Study On The Mechanism Of RD19 Interacting With PAD4 To Regulate Plant Innate Immunity

PAD4（phytoalexin deficiency 4）and EDS1（enhanced diseases susceptibility 1）are essential regulators in plant immunity.EDS1 and PAD4 form a heterodimer to promote salicylic acid synthesis and activate immune responses.EDS1 and SAG101 also form a heterodimer in plant immunity,but its loss is largely compensated for by PAD4.To explore the signaling pathway mediated by PAD4-EDS1.Co-IP followed with mass spectrum experiments were performed and identified RD19（responsive to dehydration 19）as a candidate interacting protein with PAD4.RD19was reported that it associates with Pop P2 to form a nuclear complex that is required for activation of the RRS1-R mediated resistance.Using Co-IP,BIFC and Split-LUC assays,we confirmed the interaction between RD19 and PAD4.We found the double mutant rd19lrd19 was more sensitive to pst DC3000 Avr RPS4 than wild type Col-0.In addition,RD19 overexperession in Col-0 background（OE RD19）lead to autoimmunity phenotypes including constitutive expression of ICS1,PR1,PR2 genes,enhance resistance to virulent bacteria strain pst DC3000.In order to further explore the genetical relationships among EDS1,PAD4,SAG101 and RD19,we crossed eds1-2,pad4 sag101 with OE RD19,and obtained homozygous progeny.The OE RD19 eds1-2,OE RD19 pad4,OE RD19 pad4 sag101 plants grew normally,and were as sensitive as eds1 plant to pst DC3000 infection.While,OE RD19 sag101plants showed similar phenotypes as OE RD19 Col-0.These results suggested that RD19-mediated resistance is dependent on EDS1 and PAD4,but not SAG101.In addition,we proved RD19 being an active cysteine protease.RD19 ^C159Aand RD19 ^N329Amutations abolish its cysteine proteases activity and ability to enhance plant resistance to virulent bacteria.Taken together,our study shows that RD19 specifically interacts with PAD4 to regulate plant immunity.Furthermore,the RD19-meidated resistance is dependent on EDS1 and PAD4.Moreover,RD19 have cysteine proteases activity,and the activity is required for its function in immunity.Therefore,our results provide important mechanistic insight into the regulation of plant immunity.