Tomato leaves infected by the fungal pathogen Cladosporium fulvum contain several types of intracellular and extracellular pathogenesis-related (PR) proteins. Previously, we reported the purification and serological characterization of five extracellular PR proteins: P2, P4, P6, a chitinase and a beta-1,3-glucanase [22, 23]. Here we describe the purification of a basic intracellular 33 kDa beta-1,3-glucanase and the isolation and characterization of cDNA clones encoding the two extracellular P14 isomers P4 and P6, the extracellular acidic beta-1,3-glucanase and a basic 35 kDa beta-1,3-glucanase, different from the purified 33 kDa protein. Southern blot analysis demonstrated that tomato PR proteins are not encoded by large gene families, as is the case in tobacco. The number of genes corresponding to each protein was estimated to vary between one and three. A northern blot analysis indicated that the mRNAs for the extracellular PR proteins (P4, P6 and acidic beta-1,3-glucanase) accumulate to similar levels in compatible and incompatible tomato-C. fulvum interactions, although the maximum level of expression is reached much faster in the incompatible interaction. On the other hand, the mRNA for the basic 35 kDa beta-1,3-glucanase is induced rapidly to high levels in both interactions, but declines in time to background levels only in the incompatible interaction. The relevance of this difference in relation to plant defence is discussed.

Pathogenesis-related (PR) proteins form a heterogeneous family of plant proteins that are likely to be involved in defense and are inducible by pathogen attacks. One group of PRs, represented by the subfamily PR-1, are low-molecular-weight proteins of unknown biochemical function. Here we describe the cloning and characterization of two closely related genes encoding a basic and an acidic PR-1 protein (PR1b1 and PR1a2) from tomato (Lycopersicon esculentum). We present a comparative study of the mode of transcriptional regulation of these two genes in transgenic tobacco plants using a series of promoter-GUS fusions. Unexpectedly, the chimeric PR1a2/GUS gene is not induced by pathogenic signals but instead shows constitutive expression with a reproducible developmental expression pattern. It is expressed in shoot meristems, trichomes, and cortical cells as well as in vascular and nearby tissues of the mature stem. This constitutive expression pattern may represent preemption of plant defenses against potential pathogens. Conversely, the chimeric PR1b1/GUS gene does not show any constitutive expression in the plant, but it is transcriptionally activated following pathogen attack. Upon infection by tobacco mosaic virus, the PR1b1 gene is strongly activated locally in tissues undergoing the hypersensitive response but not systemically in uninoculated tissues. Furthermore, its expression is induced by both salicylic acid and ethylene precursors, two signals that coexist and apparently mediate the activation of local defenses during the hypersensitive response. We speculate that the different mode of expression of the two genes presented here, together with that reported previously for the induction of other PR-1 genes in systemic, uninoculated tissues, may all be complementary and necessary for the plant to acquire an efficient refractory state to resist pathogen attacks.

Many reports point to the existence of a network of regulatory signalling occurring in plants during the interaction with micro-organisms (biotic stress) and abiotic stresses such as wounding. However, the focus is on shared intermediates/components and/or common molecular outputs in differently triggered signalling pathways, and not on the degree and modes of effective influence between abiotic and biotic stresses nor the range of true plant-pathogen interactions open to such influence. We report on local and systemic wound-induced protection in tomato (Solanum lycopersicum L.) to four pathogens with a range of lifestyles (Botrytis cinerea, Fusarium oxysporum f.sp. lycopersici, Phytophthora capsici and Pseudomonas syringae pv. tomato). The role of ethylene (ET) in the phenomenon and in the induction by wounding of several markers of defense was investigated by using the never-ripe tomato mutant plants impaired in ET perception. We showed that PINIIb, PR1b, PR5, PR7 and peroxidase (POD) are influenced locally and/or systemically by wounding and, with the exception of POD activity, by ET perception. We also demonstrated that ET, although not essential, is positively (B. cinerea, P. capsici) or negatively (F. oxysporum, P. syringae pv. tomato) involved not only in basal but also in wound-induced resistance to each pathogen.

Bacterial speck disease, which is caused by Pseudomonas syringae pv. tomato, is an economically important disease on tomato. In the present study, we show that P. syringae pv. tomato DC3000 is a pathogen of tomato seedlings, an aspect of pathogen biology that has not been previously investigated. This resulted in the development of a virulence assay on tomato seedlings that has several advantages over labor-intensive foliar assays, including a shorter growth and incubation period, ease of inoculation and handling, and rapid generation of larger sample sizes per experiment. The utility of this assay was investigated by exploring the virulence function of coronatine (COR) on tomato seedlings. Using the COR- mutant DB29 and a MAPMAN display of transcript data from TOM1 microarrays, COR-dependent expression of genes involved in secondary metabolism, polyamine biosynthesis, reactive oxygen species homeostasis, and the novel transcription factor SlNAC2 were identified. Furthermore, during pathogenesis, genes involved in photosynthetic light reactions and the Calvin-Benson cycle were strongly repressed by COR. In conclusion, we show that P. syringae pv. tomato infects tomato seedlings and that COR is required for virulence in seedlings. The seedling assay can be used in high-throughput screens for the identification of molecular targets for COR and for the identification of genes involved in pathogenesis.

The aerial organs of plants are covered by the cuticle, a polyester matrix of cutin and organic solvent-soluble waxes that is contiguous with the polysaccharide cell wall of the epidermis. The cuticle is an important surface barrier between a plant and its environment, providing protection against desiccation, disease, and pests. However, many aspects of the mechanisms of cuticle biosynthesis, assembly, and restructuring are entirely unknown. To identify candidate proteins with a role in cuticle biogenesis, a surface protein extract was obtained from tomato (Solanum lycopersicum) fruits by dipping in an organic solvent and the constituent proteins were identified by several complementary fractionation strategies and two mass spectrometry techniques. Of the approximately 200 proteins that were identified, a subset is potentially involved in the transport, deposition, or modification of the cuticle, such as those with predicted lipid-associated protein domains. These include several lipid-transfer proteins, GDSL-motif lipase/hydrolase family proteins, and an MD-2-related lipid recognition domain-containing protein. The epidermal-specific transcript accumulation of several of these candidates was confirmed by laser-capture microdissection and quantitative reverse transcription-PCR (qRT-PCR), together with their expression during various stages of fruit development. This indicated a complex pattern of cuticle deposition, and models for cuticle biogenesis and restructuring are discussed.

When the biocontrol agent Pythium oligandrum (PO) colonizes the rhizosphere, it suppresses bacterial wilt disease in tomato (Solanum lycopersicum cv. Micro-Tom) caused by Ralstonia solanacearum, and a homogenate of its mycelia exhibits elicitor activity, inducing an ethylene (ET)-dependent defence response in Micro-Tom. Since salicylic acid (SA) and jasmonic acid (JA) play an important role in plant defence responses to pathogens, the involvement of SA- and JA- dependent signal transduction pathways in resistance to R. solanacearum was investigated in tomato roots treated with a mycelial homogenate of PO. Bacterial wilt disease was also suppressed in tomato cv. Moneymaker treated with the PO homogenate. However, the SA-inducible PR-1(P6) gene was not up-regulated in either Micro-Tom or Moneymaker. SA did not accumulate in homogenate-treated roots in comparison with distilled water-treated controls, even 24 h after inoc- ulation. Induced resistance against R. solanacearum was not compromised in SA-non-accumulating NahG transgenic plants treated with the PO homogenate. On the other hand, the expression of the JA-responsive gene for the basic PR- 6 protein was induced in both tomato cultivars treated with the PO homogenate. Furthermore, quantitative disease assays showed that the induced resistance against R. solanacearum was compromized in PO homogenate-treated jai1-1 mutant plants defective in JA signalling. These results indicated that the JA-dependent signalling pathway is required for PO-induced resistance against R. solanacearum in tomato.