Biophysical characterization of Hpa1 protein from Xanthomonas orzyae

Xanthomonas oryzae pv. oryzae (Xoo), the causative agent of bacterial blight in rice, secretes a suite of effectors via the Type III Secretion System (T3SS), among which harpin proteins like Hpa1 are key modulators of plant immunity. Unlike other T3SS effectors, Hpa1 lacks enzymatic activity and instead acts as a biostimulant, promoting growth and defense responses. In this study, the hpa1 gene was cloned from Xoo BXO1 strain and successfully expressed in E. coli Rosetta cells. The recombinant protein was purified using Ni-NTA chromatography, and SDS-PAGE, Western blotting, and MALDI-TOF analysis confirmed its molecular weight (∼16.5 kDa). Biophysical characterization revealed a predominance of α-helical content, with CD spectroscopy. Thermal and chemical unfolding of Hpa1 was evaluated using CD spectroscopy (with GuHCl) and ANS fluorescence (with GuHCl and urea), revealing moderate stability. pH-dependent stability assessed by fluorescence showed optimal structural retention at pH 5. TFE-induced structural modulation, studied by CD, demonstrated concentration-dependent α-helix enhancement. Thermal stability was assessed through four approaches: SDS-PAGE, CD at 222 nm, SYPRO Orange-based thermal shift assay, and HR-inducing activity in Nicotiana benthamiana. These results collectively confirmed that native Hpa1 conformation is not necessary for its function. These findings highlight its potential as a protein-based biostimulant for agricultural applications and its utility as a model for studying intrinsically disordered yet functionally stable proteins.