A Study On The Interaction Between A PIP Protein And Harpin Protein Hpa1 Of The Bacterial Blight Pathogen

Harpins,produced by gram-negative plant pathogenic bacteria,are virulence accessory proteins with multiple functions in a variety of plants.External application of harpins to plants activates distinct signaling pathways,which regulate several beneficial effects,such as plant growth enhancement,disease resistance,and drought tolerance.Previous studies have shown that the multiple functions of harpins depend on their interactions with recognition sensors present in plant plasma membranes.A rice plasma membrane aquaporin,OsPIP1;3,has been shown to interact with Hpa1,a harpin protein produced by bacterial blight pathogen of rice.However,the structural basis of the molecular interaction is unknown.To identify amino acid residues or functional motifs in the OsPIP1;3 sequence that are responsible for the OsPIP1;3 interaction with Hpa1,we generated mutant versions of OsPIP1;3 by fragment deletion and site-directed mutagenesis,respectively.When a split-ubiquitin-based yeast two-hybrid system was employed to analyze protein combinations of Hpa1 with the canonical form and mutant versions of OsPIP1;3,interaction was found between Hpal and the canonical form,but not in the combination of Hpa1 with the mutant protein generated by deleting a short intracellular region called connecting loop E and transmembrane domain 6.Amino acid residues 246 to 279 were found to be critical for the Hpal-OsPIP1;3 interaction,as evidenced by the absence of interaction under the circumstance of residue deletions.Analyses of additional substitutive protein mutants indicated that residues 276 to 279 in transmembrane domain 6 were also important for the molecular interaction.These data suggest that loop E and transmembrane domain 6 are determinants of OsPIP1;3 interaction with Hpal while particular residues play crucial roles in the molecular interaction.AtPIP1;4,A Arabidopsis thaliana plasma membrane aquaporin has been shown to interact with Hpa1,a harpin protein produced by bacterial blight pathogen of rice in plant cell membrane.Our studies found that AtPIP1;4 could enhance plant growth.We found AN and gm in atpipl;4 were significantly decreased compared to WT plants,but the complement plants can be restored to wild-type levels while the overexpression plants were significantly enhanced.This shows that AtPIPl;4 effect on the photosynthesis of plants by increasing the CO₂ transport across the membrane.We also found that AN and gm in Arabidopsis thaliana were significantly increased due to AtPIP1;4-Hpal protein interactions.These results suggest that the role of Hpal in enhancing plant growth may due to its interaction with AtPIP1;4.We also cloned and conducted MY2H vectors with different harpins from different bacteria genome DNA to detect whether they can also interacted with AtPIP1;4.The results showed that other nine harpins have no interaction with AtPIP1;4.This result suggested that it may be unique to Hpal interacting with AtPIP1;4.