Self-controlled Nucleocytoplasmic Shuttling Determines RPW8.2-mediated Cell Death And Disease Resistance

The Arabidopsis thaliana RESISTANCE TOPOWDERY MILDEW8.2(RPW8.2)gene was isolated from ecotype MS-0 by map-based cloning and encode a resistance gene with transmembrane anchor and coiled-coil domain.It confer broad-spectrum resistance against powdery mildew,Hyaloperonospora parasitica and Cauliflower mosaic virus.The expression of RPW8.2 is induced by powdery mildew and it specially targets to the extra-haustorial membrane(EHM).The transportation process from Golgi to EHM for RPW8.2 is rely on cell cytoskeleton and two arginine and lysine enriched short motif(R/K-R/K-x-R/K)in itself.The RPW8.2-mediated resistance response include SA and H2O2 accumulation,enhanced PR genes expression,callose deposition and hypersensitive response with unclear mechanism.In order to confirm a predicted nuclear localization signal in RPW8.2 amino acid sequence,a construct was generated for the classical nuclear localization signal(NLS)NLSSV40 fused with single red fluorescence reporter gene(NLSSV40-RFP)and transient expressed in Nicotiana benthamiana(N.b).The localization pattern of NLSSV40-RFP is similar with RFP which localize in both of cytoplasm and nucleus,which caused by the small fusion protein simply diffuse through the nuclear pore complex(NPC).The molecular mass of NLSSV40 with two tandem RFP(NLSSV40-2×RFP)is big enough for passive transportation by NPC,and the fusion protein only localized in nucleus in leaf epidermal cells of N.b.It indicate this is a reliable method to test nucleocytoplasmic trafficking signals.Using this method,the peptide of 92-99 amino acid(aa)was confirmed as a strong NLS.The RPW8.2 was divided into 8 fragments for nucleocytoplasmic trafficking signals screening.Finally,three nuclear export signals(NESs)(1-60 aa,121-140 aa and 141-169aa)and one more NLS(65-74aa)were found in PRW8.2,and the degradation of RPW8.2 is rely on proteasome pathway.RPW8.2 initiate resistance in nucleus and induce cell death in cytoplasm.In order to find out the core sequence which induce the cell death in RPW8.2,RPW8.2 was truncated from N and C terminus follow each nucleocytoplasmic trafficking signal.The RPW8.2 variations were transient expressed in N.b for cell death.The C terminus can induce cell death in cytoplasm with the core sequence of 111-174 aa.It is the critical amino acids from 143 to 174 for C terminus function.1-60 aa and two arginine and lysine enriched short motif(20-25 aa and 91-100aa)which determined EHM targeting for RPW8.2 play negative regulators for C terminus function.The fragment of 61-81 aa play a positive regulator for C terminus function.RPW8.2 C terminus promote chloroplast degradation for inducing cell death.The senescence-associated genes such as AtWRKY53.AtSAG13,AtRPS17 and AtRBCS involve in this process and also regulate RPW8.2-mediated cell death.It will support a new directions for further research by its cell compartment specific activity in cell death and disease resistance and senescence-associated genes regulate RPW8.2-mediated cell death...