Evidence of physical interactions of puroindoline proteins using the yeast two-hybrid system

The puroindoline proteins PINA and PINB play key roles in determining wheat grain texture and also have potential antimicrobial roles. Many recent studies show that their roles in grain texture involve some interaction or interdependence, and their antimicrobial activity may also involve formation of protein complexes. The issue of whether any homo- and/or heteromeric associations occur amongst the PIN proteins is thus critical for understanding their biological functions and exploiting them for grain texture modifications or antimicrobial applications, but is as yet unresolved. This work has utilised the well-established yeast two-hybrid system to directly address this issue. The results confirm occurrence of in vivo interactions between the two PIN proteins for the first time, and show that PINB interacts with itself and also interacts, although somewhat weakly, with PINA, while PINA is a weaker interactor. The results explain the many reported observations suggesting a co-operative interaction between the two proteins and provide a rapid and efficient tool for testing the effects of various alleles/mutations on the interactions and lipid binding properties of these proteins, which are of functional significance to grain texture and antimicrobial defence functions.