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In animals, protease inhibitors of the serpin family are associated with many physiological processes, including blood coagulation and innate immunity. Serpins feature a reactive center loop (RCL), which displays a protease target sequence as a bait. RCL cleavage results in an irreversible, covalent serpin-protease complex. AtSerpin1 is an Arabidopsis protease inhibitor that is expressed ubiquitously throughout the plant. The x-ray crystal structure of recombinant AtSerpin1 in its native stressed conformation was determined at 2.2 Å . The electrostatic surface potential below the RCL was found to be highly positive, whereas the breach region critical for RCL insertion is an unusually open structure. AtSerpin1 accumulates in plants as a fulllength and a cleaved form. Fractionation of seedling extracts by nonreducing SDS-PAGE revealed the presence of an additional slower migrating complex that was absent when leaves were treated with the specific cysteine protease inhibitor L-trans-epoxysuccinyl-L-leucylamido (4-guanidino)butane. Significantly, RESPONSIVE TO DESICCATION-21 (RD21) was the major protease labeled with the L-trans-epoxysuccinyl-L-leucylamido (4-guanidino)butane derivative DCG-04 in wild type extracts but not in extracts of mutant plants constitutively overexpressing AtSerpin1, indicating competition. Fractionation by nonreducing SDS-PAGE followed by immunoblotting with RD21-specific antibody revealed that the protease accumulated both as a free enzyme and in a complex with AtSerpin1. Importantly, both RD21 and AtSerpin1 knock-out mutants lacked the serpin-protease complex. The results establish that the major Arabidopsis plant serpin interacts with RD21. This is the first report of the structure and in vivo interaction of a plant serpin with its target protease.Protease cascades are prominent mediators of rapid physiological responses in animals, playing a role in cellular immunity, blood clotting, and development. The proteolytic specificity of the serine and cysteine proteases involved dictates the fidelity of these reactions. The serpins are an important group of proteins that curb the activity of these cascades through specific irreversible inhibition of the proteases. For example, in Drosophila, the necrotic (nec) gene encodes a protease inhibitor of the serpin family. Necrotic protein controls a proteolytic cascade that activates the innate immune response to fungal and Gram-positive bacterial infections (1). In nec null mutants, Toll-mediated immune responses are constitutively activated, even in the absence of infection, implying that Nec continually restrains this immune response. As opposed to other types of protease inhibitors, serpins offer both an irreversible and tunable type of inhibition (reviewed in Ref.2). In their native conformation, serpins are in a stressed (spring-loaded) state with a solvent-exposed reactive center loop (RCL).3 Specific residues of the RCL are precisely accommodated by the target protease active site. Upon cleavage of the serpin peptide bond linking the P1 and ...

Resistance to different pathogenic races of Fusarium oxysporum f. sp. lycopersici (F. o. lycopersici) was explored at two genomic levels in tomato. Six independent Fusarium resistance loci were identified by comparing the responses of a complete set of 53 lines carrying different introgressed regions of the Lycopersicon pennellii genome in a L. esculentum background. The loci confer varying degrees of resistance to different races of the pathogen. Corresponding map positions from different tomato species were aligned and in some cases revealed parallel resistance to F. o. lycopersici with qualitative changes in race specificities. One of the loci identified corresponds to the previously characterized complex resistance locus I2, which is involved in resistance to F. o. lycopersici race 2. A novel member of this locus, I2C-5, which belongs to the NBS-LRR family of resistance genes, was cloned and shown to confer partial resistance in transgenic plants. Thus, at a particular complex locus gene members can confer full or partial resistance to F. o. lycopersici race 2. The results of our whole-genome mapping analysis underline the robust independent origin of resistance to a particular disease and demonstrate the conservation of resistance features at syntenic loci, together with the rapid diversification of genes for innate resistance within loci.

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The presence of a single resistance (R) gene allele can determine plant disease resistance. The protein products of such genes may act as receptors that specifically interact with pathogen-derived factors. Most functionally defined R-genes are of the nucleotide binding site-leucine rich repeat (NBS-LRR) supergene family and are present as large multigene families. The specificity of R-gene interactions together with the robustness of plant-pathogen interactions raises the question of their gene number and diversity in the genome. Genomic sequences from tomato showing significant homology to genes conferring race-specific resistance to pathogens were identified by systematically “scanning” the genome using a variety of primer pairs based on ubiquitous NBS motifs. Over 70 sequences were isolated and 10% are putative pseudogenes. Mapping of the amplified sequences on the tomato genetic map revealed their organization as mixed clusters of R-gene homologues that showed in many cases linkage to genetically characterized tomato resistance loci. Interspecific examination within Lycopersicon showed the existence of a null allele. Consideration of the tomato and potato comparative genetic maps unveiled conserved syntenic positions of R-gene homologues. Phylogenetic clustering of R-gene homologues within tomato and other Solanaceae family members was observed but not with R-gene homologues from Arabidopsis thaliana. Our data indicate remarkably rapid evolution of R-gene homologues during diversification of plant families.