Identification of multiple salicylic acid-binding proteins using two high throughput screens

Area of Science:

• Plant Biology
• Molecular Biology
• Biochemistry

Background:

• Salicylic acid (SA) is a crucial plant hormone regulating growth, development, and stress responses.
• SA's role in disease resistance signaling is vital but not fully understood.
• Identifying SA-binding proteins (SABPs) is key to elucidating SA's functions.

Purpose of the Study:

• To discover novel SA-binding proteins (SABPs) using high-throughput screening.
• To validate candidate SABPs (cSABPs) using multiple biochemical assays.
• To clarify the role of NPR1 as an SABP.

Main Methods:

• Developed two high-throughput screens employing photo-reactive 4-AzidoSA (4AzSA) for protein crosslinking.
• Utilized mass spectrometry and protein microarrays for initial cSABP identification.
• Confirmed SA-binding activity through SA-inhibitable crosslinking, 3-aminoethylSA binding (SPR), and [(3)H]SA binding assays.

Main Results:

• Identified nine novel SABPs meeting stringent validation criteria.
• Confirmed previously reported SABPs and assessed approximately 80 additional cSABPs.
• Demonstrated that NPR1 binds SA in all three validation assays, resolving conflicting reports.

Conclusions:

• Successfully identified nine new SABPs, expanding the known interactome of salicylic acid.
• Established robust methods for high-throughput identification and validation of SABPs.
• Provided definitive evidence for NPR1 as an SA-binding protein, crucial for plant immunity.