Elicitor from cladosporium

#Plants resistant to pathogens often are found to evoke their resistance through a mechanism which eventually yields a hypersensitive response (HR) resulting in rapid cell death of the infected plant cells. This rapid cell death or necrosis inhibits the pathogen from further growth and thus stops the infection. This mechanism is known already for a long time (Klement, Z., In: Phytopathogenic Prokaryotes, Vol. 2, eds.: Mount, M. S. and Lacy, G. H., New York, Academic Press, 1982, pp. 149-177). The HR is often confused with other lesion-like phenomena, but a typical HR gives local cell death and is associated with secondary responses such as callus deposition, generation of active oxygen species, induction of phytoalexins, changes in ion fluxes across membranes and induction of acquired resistance (AR) (Hammond-Kosack, K. E., et al., Plant Physiol. 110, 1381-1394, 1996).

#The pathogen resistance is elicited by response to elicitor compounds, which are frequently found to be of proteinaceous nature (Arlat, M., et al., EMBO J., 13, 543-553, 1994; Baker, C. J. et al., Plant Physiol. 102, 1341-1344, 1993; Staskawicz, B. J. et al., Proc. Natl. Acad. Sci. USA 81, 6024-6028, 1984; Vivian, A. et al., Physiol. Mol. Plant Pathol. 35, 335-344, 1989; Keen, N. T., Ann. Rev. Gen. 24, 447-463, 1990; Ronald, P. C. et al., J. Bacteliol. 174, 1604-1611, 1992; Whitham, S. et al., Cell 78, 1-20, 1994;Kobe, B. and Deisenhofer, J., Trends Biochem. Sci. 19, 415, 1994; and Honée G. et al., Plant Mol. Biol. 29, 909-920, 1995). These elicitor proteins (encoded by avirulence genes) are produced by the pathogen and are thought to interact with a resistance protein available in the plant, therewith starting a cascade of events resulting in the HR-response. The elicitor proteins are characterized by that they are (race-)specific and only are able to elicit the response with a corresponding (also specific) resistance protein. The concept of avirulence-gene based resistance is also known under the name of the gene-for-gene response. Avirulence genes have been cloned from bacterial pathogens (such as Pseudomonas and Xanthomonas) and from fungal pathogens (such as Cladosporium fulvum, Rhynchosporium secalis and Phytophthora parasitica). Also plant genes coding for some of the corresponding resistance genes have been cloned (such as the tomato gene Cf9 corresponding to the avirulence gene avr9 fronm Cladosporium fulvum, and the tomato Pto-gene corresponding to the aviruience gene avrPto from Pseudomonas).

#In recent years, a number of extracellular elicitor proteins of Cladosporium fulvum has been identified (Knogge, 1996, Lauge and De Wit, 1998). Cladosporium fulvum is a fungus which in nature is able to infect tomato plants. Corresponding with the elicitor molecules from the fungus there are a number of resistance proteins found, on basis of specific interactions between the diverse pathotypes of the fungus with the corresponding diverse varieties of tomato.

#FIG. 1 Symptom of leaves toothpick inoculated with Agrobacterium containing pSfinx:: 43-7G (A-J, L) or pSfinx::Avr4 (K). A: N. benthamiana, B: N. clevelandii, C: N. glutinosa, D: N. cordifolia, E: N. rustica, F: N. langsdorfii, G: N. tobacum (cv. Samsun), I: N. paniculata, J: N. sylvestris, K and L: N. clevelandii.

#FIG. 2 Symptom of plants toothpick inoculated with Agrobacterium transformed with plasmids containing 5′ deletion constructs. Left side of tomato (A,B) and tobacco (C, D) leaves were inoculated with Agrobacterium transformed with plasmids containing the full length DNA, while the right side was inoculated with deletion Δ79 (A,C) or deletion Δ256 cDNA (B, D). The entire leaf of Nicotiana clevelandii (E-H) was inoculated with Agrobacterium containing a plasmid with the full length DNA (E), deletion Δ79 (F) or Δ256 (G). H shows the mosaic symptom of an uninoculated leaf of N. clevelandii plants that had been inoculated with Agrobacterium transformed with plasmids containing the 5′ deletion constructs.

#Surprisingly now a protein produced by Cladosporium fulvum has been found which is capable of giving an induction of the HR response The experiments reported in the experimental section show that the protein is able to elicit a response in both Cf4- and Cf9-containing tomato plants, and in a large number of tobacco species, indicating that the protein is not of the same type as the Avr-proteins (like Avr4 and Avr9) which cause a clear pathogen-host specificity. Further, from the molecular data disclosed in this application it can be derived that the protein of the invention is dissimilar to the Avr-proteins in a second way: it is a putative transcription factor (containing a bZIP motif sequence). The suspected mode of action is that, when expressed in a plant cell, this protein triggers the hypersensitive response through ectopic expression of genes involved in the execution of this defense reaction. Although it may thus not be a true ‘elicitor’ in the way this word is conventionally used, for ease of reference the term Cladosporium elicitor is used throughout this specification. It is well known to those skilled in the art that the hypersensitive defense response is an active response. This is clearly illustrated by the fact that protein elicitor-mediated induction of the hypersensitive response can be inhibited by alpha-amanitin, a powerful inhibitor of eukaryotic RNA polymerase, or by cycloheximide, a known inhibitor of eukaryotic protein synthesis (He, S. Y. et al. Cell 73 (7)1255-1266(1993)). Inhibition by these inhibitors illustrates very well the need for de novo transcription and protein synthesis, respectively, for the ‘execution’ of the hypersensitive response. Most measurable cellular responses associated with the hypersensitive response are suppressed effectively upon transcription inhibition, including cell death induction. Although the mechanism by which recognition of a pathogen avirulence protein by the plant is converted into an altered transcription pattern is not understood in much detail, it is likely that transcription factors mediate this effect. Since the hypersensitive response is a phenomenon found in almost all plant species, it is believed that at least the steps involving de novo transcription and protein synthesis are common between those plants and will be effected by tightly regulated transcription factors. It is believed that the transcription factor of the present invention can replace the endogenous plant transcription factors without being regulated. In this way, the presence or absence of the transcription factor of the invention acts as an on/off signal for the generation of an HR.

#Transcription factors regulate gene expression by exerting their effects on a promoter. What most people would call ‘promoters’ consists of basically two different elements. The minimal promoter is the area where the basic transcription machinery, RNA polymerase and associated proteins bind, unwind the DNA and start transcription.

#Flanking that minimal promoter, but in plants usually upstream from the minimal promoter (measured from the transcribed area), many different binding sites for transcription factors are found. This area is usually called ‘enhancer’, but also other descriptions, such as ‘silencer’ can be used (often based on the nature of the influence of the transcription factors).