Functional Analysis On Receptor-like Kinase OsWAK50 And Intracellular Interacting Protein OsAHP2 In Rice

The wall-associated kinases(WAKs)belong to a unique subfamily of the receptor-like kinases(RLKs)in plants,named for its tight association with the cell wall.WAKs play important roles in pathogen resistance,heavy-metal responses and cell expansion in Arabidopsis(Arabidopsis thaliana).There are 125 OsWAK genes in rice(Oryza sativa),of which OsWAK50 is a typical RLK with an extracellular domain,a transmembrane domain and a cytoplasmic kinase domain,and shares higher similarity to AtWAKs than others Previous works in our lab have shown that OsWAK50 overexpression transgenic rice plants displayed pleiotropic phenotypes during development and enhanced salt tolerance.The aim of this work is to further identify the biochemical function,expression pattern and intracellular interacting protein of OsWAK50.Our results show that OsWAK50-GFP fusion protein is localized on the cell surface and associated with the cell wall when plasmolyzed.Further cell fractionation combined with western blotting analysis confirmed the cell wall-associated feature of OsWAK50.Histochemical analyses of the OsWAK50 promoter fused with the?-glucuronidase(GUS)reporter gene show that OsWAK50 expresses at shoot root junction,leaf tip,lamina joint,lateral root initiation site,root tip and spikelet.Quantitative real-time PCR analysis and western blot analysis show that both the RNA level and the protein level of OsWAK50 can be induced by salt and drought stress.By the yeast two-hybrid screen,we identified OsWAK50 intracellular interacting proteins,among which a shikimate kinase protein(OsSK)and a histidine-containing phosphotransfer protein(OsAHP2)were further confirmed for interacting with OsWAK50 by in vivo BiFC assay.Arabidopsis histidine-containing phosphotransfer proteins(AHPs)are mediators in a multistep phosphorelay pathway of cytokinin signaling.According to the analysis on conserved histidine amino acid,OsAHP1 and OsAHP2 are the only two functional HPs with high similarity in rice.To further obtain genetic evidence for the interaction between OsWAK50 and OsAHP2,we generated OsAHP2 overexpression and RNAi transgenic rice plants.OsAHP2 overexpression rice plants show no obvious phenotype under normal growth condition or salt stress.Whereas OsAHP RNAi rice plants show various abnormalities in growth and development,including semi-dwarf,early leaf senescence,decreased number of tillers and reduced fertility.Moreover,OsAHP RNAi rice plants are more sensitive to salt stress which is consistent with OsWAK50 loss-of-function mutant.Histochemical analyses of the OsAHP2 promoter fused with the GUS reporter gene show that OsAHP2 expresses at seminal root,adventitious root,lateral roots,lamina joint,basal node and spikelet,which is similar to OsWAK50 tissue expression pattern.Especially when induced by salt treatment,the expression pattern of OsWAK50 in root is very similar to that of OsAHP2.OsAHP2-GFP was localized on the cell surface as well as in the cytoplasm and nucleus.Our results indicate that OsWAK50 and OsAHP2 both are essential and may interact within cytoplasmic of root cells under salt stress.OsAHP RNAi seedlings displayed reduced sensitivity to cytokinin inhibition of lateral root formation and retardation of leaf senescence,and reduced cytokinin primary response gene expression.While OsAHP2 overexpression rice plants displayed increased cytokinin primary response.The above results indicate that OsAHPs function as positive regulators in cytokinin signaling.The enhanced salt tolerance of OsWAK50 overexpression rice is possibly caused by the interaction with OsAHP2 to affect the cytokinin signaling.Further downstream genes expression is being analyzed.