Characterization of Recombinant CDR1, an Arabidopsis Aspartic Proteinase Involved in Disease Resistance

Abstract

The Arabidopsis thaliana constitutive disease resistance 1 (CDR1) gene product is an aspartic proteinase that has been implicated in disease resistance signaling (Xia, Y., Suzuki, H., Borevitz, J., Blount, J., Guo, Z., Patel, K., Dixon, R. A., and Lamb, C. (2004) EMBO J. 23, 980 -988). This apoplastic enzyme is a member of the group of "atypical" plant aspartic proteinases. As for other enzymes of this subtype, CDR1 has remained elusive until recently as a result of its unusual properties and localization. Here we report on the heterologous expression and characterization of recombinant CDR1, which displays unique enzymatic properties among plant aspartic proteinases. The highly restricted specificity requirements, insensitivity toward the typical aspartic proteinase inhibitor pepstatin A, an unusually high optimal pH of 6.0 -6.5, proteinase activity without irreversible prosegment removal, and dependence of catalytic activity on formation of a homo-dimer are some of the unusual properties observed for recombinant CDR1. These findings unveil a pattern of unprecedented functional complexity for Arabidopsis CDR1 and are consistent with a highly specific and regulated biological function. Plant aspartic proteinases (APs)3 are widely distributed in the plant kingdom and have been detected or purified from monocot and dicot species as well as gymnosperms (1). APs are synthesized as single chain preproenzymes and, upon activation, converted to mature two-chain enzymes. A characteristic feature of "typical" plant AP precursors is the presence of a protein domain of ϳ100 amino acids known as the plant-specific insert, which is highly similar to saposin-like proteins and is removed from the precursors upon activation into the mature form of the enzymes (1).Recently published results on plant APs with unexpected localizations, unusual sequences, and novel structural arrangements (2-10) as well as the identification of a wide variety of AP-like proteins in the Arabidopsis genome (11, 12) have triggered a redefinition of the classification of plant APs. Faro and Gal have recently proposed a new classification that takes into account the diversity of plant APs by grouping them into typical, nucellin-like, and atypical members, with the latter group comprising the majority of the newly identified Arabidopsis APs (11). Despite having low sequence identity, these atypical and nucellin-like APs share several common features such as 1) the absence of the internal segment plant-specific insert in their sequence, 2) an unusually high number of cysteines, 3) the type of amino acids preceding the first catalytic triad, and 4) unexpected localizations, which clearly differentiate them from the well studied typical plant APs. Thus far, only a rather small number of atypical and nucellin-like APs have been studied (2-10). However, the proposed roles in highly regulated processes like plastid homeostasis (tobacco CND41) (9, 10), disease resistance (Arabidopsis CDR1) (7), or programmed cell death (Barley nucellin and Arabidopsis PCS1) (2,...

Multiplicity of aspartic proteinases from Cynara cardunculus L.

“…It has been shown that the plant-speciWc insert is removed entirely or partially during maturation and processing of plant AP precursor (Guruprasad et al 1994;Frazao et al 1999;White et al 1999). There are 69 genes encoding aspartyl proteases in the Arabidopsis genome (Takahashi et al 2008), and studies on their expression at the protein level as well as their physiologic roles have already started (Simoes et al 2007).…”

Roles of Plant Proteases in Pathogen Defense

“…The gene product of A. thaliana constitutive disease resistance 1 (CDR1) is an apoplastic aspartic protease and is a member of a larger group of the atypical aspartic protease (Simões et al, 2007). The overexpression of CDR1 causes dwarfing and resistance to virulent P. syringae, and the suppression of CDR1 results in compromised resistance to avirulent P. syringae and in increased susceptibility to virulent strains compared to the wild type.…”

EAT1 promotes tapetal cell death by regulating aspartic proteases during male reproductive development in rice

“…Another Arabidopsis aspartic protease, PROMOTION OF CELL SURVIVAL1 (PCS1), has an anti-cell death role in embryonic and reproductive development 32 . A third Arabidopsis aspartic protease, CONSTITUTIVE DISEASE RESISTANCE 1 (CDR1) is associated with signalling in disease resistance 33 . S5 is the only characterized aspartic protease from rice; its gene is a major locus for indica-japonica hybrid sterility 34 .…”