CONSERVED VICTORIN-BINDING VITAL PROTEIN

Abstract: A method for alteration of physiological function in plant or other eukaryotic, or prokaryotic cells, which comprises alteration, replacement or other manipulation of a conserved victorin-binding protein and/or a multi-protein complex containing a said protein in the cells, or binding of victorin or a modified victorin molecule with a victorin-binding protein and/or a multi-protein complex containing a said protein in the cells.

Description: CONSERVED VICTORIN-BINDING VITAL PROTEIN This invention relates in general to the interactions between cells and pathogenic organisms, and in particular to plant-microbe interactions. The present invention has evolved from the identification of a conserved protein and/or multi-protein complex revealed by specific binding of the fungal metabolite, victorin, which is believed to be a component having a vital physiological function. The component is found not only in all plant cells, but in all cells, both eukaryotic and prokaryotic. More particularly, the present invention relates to the manipulation of this conserved protein or multi-protein complex in order to obtain an alteration of the physiological function within the cells. By way of example, the invention extends to alteration or replacement of this conserved victorin- binding protein and/or a multi-protein complex containing this protein in plant cells in order to obtain a desired alteration in the physiological function of the protein or complex, such as altered disease resistance of plants. The invention further extends to use of a modified victorin molecule in order to alter the binding of this molecule with the conserved victorin-binding protein and /or multi-protein complex again leading to alteration in the physiological function of the protein or complex such as inhibition of pathogenic organisms or selected cells (e.g. diseased cells), or as toxicity to plant cells (e.g. as a herbicide). The fungal toxin victorin has aroused great interest ever since its discovery in 1948 because of the toxin's unusual connection to plant disease resistance genes 24 ' 25 . The toxin was discovered when a new disease appeared in oats following the introduction of a previously unknown susceptibility which was genetically linked to resistance against crown rust, a completely different disease. The rust resistance gene, Pc-2, was first incorporated into North American oat cultivars from crosses with the variety, Victoria, from Uruguay 12,24,25 . The gene provided strong resistance to crown rust caused by several races of a fungal pathogen (Puccinia coronata). Selections from crosses with Victoria were found to be so hypersensitive to the prevalent races of P. coronata that cells immediately surrounding the infection loci collapsed before the fungus could produce uredospores 12,18,22 . After wide deployment of those cultivars in the United States in the early 1940s, it soon became evident that they alone were susceptible to a completely new disease, victoria blight, named after the parent cultivar, Victoria 24 . Investigation proved the disease to be caused by a toxin (named victorin) secreted by Cochliobolus (Helminthosporium) vctor/ae 25,26,34 , a soil fungus which previously had not been characterized. Toxin susceptibility was traced to a dominant gene in oats which was named the Vb locus (after victoria blight) 27,29,30,31,35,36,37 . Breeding studies indicated that Pc-2 and Vb were closely linked 16,17,19 . Because of the demand for effective crown rust resistance, an intense effort was made to separate rust resistance from toxin sensitivity, but the linkage has never been broken 12,19,28 . The strong possibility exists that the two traits are encoded by the same gene or are members of a complex locus. The remarkable properties of this locus provide a unique opportunity for the isolation of both a strong plant resistance gene and a plant toxin receptor by using the toxin as a probe 4,5,8,20,21 ,23 . Thus, although victoria blight is no longer an economically important disease, interest in victorin and its properties have not waned. Victorin has been purified and found to be present in fungal culture filtrates as a predominant form, victorin C, and four minor, closely related forms 38,39 . All forms are chlorinated, partially cyclic pentapeptides 7,9,21,23,33,38,39,40,4 . Wolpert and Macko have derivatized victorin C with I 125 !] Bolton-Hunter reagent and used it to identify a 100 kD protein as a specific victorin-binding protein 42,43 . They reported that in vivo, the 100 kD was labelled in the susceptible plant but not in the resistant plant. By contrast, under in vitro conditions, the 100 kD protein was labelled in both types of plants 42 . Akimitsu et al., using polyclonal antibodies raised against pure victorin C, have recently reported finding 100 and 45 kD victorin-binding proteins in vivo and 100, 65 and 45 kD victorin-binding proteins in vitro. They found no difference between resistant and susceptible plants 1 . Recent years have seen an increasing interest in the identification of receptors in plant systems to phytohormones and various molecular signals. Oats provide a promising opportunity for such a study in that the dominant Vb allele of oats confers susceptibility to the fungal toxin victorin and is genetically inseparable from Pc-2, a dominant disease resistance gene to oat crown rust. Victorin, one of the most potent and specific plant toxins known, can be derivatized with a radioactive tag without destroying its toxicity or specificity, and the toxin binds via a covalent bond with target oat proteins. In work leading to the present invention, two different radioactive compounds have been used to label victorin and the tagged victorin molecule then used to investigate how the fungal toxin associates with proteins first in oats and then in different organisms. The results have clearly shown multiple binding proteins in oats and other plant cells. In addition, it has been shown that apparently all living cells contain at least one low molecular weight protein which specifically binds victorin. Victorin C labelled with either f 125 !] or S] has been used to identify the victorin-binding proteins in resistant and susceptible oats. Specific victorin-binding proteins of 98, 69, 45, 26, 21 and 15 kD and doublets at 14 and approximately 12.5 kD have been identified. They are present in both resistant and susceptible oats. It is now shown for the first time that there is a difference in vitro between the binding properties of several victorin-binding proteins in a resistant and susceptible plant. Further, it is shown that all plants contain a set of proteins which specifically bind victorin and are similar to those in oats. Even more surprising, the specific binding of victorin reveals a conserved low molecular weight protein which is found in all cells, both eukaryotic and prokaryotic. The genetics of susceptibility to victorin toxicity in oats suggest the presence of a functional toxin receptor in the susceptible plant 31 . Using f 25 .] labelled victorin C, Wolpert and Macko 42 reported results from in vivo binding tests which conformed to that expectation. However, by using antibodies to victorin, Akimitsu et a/. 1 found no difference in binding of native victorin C in vivo between resistant and susceptible plants. The present inventors also have found no difference for specific proteins in vivo between resistant and susceptible plants. It has been consistently found that the resistant plant binds more label uniformly to all of the binding proteins than does the susceptible plant, but there is no protein/s which uniquely binds labelled victorin in vivo in either genotype. In vitro experiments have given consistent results. In oats, the 21 and 14 kD proteins bind the great majority of the label seen on victorin- binding proteins (about 90%). The 98 kD protein binds much less (about 10%), and the other victorin-binding proteins capture only a trace amount in relative terms. It has not yet been determined whether all of the proteins are present as functional entities in living cells or whether some are proteolytic degradation products or artefacts of the isolation procedure. Based upon the strength of the signals and the repeatability of the experiments, it is suspected that the 98, 21 and 14 kD proteins are functional members of the cell. Even though the 45 kD protein is very weak in vitro, its absolute dominance in vivo suggests that it too may be functional in that form. The strong signal obtained for the low molecular weight proteins suggests that they may be present in the highest molar quantities. An alternative explanation, that they possess a greater affinity for victorin than the other proteins, seems less likely since the binding is covalent and since they are out-competed more rapidly by unlabelled victorin than are the 98 and 69 kD proteins. The difference in in vitro binding properties of the 14 kD protein between resistant and susceptible oats suggests that the 14 kD protein may be the product encoded by the Vb and its allele the vb gene. However, it should be noted that this protein appears to associate as a member of a multiprotein complex. It is equally possible that another member of that complex which does not bind victorin could have a mutant form which affects the tertiary structure of the complex in such a way as to affect the binding accessibility of other members of the complex which do bind victorin. Structural complexity is also hinted at by the slower binding to the 98 kD protein compared to the 21 and 14 kD proteins. A working hypothesis for oats is that both genotypes contain physiologically functional and equivalent structures which contain the product of the Vb or vb aileles. The function of that structure is unknown but is assumed to be a vital one. In the case of resistant plants, victorin can bind to members of the complex but does not seriously impair physiological functioning. In the case of susceptible plants, the mutated form of one member of the complex, the Vb gene product, permits normal physiological function in the absence of victorin but causes disruption of function when victorin binds. As already pointed out, it is not necessary for the Vb gene product itself to bind victorin. Its mutated form may destabilize the structure in such a way that function is blocked when other members of the complex bind victorin. Accordingly, in one aspect the present invention provides a method for alteration of physiological function in plant or other eukaryotic, or prokaryotic, cells, which comprises alteration, replacement or other manipulation of a conserved victorin-binding protein and/or a multi-protein complex containing a said protein in the cells. In this aspect, this invention further provides plant or other eukaryotic, or prokaryotic, cells having altered physiological function, characterised in that a victorin-binding protein and/or a multi-protein complex containing a said protein in the cells is altered, replaced or otherwise manipulated. As used throughout this specification, the term "a victorin-binding protein" refers to one or more of the proteins which are disclosed herein as binding to victorin, particularly victorin C. This aspect of this invention is demonstrated, by way of example, by the work described herein where in the one single instance provided by nature in which a mutation results in a detectable physiological response to victorin in living cells (oat cells with the Vb locus), that response is rapid, cell death. This example illustrates that a victorin-binding protein or multi-protein complex can be manipulated without altering its binding to the fungal metabolite, but with alteration to the physiological function of the protein or complex which can lead to cell death or other secondary cell responses. Thus, in plants, a victorin-binding protein or multi-protein complex may form part of the defence mechanism of the plants, for example to invading pathogenic organisms such as viruses, fungi, bacteria, mycoplasmas, protozoa, insects or nematodes. ln addition, it has been shown that victorin C has a concentration dependent, growth inhibition effect exhibited to differing degrees on several strains of fungi thus showing that victorin can interact with the physiology of eukaryotic organisms besides the sensitive strain of oats. In another aspect, the present invention provides a method for alteration of physiological function in plant or other eukaryotic, or prokaryotic, cells, which comprises binding victorin or a modified victorin molecule with a victorin-binding protein and /or multi-protein complex containing a said protein in the cells. The use of a modified victorin molecule alters the binding to the victorin-binding protein. The modified molecule may be, for example, labelled victorin or a chemical derivative or analogue of victorin. This aspect of the invention is demonstrated by the competition studies on soluble proteins from victorin-susceptible and victorin-resistant oats described herein. These studies show differences in binding patterns for I 35 S] -labelled victorin between the susceptible and resistant oats in the presence of competing unlabelled victorin. In this aspect, the present invention extends to the use of victorin or a modified victorin molecule as an antibiotic or toxic agent or as an inhibitory agent having selective activity in relation to specific cell types, such as parasitic or pathogenic disease organisms, or differently differentiated cells such as oncogenic or cancerous cells. Particular examples of such use of victorin or a modified victorin molecule include, but are not limited to: use as an agent which is toxic or inhibitory to certain protozoans but not toxic or less toxic to mammalian cells and which may be used as an antibiotic against malaria or sleeping sickness or other protozoan caused diseases; use as an agent which is toxic or inhibitory to certain fungi but not toxic or less toxic to mammalian cells and which may be used as an antibiotic against human or animal parasitic fungal diseases; use as an agent which is toxic or inhibitory to certain plant cells and which may be used as a herbicide; use as an agent which is toxic or inhibitory to human cancer cells but not toxic or less toxic to normal human cells and which may be used as an anticancer drug. It should be noted that although the victorin-binding proteins and/or multi-protein complex containing these proteins are well conserved across species, small differences are expected to exist between species and between different states of differentiation within multicellular organisms. Such small differences may be targeted and exploited to provide, for example, specific antibiotic effects against protozoa! or fungal parasites or other pathogenic organisms or cells as described above. The victorin-binding proteins and associated proteins are conserved in plants. The patterns on IEF gels are remarkably similar between oats, a monocotyledon and the two dicotyledons, Arabidopsis and tobacco. Since the net isoelectric point of a protein complex is affected by its charge composition, it can be concluded that there is general similarity in the structures revealed in plants by victorin-binding. The universal conservation across taxonomic boundaries of a protein with a specific victorin-binding site was very surprising. Structural conservation of a protein is partially enforced by an indispensable role for that protein and partially by that protein's association as a member of a multiprotein complex which serves some vital function. In this case, the strongly binding low molecular weight protein is found in both prokaryotes and eukaryotes. Differences between IEF and SDS PAGE gels show that it is associated with other proteins as extracted in the tissue supernatant. The similarity of the pattern of mammals, fungi and bacteria seen on IEF gels is unexpected. In comparing the eukaryotes to the pattern in E.coli, it is easy to speculate that the low molecular weight victorin-binding protein originates in the mitochondria of eukaryotic cells. In plants, both the mitochondria and the chloroplast may contribute forms of the protein. If the conserved set of high molecular weight victorin-binding proteins (45 kD and above) found in plants are involved in a unique plant function and if one of those proteins is in fact the Vb product, it is possible that those proteins may be involved in the function of disease resistance 4,5,8 . Since the Vb and Pc-2 genes are nuclear inherited, it may be that the victorin- recognition domains of those proteins are encoded by a gene segment captured earlier from a universal mitochondria! gene. Further details of the present invention will be apparent from the following Example, and the accompanying drawings. In the drawings: FIG 1 Specificity of Binding: Isoelectric focussing gel containing: Lane 1 - I 95 S] -victorin C only, Laofi_2 - oat soluble proteins incubated with SJ-victorin C plus an excess of unlabelled victorin C and Lane 3 - oat soluble proteins with C only. Lane 4 contains an additional control, oat soluble proteins incubated with the product of a mock labelling reaction in which only the labelling reagent (no victorin) was present. Some contamination or breakdown products of the labelling reagent as supplied by the manufacturer co-chromatograph with labelled victorin C which necessitated testing those products for protein binding. In all examples, a large excess of labelled victorin C was included so that all potential binding sites could be labelled. Panel B is a greatly overexposed print of Panel A to show that no faint bands are present in Lanes 2 or 4. FIG 2 Comparison of oat microsomal membrane proteins and soluble proteins: Comparison on an isoelectric focussing gel of f^S]- victorin-binding proteins in oat leaf extracts and in microsomal membranes derived from those extracts. Lane 1 - susceptible oat microsomal membranes and Lane 2 - resistant oat microsomal membranes. Lane 3 - susceptible oat soluble proteins and Lane 4 - resistant oat soluble proteins. Soluble proteins are the supernatants of leaf tissue extracts from which the microsomal membranes have been removed. Resistant and susceptible plants are compared as equal aiiquots normalized to total protein and the microsomal membrane lanes have been overloaded by a four fold excess (relative to their respective supernatants) to enhance visibility. All preparations were incubated for two hours at room temperature with equal amounts of I 95 S] -victorin C. FIG 3 Rapidity of labelling and stability of [^-victorin-binding proteins: This is an SDS PAGE gel with molecular weights in kD on the left and time of incubation in hours indicated on the bottom. All preparations were equal aiiquots of oat soluble proteins (see Fig.2 legend) incubated with an equal amount of pSJ-victorin C at room temperature. The 0 time point was frozen in liquid Ng immediately upon mixing. FIG 4 Competition Series - SDS: SDS PAGE gels showing competition series on susceptible and resistant oat soluble proteins. Conditions are the same as in Fig.3. FIG 5 Competition Series - Enlargement: An enlargement of portions of the SDS gels of Fig.4. The two top panels show the high molecular weight victorin-binding proteins which behave in an essentially equivalent manner and serve as useful internal controls. The two bottom panels show that the low molecular weight proteins do not behave in an equivalent manner for competition for victorin-binding sites. The small inset to the bottom left shows the new band at 15 and the doublets at 14 and 12.5 kD which appear after a substantial number of victorin sites are filled (.2 μg/ml unlabelled victorin). The doublets are not visible when no competing unlabelled victorin is present. FIG 6 Comparison of soluble proteins which specifically bind [^-victorin C in eight different organisms, eukaryotic and prokaryotic: Isoelectric focussing gel patterns show soluble proteins which specifically bind labelled victorin in from left to right: oats, arabidopsis, tobacco, mouse, human, Saccharomyces pombe, Saccharomyces cerevisiae and Escherichia coli. In all cases, all binding of radioactive victorin is prevented by the presence of an excess of unlabelled victorin C. Comparison of IEF patterns to SDS patterns in Fig.7 shows that the IEF patterns are of victorin-binding proteins associated non-covalently (except possibly by a disulfide bond) with other proteins forming complexes with different net isoelectric points. FIG 7 Comparison of soluble proteins which specifically bind [^J-victorin C in six different eukaryotic organisms: This SDS PAGE gel shows proteins of different molecular weights (indicated on left in kD) which specifically bind labelled victorin in from left to right: oats, arabidopsis, tobacco, mouse, Saccharomyces pombe and Saccharomyces cerevisiae. Panel A is a darker exposure to show the more weakly binding high molecular weight proteins. Panel B is a 12.5 fold lighter exposure to show the more intensely binding low molecular weight proteins. -EXAMPLE 1 Toxin preparation: Victorin C was purified from culture filtrates of Cochliobolus wctor/ae 38,39 with a final passage through a preparative C, 8 reverse phase HPLC column. The toxin was labelled with f 125 !] Bolton-Hunter reagent or with S] protein labelling reagent (a Bolton-Hunter analogue) (Amersham) following a published procedure 40 . Our victorin C co-chromatographed with a reference sample of victorin C kindly supplied by Dr.Tom Wolpert, Oregon State University. A test labelling of that reference sample with f 25 .] yielded identical protein binding results as our own preparation of victorin C. Finally, [ 125 l] labelled victorin C resulted in identical binding patterns as f^S] labelled victorin C except that the sulfur label resulted in much sharper, clearer bands showing in several cases clear doublets where the iodine label showed only one large blurred band. For increased sensitivity and quantitative comparisons, all radiographic imaging was performed with a model 400 Phosphorlmager from Molecular Dynamics. Labθlled-victorin specificity: Specificity of the labelled toxin was verified by comparisons on isoelectric focusing gels of preparations of oat plant extract incubated with labelled victorin C plus an excess of unlabelled victorin C, or incubated with labelled victorin C only. For an additional control, oat extract was incubated with the product of a mock labelling reaction in which only the labelling reagent (no victorin) was present, (see Fig.1). Some contamination or breakdown products of the labelling reagent, as supplied by the manufacturer, co-chromatograph with labelled victorin C making it necessary to test those products for protein binding. In all examples, a large excess of labelled victorin was included so that all potential binding sites could be labelled. Even when the image was greatly overexposed, (Fig.1 B), it is clear that only the lane containing labelled victorin C with oat extract shows labelled protein bands. Presence of excess unlabelled victorin blocks binding of labelled victorin and no binding to protein takes place in the mock reaction lane. Examination of Coomassie stained gels shows that the radioactive victorin-binding bands are embedded in the midst of numerous prominent protein bands throughout the lane which do not bind victorin. In vivo binding studies: By using either S] or f 25 .] labelled victorin C, we were not able to repeat an earlier report which described specific binding of f 25 1] -victorin C to a 100 kD protein in susceptible oats but not in resistant oats. In this point, our results agree with the recent results of Akimitsu et al. who also found no binding differences in vivo between resistant and susceptible genotypes with their polyclonal antibodies to Victorin C 1 . With the f 125 1] -victorin C we observed labelling of a 98, 45, 26, 21 and 14 kD protein (not shown) in vivo following the Wolpert and Macko procedure 42 . All of the proteins appeared to bind victorin in the same manner when the respective proteins were compared between susceptible and resistant plants. With the S] -victorin C we observed the same pattern as with radioactive iodine except that we also saw a 69 kD protein and in addition, saw extra binding-proteins which varied from experiment to experiment (not shown). The experiments were repeated many times showing always that the 45 kD was invariably laoelled most strongly and that the resistant plant consistently bound more labelled victorin to all victorin-binding proteins than did the susceptible plant. To more closely mimic a natural interaction of victorin with oat leaf, we placed whole leaves cut at the base into solutions of I 35 S] -victorin C. The label was quickly taken up into the entire leaf and exposure of the whole leaf to a Phosphorlmager screen showed a uniform and intense distribution of the label throughout the leaf within 30 minutes. However, after 2 and 4 hours of exposure, only the 45 kD protein was labelled intensely. Other victorin-binding proteins showed only a trace of label. In vitro Binding studies: The in vitro experiments proved to be completely repeatable. Oat extracts were prepared by grinding freshly frozen leaves of 7 to 8 day old plants in liquid nitrogen, mixing with buffer A (50 mM HEPES-KOH, pH7.5, 0.3M sucrose, 20 mM 2-mercaptoethanol, 10 mM KCI, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride) 2 ,3,10,11 (3:1 of Buffer A in mis to leaf weight in grams), straining through cheese cloth and removing debris by centrifugation at 20,000 g for 15 min. Microsomal membranes were separated from the extract by centrifugation at 100,000 g for 1 hour. The membrane pellets were washed by resuspension in 10 mis of fresh buffer A and recentrifuged. Washed microsomal membrane pellets were then resuspended in minimal volumes of buffer A (1 :1 of estimated pellet volume to buffer volume) and compared in labelling studies with the remaining portion of the oat extract from which they were originally derived, hereafter called the supernatant. Both microsomal membrane and supernatant proteins were incubated at room temperature for two hours with a constant amount of labelled victorin C. Aiiquots were normalized on the basis of total protein. Results were analyzed on both IEF (Pharmacia) and SDS PAGE (Gradipore) gels. When washed microsomal membranes from both resistant and susceptible oats were compared with their respective supernatants, almost all of the victorin-binding activity was found in the supernatant (Fig.2). The small amount of binding activity in the microsomal membranes is exaggerated in the figure by four-fold since the microsomal membrane lanes were overloaded by four times to make the signal visible. By inspecting different exposure levels of both [ 12 l] and pSJ-labelled proteins in SDS PAGE gels, 10 proteins of oats have been identified as specific victorin-binding proteins; 98 kD, 69 kD, 45 kD, 26 kD, 21 kD and 15 kD and doublets at 14 kD and approximately 12.5 kD 13,14,15 . When examined on IEF gels, these labelled proteins segregated into seven major bands with six to eight lesser bands depending upon the intensity of the exposure. Comparison of IEF and SDS PAGE gels suggests that on IEF gels, the victorin-binding proteins are associated with one or more other proteins in different complexes or subcomplexes each with different net isoelectric points. In comparing in vitro binding between resistant and susceptible plants, there is no pronounced difference between the two when examining IEF gels either of the supernatants or microsomal membranes. SDS gels reveal a slightly stronger binding of the lowest molecular weight proteins in resistant plants over susceptible plants. A distinct difference only appears during serial competition with unlabelled victorin (see below). The victorin-binding proteins are quite stable, and the binding reactions are very fast, (Fig.3). Labelling of all but the 98 kD protein is as immediate as we can detect with binding to the 98 kD protein almost complete in 30 minutes. The stability of the proteins are such that a single freeze-thaw cycle does not apparently damage the binding activity and a 4 minute boiling step reduces subsequent binding activity to approximately a third (not shown. After 10 hours at room temperature, the 21 kD protein is lost, apparently broken down into a 14 kD form, (Fig.3). Serial competition studies: Serial competition studies on supernatants from both resistant and susceptible plants show differences in binding properties for the low molecular weight proteins, particularly the 14 kD protein, (Fig.4). In the competition series, the 98 and 69 kD proteins behave essentially the same way in resistant and susceptible plants and serve as useful internal controls, (Fig.5). However, in the case of the 14 kD protein, the C signal persists to approximately a 10-fold greater concentration level of competing unlabelled victorin C in the susceptible plant as compared to the resistant plant, (Fig.5). To our knowledge, this is the first reported difference in in vitro victorin-binding behaviour between a resistant and susceptible plant. Close examination of the low molecular weight region shows that there is a protein at 15 kD, a doublet at 14 kD and a doublet at approximately 12.5 kD, (see small bottom-left inset of Fig.5). These details only appear when a substantial number of victorin-binding sites are occupied (at about 0.2 μg/ml of competing unlabelled victorin C). Victorin-binding activity in other organisms: As controls, a number of other species were tested for victorin-binding activity. Tissue extracts and supernatants were prepared exactly as described for oats. A specific victorin-binding protein or proteins were found in all organisms tested. In all cases binding was specific in that binding of f^SJ-labelled victorin C was prevented by an excess of unlabelled victorin C. A comparison of IEF binding patterns from oat, Arabidopsis thaliana, tobacco, mouse live, human fibroblasts, Saccharomyces pombe, Saccharomyces cerevisiae and Escherichia coli is shown in Fig.6. The pattern on IEF gels is remarkably similar in the plants between the monocotyledon, oats, and the two dicotyledons, Arabidopsis and tobacco. The pattern is clearly different in mammals and fungi but the binding is distinct and clear. The bacterial pattern looks much like the mammalian pattern except that it is shifted to a slightly lower isoelectric point. In view of the fact that simple charge differences on proteins can cause position shifts on IEF gels, the degree of conservation of the patterns is extraordinary. The pattern of individual protein molecular weights detected on SDS PAGE gels (Fig.7) is similar in the plants tested. For the mammalian and fungal samples, higher molecular weight proteins show a different pattern. Most significantly, however, all of the organisms have a low molecular weight protein in the 12 to 14 kD range which binds radioactive victorin far more intensely than all of the other victorin-binding proteins in each organism. In that sense, this protein appears to be the most highly conserved. In addition to the victorin-binding proteins already demonstrated in the eukaryotes oat, Arabidopsis thaliana, tobacco, mouse, human, Saccharomyces pombe, Saccharomyces cerevisiae and the prokaryote, Escherichia coli, the following organisms have been tested by the same methods and it has been confirmed that they also possess victorin-binding proteins which are similar to and consistent with those previously described. Eukaryotic: Neocallimastix sp. Caeomyces sp. Giardia duodenalis Chlamidymonas reinhardii Prokaryotic: Rhizobium legυminosarium Archeobacter halophilae Observation of victorin-binding proteins in bacteria (E.coli ) heightened the possibility that all or some of the victorin-binding proteins of eukaryotes may be located in the mitochondria and chioroplasts. Fractionation of yeast mitochondria and subsequent testing with ^S-radioactivity labelled victorin C showed that the greater proportion of victorin-binding proteins were cofractionated with the mitochondria. However, victorin-binding proteins were cofractionated with the mitochondria. However, victorin-binding proteins were also found in the other cellular fractions and so it cannot yet be stated where those victorin-binding proteins crucial to the physiological response are located in eukaryotes. The low molecular weight victorin-binding protein has been found in Neocallimastix and Caecomyces which are anaerobic fungi and in the anaerobic protozoan, Giardia . These three organisms, which are classified as eukaryotes, possess no mitochondria but instead a unique organelle called hydrogenosome. An unusual "primitive'' bacterial species, an archeobacteria which is a halophile, has also been tested and a strong signal for the victorin-binding proteins has been found in this primitive bacterium as well. In addition, a unicellular plant (an alga), Chlamidymonas reinhardii , was tested to see whether the isoelectric pattern of the victorin-binding proteins was different or similar to that of higher plants. The pattern was in fact similar to the higher plants tested which confirms even more strongly a conservation of multiprotein complex associated with victorin-binding proteins of plants. EXAMPLE 2 Physiological Effect of Victorin on Organisms other than Oats. Two preparations of victorin were tested for a growth inhibiting effect on selected microorganisms. Preparation "A" was purified victorin C. Preparation "B" was purified victorin but containing predominantly victorin C with lesser amounts of the other naturally occurring victorin forms. Victorin was purified as described in Example 1. Cultures of the test organisms were grown on complex YEPD media (CM) or on minimal media (MM) with glucose or gtycerol as the carbon source, although it was observed that the use of glyceroi as a carbon source did not seem to enhance the toxic effect. Cultures were grown at 30° with shaking. A. Using preparation "B" and either CM or MM media, a number of fungi were screened for growth in the presence of victorin (5 micromolar initial concentration) using optical density as a measure of growth. Measurements were made at 20, 40 and 60 hours after addition of the toxin. Results: (at 60 hours) • Saccharomyces cerevisiae (AH22): minor growth inhibition of approximately 5% observed in both types of media • S. cerevisiae (AH22 Rho°); growth inhibition of 10 to 20% with the inhibition more pronounced in minimal medium (MM) • S. cerevisiae OP-1 mutant: approximately 25 to 30% growth inhibition • S. cerevisiae D13.1A Rho + : no detectable inhibition S. cerevisiae D13.1A Rho°: approximately 25 to 30% growth inhibition • Candida glabrata (wild type): approximately 5 to 10% growth inhibition with the inhibition more pronounced in minimal medium (MM) • Candida glabrata (respiratory deficient mutant): growth inhibition of at least 30% in complex medium (CM) and more than 40% in minimal medium (MM) • S. pombe (fission yeast, wild type): growth inhibition of approximately 10% in both types of media • Kluyveromyces lactis (budding yeast, wild type): no detectable inhibition on both types of media • Neurospora crassa (wild type): no detectable inhibition. Using preparation "A" and CM media, two strains of yeast (Saccharomyces cerevisiae ) were tested for growth in the presence of toxin (40 micromolar or 4 micromolar initial concentration). Strain AH22 is a standard laboratory strain often used as a "wild type" reference strain. Strain OP-1 carries an ADP-ATP translocase mutation. Growth was measured by performing actual cell counts. Measurements were made at 6, 20, 32 and 36 hour time points after addition of victorin to the growth medium. The viability of counted cells was tested in strain AH22 by measuring colony plaque forming units (CFU). Results: • Saccharomyces cerevisiae (AH22): (a) at 40 micromolar victorin - growth inhibition of 78% at 6 hours with the inhibition difference declining progressively at later times (b) at 4 micromolar victorin - growth inhibition of 25% at 6 hours with the inhibition difference declining progressively at later times • S. cerevisiae (OP-1): (a) at 40 micromolar victorin - growth inhibition of 75.5% at 6 hours with the inhibition difference declining progressively at later times. (b) at 4 micromolar victorin - growth inhibition of approximately 10 to 15% at 6 hours. NOTES: (a) Counts of colony forming units (CFU) verify that the number of viable cells corresponds to the cell count data. (b) The morphology and presumably the physiology of mutant OP-1 is affected in that sedimented toxin-treated cells are brownish in colour rather than pink to red as are untreated sedimented OP-1 cells. 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Claims: C LAI MS: 1. A method for alteration of physiological function in plant or other eukaryotic, or prokaryotic, cells, which comprises alteration, replacement or other manipulation of a conserved victorin-binding protein and/or a multi- protein complex containing a said protein in the cells. 2. A method according to claim 1 wherein said alteration, replacement or other manipulation results in cell death or other secondary cell response to victorin, without altering the binding of said protein or complex to victorin. 3. A method according to claim 1 or claim 2 wherein said cell is a plant cell, and said alteration, replacement or other manipulation alters the defence mechanism of said plants to invading pathogenic organisms. 4. A plant or other eukaryotic, or prokaryotic, cell having altered physiological function, characterised in that a victorin-binding protein and/or a multi-protein complex containing a said protein in the cells is altered, replaced or otherwise manipulated. 5. A plant or other eukaryotic, or prokaryotic, cell according to claim 4, wherein said altered physiological function leads to cell death or other secondary cell response to victorin, without altering the binding of said protein or complex to victorin. 6. A plant cell according to claim 4, wherein said altered physiological function forms part of the defence mechanism of said plant cell to invading pathogen organisms. 7. A method for alteration of physiological function in plant or other eukaryotic, or prokaryotic, cells, which comprises binding victorin or a modified victorin molecule with a victorin-binding protein and /or a multi- protein complex containing a said protein in the cells. 8. A method according to claim 7 wherein said modified victorin molecule comprises labelled victorin or a chemical derivative or analogue of victorin. 9. A method according to claim 7 wherein said binding of victorin or a modified victorin molecule with said protein or complex has an antibiotic or toxic effect in said cells. 10. A method according to claim 7 wherein said binding of victorin or a modified victorin molecule with said protein or complex has an inhibitory effect in said cells. 11. A method according to claim 9 or claim 10 wherein antibiotic or toxic effect, or said inhibitory effect is selective in relation to specific cell types, such as parasitic or pathogenic disease organisms, or oncogenic or cancerous cells. 12. A method according to claim 7 wherein said alteration of physiological function comprises inhibition of growth of said cells. 13. A method according to claim 1 wherein said alteration of physiological function provides resistance to toxic or inhibitory effects of victorin or a modified victorin molecule. 14. A method according to claim 7 wherein said alteration of physiological function comprises a toxic effect in plant cells whereby said victorin or modified victorin molecule acts as a herbicide in said cells.

Inventors: LOSCHKE DAVID CARL, ROLFE BARRY GARTH, LOSCHKE, DAVID, CARL, ROLFE, BARRY, GARTH
Applicants: LOSCHKE DAVID CARL, ROLFE BARRY GARTH, UNIV AUSTRALIAN, THE AUSTRALIAN NATIONAL UNIVERSITY
Application year: 1993
Main classification: A01N63
IPC codes: A01H3, A01N63, A61K38, C07K14, C12N1, C12N5, C12R1