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

Isaura Simões,Rosário Faro,Daniel Bur,Carlos Faro

doi:10.1074/jbc.m702477200

Isaura Simões, Rosário Faro + Show 2 more

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https://doi.org/10.1074/jbc.m702477200

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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.

Highlights

Ments [2,3,4,5,6,7,8,9,10] 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
We have successfully cloned, expressed, and purified Arabidopsis constitutive disease resistance 1 (CDR1), and our results indicate that this enzyme displays properties that are significantly different from those of the well studied typical plant APs
Recombinant CDR1 performs proteolytic cleavages at a higher pH compared with most APs, we clearly demonstrated the correct assignment to the family of APs by inactivating the enzyme through modification of an aspartate residue in the consensus DTG motif that is crucially required for proteolytic activity

Summary

Introduction

Ments [2,3,4,5,6,7,8,9,10] 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. 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, 8] suggest functional specialization of these particular set of plant APs as found e.g. in mammalian renin as well as tight activity regulation, in contrast to the rather unspecific housekeeping role normally ascribed to typical plant APs. there is an increasing interest on the structure and function of atypical and nucellin-like APs. Arabidopsis CDR1 is a member of the larger group of the atypical APs and was first identified by Xia and co-workers [7] while screening for gain-of-function mutants displaying enhanced resistance to bacterial pathogens. The results described, in combination with the very specific plant phenotype previously reported, strongly support the

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