Molecular Analysis of a Maize Autoactive NB-LRR Protein Rp1-D21 Identifies Precise Structural and Subcellular Requirement for Hypersensitive Response

The maize Rp1-D21 gene confers an autoactive hypersensitive response (HR) in the absence of pathogen infection. Rp1-D21 was formed by an intergenic recombination event between Rp1-D, encoding a NB-LRR protein, which confers rust resistance to corn, and Rp1-dp2, a homologue of Rp1-D. We have previously shown that H₂O₂ accumulation, the increased expression of PR1 and other defense marker genes (PRms and WIP1) were associated with Rp1-D21-mediated HR phenotype. To investigate the molecular function of Rp1-D21-regulated HR, we transiently expressed Rp1-D21 in N. benthamiana and showed that it induced clear HR in this system. A serial of deletion constructs indicated the coiled-coil domain (CC_D21) and the first 20 amino acids (AA) from N-terminus and the last 34 AA from C-terminus were indispensible for Rp1-D21-triggered HR. Rp1-D21-induced HR was P-loop dependent. Domain swaps between Rp1-dp2 and Rp1-D were used to identify precise regions/AA combinations associated with the autoactive nature of Rp1-D21-triggered HR. Subcelluar localization revealed that Rp1-D21 was predominantly localized in cytoplasm with a small amount in the nucleus. Both cytoplasmic and nuclear localizations were required for Rp1-D21- and CC_D21-triggered HR.