No abstract

Citrus exocortis viroid (CEVd) and tomato mosaic virus (ToMV), which produce a systemic non-necrotizing infection in tomato (Lycopersicon esculentum cv. Rutgers), strongly induced the accumulation of a phenolic compound that we have characterized as 2,5-dihydroxybenzoic acid (gentisic acid, GA) by nuclear magnetic resonance, following purification by high-performance liquid chromatography. Levels of free and total GA increased more than 150-fold in response to CEVd and ToMV infections. Unlike these non-necrotizing infections, the necrotizing reaction elicited by Pseudomonas syringae pv. syringae in this host did not produce any accumulation of GA. It is also shown that, in healthy leaf tissues, benzoic acid (BA) and salicylic acid (SA) were rapidly converted to GA, SA being the immediate precursor of GA, according to radiolabeling studies. Interestingly, exogenous GA elicited accumulation of the previously described CEVd-induced antifungal pathogenesis-related (PR) proteins P23, P32, and P34. These proteins were not induced by exogenous SA, which is able to elicit other CEVd-induced PR proteins in tomato. These results suggest that GA acts as a pathogeninduced signal, additional to SA, for activation of plant defense genes in tomato.

A functional genomics project has been initiated to approach the molecular characterization of the main biological and agronomical traits of citrus. As a key part of this project, a citrus EST collection has been generated from 25 cDNA libraries covering different tissues, developmental stages and stress conditions. The collection includes a total of 22,635 high-quality ESTs, grouped in 11,836 putative unigenes, which represent at least one third of the estimated number of genes in the citrus genome. Functional annotation of unigenes which have Arabidopsis orthologues (68% of all unigenes) revealed gene representation in every major functional category, suggesting that a genome-wide EST collection was obtained. A Citrus clementina Hort. ex Tan. cv. Clemenules genomic library, that will contribute to further characterization of relevant genes, has also been constructed. To initiate the analysis of citrus transcriptome, we have developed a cDNA microarray containing 12,672 probes corresponding to 6875 putative unigenes of the collection. Technical characterization of the microarray showed high intra- and inter-array reproducibility, as well as a good range of sensitivity. We have also validated gene expression data achieved with this microarray through an independent technique such as RNA gel blot analysis.

Screening of a genomic library from tomato plants (Lycopersicon esculentum) with a cDNA probe encoding a subtilisin-like protease (PR-P69) that is induced at the transcriptional level following pathogen attack (Tornero, P., Conejero, V., and Vera, P. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6332-6337) resulted in the isolation of a cluster of genomic clones that comprise a tandem of four different subtilisin-like protease genes (P69A, P69B, P69C, and P69D). Sequence analyses and comparison of the encoded proteins revealed that all are closely related (79 to 88% identity), suggesting that all are derived from a common ancestral gene. mRNA expression analysis as well as studies of transgenic plants transformed with promoter-␤-glucuronidase fusions for each of these genes revealed that the four genes exhibit differential transcriptional regulation and expression patterns. P69A and P69D are expressed constitutively, but with different expression profiles during development, whereas the P69B and P69C genes show expression following infection with Pseudomonas syringae and are also up-regulated by salicylic acid. We propose that these four P69-like proteases, as members of a complex gene family of plant subtilisin-like proteases, may be involved in a number of specific proteolytic events that occur in the plant during development and/or pathogenesis.Proteolysis is fundamental for the normal functioning of multicellular organisms and plays key roles in a variety of processes such as development, physiology, defense and stress responses, and adaptation to the changing environment. In plants, despite the importance of all these processes and involvement of different classes of proteinases (Refs. 1-5, and references therein), it still remains to be defined more precisely what components and molecular mechanisms are responsible for regulating specific aspects of protein degradation/processing. A major task for research will be to determine which pathway of proteolysis is responsible for the degradation of particular proteins.The serine proteases are one of the best characterized groups of proteolytic enzymes in higher organisms. They can be grouped in six clans, of which one of the largest is the subtilisin-like clan (EC 3.4.21.14) that includes over 200 different members. Despite this wealth of knowledge, very little is know about subtilisin-like proteases in plants. Recently, we and others have shown the existence of members of this clan in plants, including Arabidopsis (6), tomato (7, 8), melon (9), and Lilium plants (10). According to a recent classification (11), the subtilisin-like proteases from plants can be grouped within the Pyrolysin subfamily, which is highly related to the Kexin subfamily of proteases involved in the posttranslational processing of peptide hormones (12, 13). Comparative molecular, biochemical, and cellular studies indicate that the subgroup of plant subtilisin-like enzymes are characterized by the presence of a large polypeptide sequence insertion preceding the reactive Ser residue and/or lon...