic plants,SIM are overexpressed,exibiting dwarf and serrated leaves which are similar to the phenotype of RNA interference of Cyclin-Dependent kinase(CDKA1).This indicates the SIM is a CDK inhibitor.In the sim smr1/35S:SoSIM transgenic plants,the phenotype are restored to the WT and the CDKA1 RNAi with different expression of SoSIM.On the other hand,the resistance and PCD of sim smr1/35S:SoSIM transgenic plants with WT phenotype were also restored to the WT,indicating that SoSIM activates the plant immunity and PCD.These data indicate that SoSIM functions as its counterpart AtSIM to negatively control cell proliferation,and to positivily regulate the plant immunity and PCD.Our data verify that SoCPR5 and SoSIM function as their Arabidopsis counterpart in plant immunity and plant development,and they are conserved between plant species.Therefore,SoCPR5 and SoSIM could be genetically applied to control the plant resistance and improve the qulity and yield of spinach.