# Patent

## Title
Biological control of pathogenicity in microbes that use alpha, beta unsaturated fatty acid signal molecules

## Abstract

DSF-based microbial pathogens may be controlled by certain methods and compositions. A method of controlling diseases caused in a host organism by pathogenic microbial expression of α,β unsaturated fatty acid signal (DSF) molecules involves inoculating the host organism with a non-pathogenic microbe that disrupts α,β unsaturated fatty acid signal molecule-mediated cell-cell communication by the pathogenic microbe in the host organism. Microbes having protein expression that interferes with α,β unsaturated fatty acid signal molecule-mediated cell-cell communication by the pathogenic microbe in the host organism can be identified and isolated or engineered by transformation with DSF-interference (inhibition or activation) gene or genes. Genes conferring DSF-interference activity can also be expressed in organisms (e.g., plants) susceptible to diseases caused by microbes that use DSF, resulting in genetically resistant organisms.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. application Ser. No. 11/157,469, filed Jun. 20, 2005, which claims priority to U.S. Provisional Patent Application No. 60/580,786, filed Jun. 18, 2004, titled BIOLOGICAL CONTROL OF PATHOGENICITY IN MICROBES THAT USE ALPHA, BETA UNSATURATED FATTY ACID SIGNAL MOLECULES, each of which is incorporated herein by reference for all purposes.


BACKGROUND OF THE INVENTION
The present invention relates generally methods and compositions for control of microbial pathogens.
Species of Xanthomonas (Xanthomonas spp.) are responsible for major agricultural diseases. For example, members of the Brassicaceae, which includes broccoli, cabbage and other plants, are susceptible to black rot, which is caused by Xanthomonas campestris pathovar campestris (Xcc); members of the Solanaceae, which includes tomatoes and peppers, are susceptible to bacterial spot, which is caused by X. campestris pathovar vesicatoria (Xcv); and rice is subject to bacterial leaf blight, which is caused by X. oryzae pathovar oryzae (Xoo). Many other important commercial crops are susceptible to diseases caused by Xanthomonas spp., including wheat, soybean, walnut, mango, peach and many ornamental crops.
Xcc is also known to be closely related to another pathogenic bacterial species, Xylella fastidiosa (Xf), which causes citrus variegated chlorosis, almond leaf scorch and Pierce's disease of grape. Again, several other important commercial crops are susceptible to diseases caused by Xylella spp., including almonds, peaches, plums, alfalfa, citrus, oak, maple, coffee and oleander. Pierce's disease, as a particular example, is a chronic problem in the grape industry in California, greatly exacerbated due to the introduction of the glassy-winged sharpshooter, which is a very effective vector of the Xf pathogen. The management of this disease is particularly problematic since vector control has not proven effective, and the nature of the colonization of grapevines by the pathogen limit the utility of bactericides in killing the pathogen and make strategies of disease control based on other bacterial diseases ineffective.
Improved methods and compositions for control of Xanthomonad, Xylella, and related microbial pathogens are needed.
SUMMARY OF THE INVENTION
The present invention relates generally methods and compositions for control of microbial pathogens by disrupting their ability to communicate via a particular class of small molecules.
Species of Xanthomonas and Xylella communicate using a class of small molecules, α,β unsaturated fatty acid signal molecules, which are termed diffusible signal factors (DSF), as signals. The signals, which increase in concentration with population density, typically coordinate the expression of genes involved in exploitation of a host organism. The virulence of the microbial pathogens is greatly reduced when the ability to produce signaling compounds is disrupted by mutation. The disruption may be by way of inhibition of DSF by degradation or blocking with an analog, or, since over-expression of DSF has also been shown to reduce virulence, activation/over-expression of DSF (e.g., with DSF synthase).
The present invention provides methods and compositions for control of DSF-based microbial pathogens. The methods and compositions of the invention draw on the findings that blocking cell-cell signaling in DSF-based pathogenic organisms reduces virulence (in the case of the Xanthomonads, for example) or spread (in the case of Xylella, for example) of the pathogen. Methods and materials for identifying DSF-interfering strains of bacteria have been developed and control of Xcc and Xf by inoculation of plants with the DSF-interfering strains has been established. In addition, construction of DSF transgenes and heterologous expression of DSF has been demonstrated. The elucidation of the bacterial mechanisms of Xf pathogenicity and the establishment of an associated DSF-based cell-cell signaling requirement allows extension of the principles of control based on DSF-based signal blocking from Xanthomonads to Xylella, even though the mechanisms are different.
In one aspect, the invention relates to a method of controlling diseases caused in a host organism by pathogenic microbial expression of α,β unsaturated fatty acid signal molecules. The method involves inoculating the host organism with a non-pathogenic microbe that disrupts α,β unsaturated fatty acid signal molecule-mediated cell-cell communication by the pathogenic microbe in the host organism.
In another aspect, the invention relates to the identification and isolation or manufacture of microbes having protein expression (express one or more proteins) that interferes with α,β unsaturated fatty acid signal molecule-mediated cell-cell communication by the pathogenic microbe in the host organism. The invention provides for the identification and isolation of naturally occurring microbes that have been identified as having the required DSF-interference property. The invention also provides for the construction of transgenic microbial cells having the required DSF-interference property. In the latter case, robust species particularly well-suited for large scale implementation biological control (i.e., vigorous non-pathogenic colonists of the host plant (the plant being protected)) may be transformed with DSF-interference gene or genes (i.e., coding sequence(s)).
In a further aspect, the invention relates to a host organism having cells transformed to have protein expression (express one or more proteins) that interferes with α,β unsaturated fatty acid signal molecule-mediated cell-cell communication by a pathogenic microbe in the host organism. Thus, genes (coding sequences) conferring DSF-interference activity can be expressed in organisms (e.g., plants) susceptible to diseases caused by microbes that use DSF, resulting in genetically resistant organisms.
These and other features of the invention are further described and exemplified in the detailed description below.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1, 2A and 2B illustrate the results of DSF biosensor assays in accordance with aspects of the present invention.
FIG. 3 illustrates control of black rot in cabbage by DSF-interfering bacteria in accordance with one aspect of the present invention.
FIG. 4 illustrates the effects of treatments of Pierce's disease in plants inoculated with Xylella fastidiosa or Xf plus a signal interference strain in accordance with one aspect of the present invention.
FIG. 5 illustrates the result of DSF biosensor assay in accordance with one aspect of the present invention.
FIG. 6 illustrates the structure of a diffusible signal factor (DSF) (alpha,beta unsaturated fatty acid) in accordance with one aspect of the present invention.
FIGS. 7 and 8 illustrate the results of an experiment showing that the severity of Pierce's disease in plants was greatly reduced when rpfF was over-expressed in Xf under the control of a high and constitutive promoter in accordance with one aspect of the present invention.
FIG. 9 illustrates a model of DSF regulation of genes required for movement of Xf from colonized vessels.
FIG. 10 illustrates the experimental result that leafhoppers fed on rpfF mutant-infected plants ingested rpfF cells but were able to rapidly clear themselves whereas the wild type is never cleared.
FIG. 11 illustrates that the incidence of Pierce's disease was greatly reduced by all of the DSF-interfering strains that were tested in accordance with one aspect of the present invention.
FIG. 12 illustrates disease control by topical inoculation of DSF-producing bacteria to the foliage of plants as well as by pre-treatment of plants by injection before inoculation with Xf in accordance with one aspect of the present invention.
FIG. 13 illustrates the results of topical applications of bacterial suspensions to plants in solutions of organosilicone surfactants having very low surface tensions in accordance with one aspect of the present invention.
FIGS. 14-16 illustrate control of Pierce's disease of grape with bacterial strains that produce DSF in accordance with one aspect of the present invention compared with the control seen with mutants of these strains that can no longer produce DSF.
FIG. 17 illustrates that a mutant strain that no longer can degrade DSF is not as effective as its parental strain in controlling Pierce's disease of grape.
FIGS. 18 and 19 illustrate DSF degradation reduces pathogenicity of Xcc and virulence of Xf, respectively.



DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS
Reference will now be made in detail to specific embodiments of the invention. Aspects of the specific embodiments are illustrated in the accompanying drawings. While the invention will be described in conjunction with these specific embodiments, it will be understood that it is not intended to limit the invention to such specific embodiments. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The present invention may be practiced without some or all of these specific details. In other instances, well known process operations have not been described in detail in order not to unnecessarily obscure the present invention.
INTRODUCTION
In Xanthomonas campestris pathovar campestris (Xcc), the expression of pathogenicity genes is controlled by the Rpf system of cell-cell communication, enabling a population of cells to launch a pathogenic attack in a coordinated manner. Barber, C. E. et al. A novel regulatory system required for pathogenicity of Xanthomonas campestris is mediated by a small diffusible signal molecule. Molecular Microbiology 24, 555-66 (1997). A cluster of genes (called rpf for regulation of pathogenicity factors) of Xcc encode the components of a cell-cell communication system. The gene rpfF is involved in the production of a class of small molecules, α,β unsaturated fatty acid signal molecules, which are termed diffusible signal factors (DSF), as signals. DSF is released from cells and is required for production of an extracellular polysaccharide (EPS) and extracellular enzymes, which play a major role in pathogenicity, and therefore rpfF mutants are completely avirulent. As the population grows, the local concentration of DSF increases. Other Rpf proteins are thought to sense the increase in DSF concentration and transduce a signal, resulting in expression of pathogenicity factors involved in exploitation of a host organism. Whitehead Neil, A., Barnard Anne, M. L., Slater, H., Simpson Natalie, J. L. & Salmond George, P. C. Quorum-sensing in Gram-negative bacteria. FEMS Microbiology Reviews 25, 365-404 (2001). This coordinate expression of virulence traits in a cell-density dependent fashion, referred to as quorum sensing, presumably occurs because cells would not benefit from expression of virulence traits when in low numbers in a host. The expression of such traits by solitary cells would be futile and a deleterious waste of metabolic resources. Conversely, when part of a group, the protection afforded by expression of virulence traits would be mutually beneficial and hence cells express such traits only when in high numbers.
There is a very close phylogenetic relationship between plant pathogenic bacteria in the genus Xanthomonas and Xylella fastidiosa (Xf). Xf shares sequence similarity with Xcc; the Xf genome not only contains homologs of the rpf genes most essential for cell-cell signaling in Xcc, but also exhibits striking colinearity in the arrangement of these genes on the chromosome. Dow, J. M., and M. J. Daniels. 2000. Xylella genomics and bacterial pathogenicity to plants. Yeast 17:263-71; Simpson, A. J. G., F. C. Reinach, P. Arruda, et al. 2000. The genome sequence of the plant pathogen Xylella fastidiosa. Nature 406:151-157. The Xf genome lacks homologs of genes shown to be involved in production and perception of other known cell-cell signaling systems, such as the luxI/luxR genes and other genes shown to be involved in production and perception of AHLs. Crossman, L., Daniels, M. J. & Dow, M. in Molecular Plant-Microbe Interactions (Madison, Wis., 2001). Xf has also been shown to produce a diffusible signal that is recognized by Xcc. Scarpari, L. M., Lambais, M. R., Silva, D. S., Carraro, D. M. & Carrer, H. Expression of putative pathogenicity-related genes in Xylella fastidiosa grown at low and high cell density conditions in vitro. FEMS Microbiol Lett 222, 83-92 (2003).
It has now been established that the existence of a DSF-mediated cell-cell signaling system in Xf and the mechanism by which it operates. Unlike Xanthomonads, Xf requires insect vectors for spread from plant to plant. DSF signaling has been found to play a role in virulence and transmission of Xf. Xf rpfF mutants exhibit increased virulence to plants, however, they are unable to be spread from plant to plant by their insect vectors. Newman, K. L., Almeida, R. P. P., Purcell, A. H. & Lindow, S. E. Cell-cell signaling controls Xylella fastidiosa interactions with both insects and plants. Proc. Natl. Acad. Sci. USA 101, 1737-1742 (2004).
The present invention relates generally methods and compositions for control of microbial pathogens by disrupting DSF-mediated cell-cell communication. Disruption or interference of DSF-mediated cell-cell communication may be achieved by DSF inhibition or activation/over-expression. Inhibition may be achieved by the expression, e.g., by an isolated or engineered cell, of a protein that degrades DSF or expresses a non-functional DSF analog. Activation/over-expression may be achieved by providing additional DSF or DSF synthase, e.g., by isolated, mutated and/or engineered cells that over-expresses DSF.
The methods and compositions of the invention draw on the findings that blocking cell-cell signaling in DSF-based pathogenic organisms reduces virulence (in the case of the Xanthomonads, for example) or spread (in the case of Xylella, for example) of the pathogen. Methods and materials for identifying DSF-interfering strains of bacteria have been developed and control of Xcc and Xf by inoculation of plants with the DSF-interfering strains has been established. In addition, construction of DSF transgenes and heterologous expression of DSF has been demonstrated. The elucidation of the bacterial mechanisms of Xf pathogenicity and the establishment of an associated DSF-based cell-cell signaling requirement allows extension of the principles of control based on DSF-based signal blocking from Xanthomonads to Xylella, even though the mechanisms are different.
Disease Control Methods
In one aspect, the invention relates to a method of controlling diseases caused in a host organism by pathogenic microbial expression of α,β unsaturated fatty acid signal molecules. The method involves inoculating the host organism with a microbe that disrupts α,β unsaturated fatty acid signal molecule-mediated cell-cell communication by the pathogenic microbe in the host organism.
The method is particularly applicable in agricultural biological control applications where the host organism is a plant and the pathogenic microbe is bacterial. For example, members of the Brassicaceae, which includes broccoli, cabbage and other plants, are susceptible to black rot, which is caused by Xanthomonas campestris pathovar campestris (Xcc). Members of the Solanaceae, which includes tomatoes and peppers, are susceptible to bacterial spot, which is caused by X. campestris pathovar vesicatoria (Xcv). Rice is subject to bacterial leaf blight, which is caused by X. oryzae pathovar oryzae (Xoo). And Xylella fastidiosa (Xf) causes citrus variegated chlorosis, almond leaf scorch and Pierce's disease of grape.
Method of Identifying Interference Microbial Strains
Suitable bacterial strains for inoculation of host plants may be obtained from a variety of techniques. Naturally occurring microbial strains that have been identified as having the required DSF-interference property can be identified and isolated. For example, bacterial strains that can interfere with DSF signaling can be identified using a biosensor. Suitable biosensors include a bacterial strain that expresses its own DSF and emits a signal (e.g., fluoresces) unless DSF signaling is disrupted. Bacterial strains are applied to appropriate growth media with the biosensor and those strains possessing a DSF-inhibition property may be identified by weakening of the fluorescence signal. An example of this is illustrated with reference to FIG. 2B in Example 1, below.
DSF-activating strains can be identified by a second biosensor that cannot express DSF and can only emit a signal (e.g., fluoresce) if exogenous DSF is provided by the activating strain.
This second biosensor strain can also be used to verify that DSF-inhibiting strains can degrade DSF. Lack of fluorescent signal when DSF is added to a culture of the identified inhibition strain and subsequently tested with this biosensor confirms that the inhibitor cells were able to degrade the DSF that was added to the culture.
Such identified strains may then be isolated and cultured according to procedures well know to those of skill in the art for further use, for example in inoculation of plants for disease control.
Method of Constructing Interference Microbial Strains
Transgenic bacteria having the required DSF-interference property may also be constructed. Initially genes conferring DSF interference are isolated from wild bacterial strains. The genes may be isolated by a number of techniques, such as by transposon mutagenesis and screening for loss of DSF degradation ability followed by identification of the gene disrupted by the transposon. According to one example of this technique, a library of mutants can be obtained by diparental mating of each interference strain with Eschericia coli cells harboring pRL27, a suicide vector carrying a Tn5-derivative transposon. Larsen, R. A., Wilson, M. M., Guss, A. M. & Metcalf, W. W. Genetic analysis of pigment biosynthesis in Xanthobacter autotrophicus Py2 using a new, highly efficient transposon mutagenesis system that is functional in a wide variety of bacteria. Arch Microbiol 178, 193-201 (2002). Transposon insertion events confer kanamycin resistance to the host cell, allowing efficient selection of mutated cells. The resulting mutant collections can be screened for a loss of the ability to interfere with DSF signaling using a DSF biosensor-based screen, such as that described above. Once a DSF-interference mutant is identified, the gene disrupted by the Tn5-derivative transposon can be cloned and sequenced using the one-step cloning strategy described by Larsen et al. to identify the DSF-interference gene.
Alternatively, genes conferring DSF interference can be isolated from wild bacterial strains by construction and screening of cosmid libraries. According to this technique, for each strain, a cosmid library can be constructed in a plasmid (e.g., pLAFR3) (Huynh, T. V., Dahlbeck, D. & Staskawicz, B. J. Bacterial Blight of Soybean Regulation of a Pathogen Gene Determining Host Cultivar Specificity. Science 245, 1374-1376 (1989)) and screened for the ability to degrade DSF when introduced into E. coli, or if necessary Xanthomonas campestris pathovar campestris (Xcc), using the DSF biosensor-based screen, described above. Once a particular cosmid carrying the DSF-degrader gene is identified, the cosmid can be subjected to standard subcloning and insertional mutagenesis to narrow down which gene on the cosmid confers DSF-degradation activity. The identity of the gene can be revealed by sequencing of subcloned fragments.
Microbial Expression of DSF Interference (Activation or Inhibition)
The genes so isolated can then be used to transform suitable bacterial cells, which can then be used to inoculate the host plants. In one embodiment, in order to more effectively deliver DSF interference to the site of infection by the pathogen and facilitate large-scale application of biological control, bacteria that are vigorous non-pathogenic colonists of the plant being protected can be engineered to express DSF-degradation (inhibition) and DSF-synthesis (activation/over-expression) genes. Protection to plants threatened by Xylella fastidiosa (Xf), such as grapevines, may be best be provided by an endophytic bacterium (a bacterium that lives inside the plant body), such as Alcaligenes xylosoxidans denitrificans (Axd). Protection to plants threatened by Xanthomonas spp., such as tomato and broccoli, may be best protected by a vigorous epiphytic bacterium (a bacterium that lives on the surface of leaves), such as Pseudomonas fluorescence A506 (PfA506).
For DSF synthesis (activation/over-expression), the rpfF gene from Xf or Xcc can be placed under the control of the kanamycin resistance gene promoter from Tn903 or another suitable promoter and introduced into the Axd and PfA506 genomes. A protocol employing a transposon-containing plasmid, pSP14, to deliver genes directly into the chromosome of Axd and PfA506 may be used. Bextine, B. R., Lauzon, C. R., Potter, S. E., Lampe, D. & Miller, T. E. Delivery of a genetically marked Alcaligenes sp. to the glassy-winged sharpshooter for use in a paratransgenic control strategy. Current Microbiology 48, 327-31 (2004). For DSF inhibition, DSF-inhibition genes under the control of a constitutive promoter, such as the Kan promoter from Tn903, can be transferred into Axd and PfA506 by the same strategy.
Isolated, mutated and/or engineered DSF-interference microbial strains may be modified in accordance with conventional techniques to introduce novel genetic capabilities. These techniques, for the most part, involve transformation or conjugation. Various genetic capabilities which can provide advantages for the DSF-interference strains include imparting antibiotic resistance, bacteriocin production, host range, growth characteristics, e.g. colicin production, nitrogen fixation, or the like. By providing for a marker which allows for selection of transformants or conjugants, the desired organisms may then be selected. Markers include antibiotic resistance, colicin resistance, heavy metal resistance, providing prototrophy to auxotrophs, or the like.
Application of Microbes Expressing DSF Interference
Depending upon the nature of the plant, the part of the plant to which the DSF-interference strain is applied, as well as other factors, a variety of methods and compositions may be employed for application of the organism to the plant. In addition, it may be desirable to use mixtures of organisms, i.e., two or more DSF-interference strains, rather than a single organism.
Xanthomonas spp. are epiphytic, lending themselves to control by agents, for example, epiphytic cells with DSF-interference capability, applied to the surface of the plant. Transformed epiphytic bacterial cells, such as Pseudomonas fluorescence A506 (PfA506), carrying genes conferring DSF-interference capability, as described above, can be effectively introduced to plant hosts by topical application of a suitable formulation to the plants.
The number of cells per unit formulation will depend on whether the formulation is a dry or wet formulation. For wet formulation, e.g. foliar sprays, suspensions, aerosols, mist, etc., the number of cells per ml will generally be from about 105 to 1010 cells per ml. Generally, it is desired to have about 104 to 1010 cells/g fr. wt. of leaves during application. For dry formulations, the number of cells will generally range from about 104 to 109 cells/g of formulated product. The cells in the formulation should provide a sufficient number of cells to allow the cells to become established on the host plant with microorganisms present in the native environment taking into consideration the mortality rate, higher with spray formulations, which are encountered with the means of administration. The number of cells should be sufficient to colonize within about one week or less for sprays.
In aqueous formulations various additives may be included in minor amounts such as surfactants, e.g. nonionic, dyes, nutrients, buffers, penetrating agents for introducing the cells into the leaf, biological or chemical pesticides e.g. herbicides, insecticides, etc. In dry formulations various additives include inert powders, bacterial stabilizing agents, salts, anticaking agents, nutrients, buffers, film forming materials, biological or chemical pesticides e.g. herbicides, insecticides, etc. The various additives will range in concentration from about 1×10−4 to 1 weight percent.
One technique is to apply the DSF-interfering cells to the seed (seed inoculum) or seed piece of a plant. The bacteria may be formulated as a dry powder formulation in accordance with conventional techniques. Of particular interest is a powder formulation containing the cells, which is derived from combining about one part by volume of a cell containing gum suspension, with 4 volumes of an inert powdered carrier, e.g., talc. The gum suspension is prepared by combining about 1 vol. of a dense suspension of cells (about 109-1011 cells/ml) with about 10 vols. of a dilute magnesium salt solution, which mixture is then combined with 10 vols of a thick aqueous suspension of a natural gum. The gum will be about 90-99% by weight of the mixture and employed initially as a 10-30 weight percent suspension. The mixture is allowed to dry and ground to a fine powdered consistency. The seeds or seed pieces in a slightly moistened state are contacted with the powder. Upon planting, it is found that the bacteria colonize the emerging stem and leaves as they emerge from the soil.
The powder formulation may also be applied as a dust application. Conveniently, the bacteria are applied to the foliage at a rate of from about 107 to 108 bacteria per gram fr. wt. of leaves being inoculated. The use of the dust powder inoculation is particularly applicable during hot weather, on bright sunny days, when applied prior to mid to late afternoon and at low relative humidities.
Another way for establishing the DSF-interfering bacteria is by foliar spray. The bacteria need only be employed as an aqueous suspension, in substantial absence of other additives, e.g., nutrients and surfactants. The application rate will generally be approximately 106 to 108 cell/ml of vegetative cells in an aqueous suspension to provide about 104 to cells/g. fr. wt. of leaves.
Since Xf is an endophytic bacterium, it is most effectively controlled by application of a control agent to the target plant host by a technique that delivers the control agent inside the plant body. Transformed endophytic bacterial cells, such as Alcaligenes xylosoxidans denitrificans (Axd), carrying genes conferring DSF-interference capability, as described above, can be introduced into grapevines by standard needle inoculation as described in Hill, B. L. & Purcell, A. H. Multiplication and movement of Xylella fastidiosa within grapevine and four other plants. Phytopathology 85, 1368-1372 (1995). While needle inoculation of individual plants may seem impractical in large scale agricultural application, the intensive nature of grape production lends itself to use of this technique, since individual plants are handled many times by workers during the course of the growing and harvesting seasons.
It has also been found that it is possible to establish large populations of bacteria within grape leaves, stems and petioles by simple topical applications of bacterial suspensions to plants in solutions of organosilicone surfactants having very low surface tensions. Non-endophytic bacterial species were also established in high numbers inside grape leaves and petioles following spray application to plants with a high concentration (e.g., about 0.2 to 1.0% V/V, for example about 0.5% to 1.0% of a silicon-based surfactant with a low surface tension (e.g., less than 25 dynes/cm), such as the product Break-Thru®, available from Goldschmidt Chemical Corporation.
Expression of DSF in Planta
In a further aspect, the invention relates to a host organism having cells transformed to have protein expression (express one or more proteins) that interferes with α,β unsaturated fatty acid signal molecule-mediated cell-cell communication by a pathogenic microbe in the host organism. Thus, genes conferring DSF-interference activity (activation/over-expression or inhibition) can be expressed in organisms (e.g., plants) susceptible to diseases caused by microbes that use DSF, resulting in genetically resistant organisms.
In one embodiment, the rpfF gene from Xf or Xcc (Genbank Accession Nos. NC—004556 (locus tag PD0407, gi:28056394) and Y09701, respectively) or other DSF-producing microbes can be placed under the control of the constitutive cauliflower mosaic virus 35S promoter or a ubiquitin promoter or other suitable promoter from the plant in a cassette on a binary vector capable of replication in both E. coli and Agrobacterium tumefasciens, pCAMBIA 2200 (The Centre for Application of Molecular Biology to International Agriculture (CAMBIA), GPO Box 3200, Canberra ACT 2601 Australia) and introduced into plant genomes using A. tumefasciens-mediated plant transformation by standard protocols, such as are described in Park, S. H., Morris, J. L., Park, J. E., Hirschi, K. D. & Smith, R. H. Efficient and genotype-independent Agrobacterium-mediated tomato transformation. J Plant Physiol 160, 1253-7 (2003).
Genbank Accession Nos. NC—004556 (locus tag PD0407, gi:28056394) (SEQ ID NO:1) and Y09701 (SEQ ID NO:2):








Xf rpfF:



translation = “MSAVHPIPHPICESSIRIIEETHRNVYWIYMHAHL


 


ARTTGAAYFSLKLIDDIMNYQSVLRQRLKEQTVQLPFVVLASDSNVFNLG


 


GDLQLFCDLIRRKEREALLDYACRCVRGAYAFHAGLNANVHSIALLQGNA


 


LGGGFEAALCCHTIVAEEGVMMGFPEVLFDLFPGMGAYSFMRQRISPKLA


 


ERLILEGNLYSSEELLAIGLIDKVVPRGKGIEAVEQIIRDSKRRQYTWAA


 


MQEVKKIAHEVSLEEMIRITELWVDSALKLSNKSLRTMERLIRAQQTHKN


 


TALKN”


 


Xcc rpfF:


translation = “MSAVQPFIRTNIGSTLRIIEEPQRDVYWIHMHADL


 


AINPGRACFSTRLVDDITGYQTNLGQRLNTAGVLAPHVVLASDSDVFNLG


 


GDLALFCQLIREGDRARLLDYAQRCVRGVHAFHVGLGARAHSIALVQGNA


 


LGGGFEAALSCHTIIAEEGVMMGLPEVLFDLFPGMGAYSFMCQRISAHLA


 


QKIMLEGNLYSAEQLLGMGLVDRVVPRGQGVAAVEQVIRESKRTPHAWAA


 


MQQVREMTTAVPLEEMMRITEIWVDTAMQLGEKSLRTMDRLVRAQSRRSG


 


LDAG”






DSF-inhibition genes under the control of the constitutive plant promoter may be transferred by the same strategy as outlined above.
EXPERIMENTAL
The invention having been generally described, may be better understood by reference to the following examples, which are provided for purposes of illustration and are not to be considered limiting of the invention.
Example 1
Identification and Effectiveness of DSF Interference Stains
A DSF signal sensing strain, or “DSF biosensor,” carrying a green fluorescent protein (gfp) gene under the control of a promoter that is up-regulated in response to the DSF signal was engineered, as described in Newman, et al. Proc. Natl. Acad. Sci. USA 101, 1737-1742 (2004). A group of bacterial strains, “inhibitor strains,” that can interfere with DSF signaling was identified using this biosensor. Results are shown in FIG. 1. The figure shows colonies of bacterial strains with (left panel) or without (right panel) DSF inhibition activity. Colonies are surrounded by a lawn of the DSF biosensor strain that expresses its own DSF and fluoresces green unless DSF signaling is disrupted. The dark halo around the colony in the left panel demonstrates inhibition of DSF signaling.
As illustrated in FIG. 2A, some of these strains interfere in signaling by degrading DSF. The figure illustrates degradation of DSF by an inhibitor strain. DSF was added to a liquid culture of the inhibitor strain (right panel) or to cell-free spent media from the same strain (left panel) and incubated overnight. The DSF remaining in the media after this treatment was extracted and plated to the right of a DSF biosensor that cannot express DSF, which can only fluoresce green if exogenous DSF is provided. The lack of fluorescence by the reporter in the right panel confirms that the inhibitor cells were able to degrade the DSF that was added to the culture.
FIG. 2B provides additional evidence indicating that DSF is enzymatically degraded by some DSF-inhibiting strains. Crude protein extracts of several DSF-inhibiting strains were able to degrade DSF, while no degradation of DSF occurred when extracts were boiled prior to incubation with DSF, indicating that, in these strains, DSF-inhibition is likely due to enzymatic degradation of the signal. Other strains were unable to degrade DSF when it was added to growing cultures or crude protein extracts, indicating the existence of an alternate mechanism of DSF inhibition, such as synthesis of a DSF analog. FIG. 2B shows the results obtained with Strain G. Green fluorescence indicates DSF remaining after incubation with a culture (a), a crude protein extract (b), or a crude protein extract that was boiled (c). In panel d, no DSF was added to the culture and in panel e, no bacteria was inoculated into the culture.
Other inhibitor strains interfere via alternate mechanisms, which may include synthesis of a DSF analog. In addition, DSF interference may be achieved by synthesis of surplus DSF (over-expression).
The effectiveness of these strains in preventing disease in plants infected with DSF-producing pathogens was preliminarily tested for both Xanthomonas and Xylella. For Xanthomonas, control of black rot in cabbage by DSF-interfering bacteria was tested by inoculating cabbage seedlings with Xcc alone or co-inoculating with Xcc and the interfering strains. Results are shown in FIG. 3. The figure illustrates control of black rot in cabbage by DSF-interfering bacteria. Xcc strain 8004 was inoculated onto leaves of cabbage seedlings by the method of clipping leaves with scissors dipped in a solution of the pathogen (Dow, J. M. et al. Biofilm dispersal in Xanthomonas campestris is controlled by cell-cell signaling and is required for full virulence to plants. Proc Natl Acad Sci USA 100, 10995-1000 (2003)) and lesion length in these leaves was measured after 13 days (Xcc). Xcc was mixed with DSF-inhibiting (A-H, K, L) or DSF-activating (I, U-Z) strains prior to inoculation. Plants co-infected with the pathogen plus a DSF-interfering strain showed significantly reduced disease symptoms. Plants infected only with the DSF-producing strain showed no symptoms. Similar results were obtained with broccoli and turnip seedlings.
FIG. 4 illustrates the effects of treatments of Pierce's disease in plants inoculated with Xylella fastidiosa (treatment Xf) or Xf plus a signal interference strain (treatments 1-13). Ten plants were inoculated with each treatment. While 90% of plants infected with the pathogen alone became diseased, significantly fewer plants were diseased when the pathogen was co-inoculated with a signal-interfering strain. Six of these strains are DSF activators (1-6), 6 are DSF inhibitors that degrade DSF (7-12), and 1 is a DSF inhibitor that does not degrade DSF (13). Similar results were seen in other repeat studies (see e.g., FIG. 12 in Example 3, below).
Both activator strains and inhibitor strains appeared effective in black rot and Pierce's disease control. These results provide a logical and practical basis for the expectation that these strains and their DSF-related activities will provide control of pathogenicity in DSF-producing disease organisms.
Example 2
Expression of DSF in Planta
DSF expression transgenes were constructed as follows. A 769 bp Hind III-Bgl II fragment containing the cauliflower mosaic virus 35S promoter was excised from pCAMBIA1201 and ligated into pCAMBIA1390 cut with Hind III and Bam HI to make pKLN1. The Xylella fastidiosa rpfF coding sequence was amplified directly from the X. fastidiosa genome using Pfu polymerase (Strategene) using primers “Xf rpfF F Nco I” (TACCATGGCCGCTGTACATCCCATTCCTCACCCCATAT) (SEQ ID NO:3) and “Xf rpfF R Bam HI” (CGGGATCCTCAGTTTTTTAGTGCTGTGTTTTTGTGA GTCT) (SEQ ID NO:4), cut with Nco I and Bam HI and ligated into pKLN1 cut with Nco I and Bgl II to make pKLN119. This plasmid carries a T-DNA that includes both hygromycin resistance and the X. fastidiosa rpfF gene driven by the CMV 35S promoter and followed by the NOS poly-A signal sequence.
pKLN119 and the empty vector pCAMBIA1390 were electroporated into Agrobacterium strain GV3101. Nicotiana benthamiana plants were transiently transformed by infiltration with suspensions of Agrobacterium harboring T-DNA construct pKLN119 or pCAMBIA1390.
Disks of infiltrated leaves were removed after two days, placed on KB agar plates and oversprayed with the DSF bioreporter strain 8525 (pKLN55). Green fluorescence indicates detection of DSF from the leaf disk. Results are shown in FIG. 5. The figure illustrates expression of DSF in Nicotiana benthamiana. In the left panel, the leaf disk infiltrated with pKLN119 is producing DSF, as shown by green fluorescence of the DSF bioreporter. In the right panel, the leaf disk infiltrated with pCAMBIA1390, the empty vector, does not cause gfp expression in the bioreporter, demonstrating that fluorescence is specific to expression of DSF.
These results demonstrate the principle that the DSF molecule can be synthesized by plant cells expressing the bacterial rpfF gene. Because expression by the plant was very robust, it is expected that expression can be achieved with ease in a variety of plant species.
Example 3
Studies of DSF Interference, and Inoculation and Transformation of Plants for Disease Control
In this example, data is provided on the virulence of the wild-type Xylella fastidiosa strain and on a strain in which the rpfF gene encoding synthesis of DSF is being over-expressed with a kanamycin resistance gene promoter. The disease is much less in the plant in which Xf is over-expressing DSF (FIGS. 7 and 8), which correlates well with other information such as that blockage of DSF production increased virulence.
Control of Pierce's disease of grape with bacterial strains that produce DSF was also shown and compared with the control seen with mutants of these strains that can no longer produce DSF (FIGS. 14-16). The data showed that the mutants that no longer can produce DSF are not as effective in control of disease. Likewise, mutants of strain “G,” a strain that no longer can degrade DSF, are not as effective as strain G in controlling Pierce's disease of grape (FIG. 17). Both of these sets of data suggest that altering levels of DSF in plants per se is what responsible for disease control.
Transgenic tomato and grape plants into which the rpfF genes of Xylella fastidiosa and Xanthomonas campestris have been introduced have also been generated.
Also, the use of an organosilicone surfactant with very low surface tension with bacterial suspensions to introduce bacteria that make or degrade DSF into plants was explored.
INTRODUCTION
DSF-mediated cell-cell signaling in Xf was investigated with the aim of developing cell-cell signaling disruption as a means of controlling Pierce's disease. It has been determined that the rpfF gene is necessary and sufficient for DSF signal synthesis and that rpfF mutants of Xf are hypervirulent and non-transmissible. Lack of transmissibility was linked to an inability of the rpfF mutant to form a biofilm in the insect foregut; while taken up by insects, the mutant strain is not retained. Xf strains that overproduce DSF produce disease symptoms in grape, but only at the site of inoculation and the cells do not move within the plant as do wild-type strains. Thus, it was hypothesized that elevating DSF levels in plants should reduce movement of Xf in the plant and also reduce the likelihood of transmission by sharpshooters. Several collections of bacterial strains isolated from plants were screened and identified bacterial strains that can interfere with Xf signaling both by producing large amounts of DSF, by degrading DSF, or by in some way interfering with recognition of DSF. When co-inoculated into grape with Xf, both DSF-producing strains and DFS degrading strains greatly reduced the incidence of disease in grape; DSF-producing strains consistently were the most effective in reducing disease.
Given that DSF appears to mediate an attenuation of virulence in Xf, it is hypothesized that it may be possible to transform grape with the rpfF gene to enable DSF production in planta in order to protect grape plants from Pierce's disease. Preliminary results indicate that transient expression of rpfF in Nicotiana benthamiana following infiltration with appropriate Agrobacterium tumefaciens strains resulted in high levels of DSF production, suggesting that it is likely that grape cells will produce DSF when transformed with the bacterial rpfF gene. Non-endophytic bacterial species were also established in high numbers inside grape leaves and petioles following spray application to plants with a high concentration of a silicon-based surfactant with a low surface tension suggesting that it may be possible to produce protective compounds such as DSF in plants by a variety of bacteria.
Two of the Rpf proteins, RpfB and RpfF, work to produce a diffusible signal factor (DSF) which has recently been described as an alpha,beta unsaturated fatty acid having the structure shown in FIG. 6. Wang, Lian-Hui, He, Y., Gao, Y., Wu, J. E., Dong, Y.-H., He, C., Wang, S. X., Weng, L.-X., Xu, J.-L., Tay, L., Fang, and R. X., Zhang, L.-H. 2004. Cell-cell communication signal with cross-kingdom structural analogues. Molecular Microbiology. 51: 903-912. As noted above, as the population grows, the local concentration of DSF increases. Other Rpf proteins are thought to sense the increase in DSF concentration and transduce a signal, resulting in expression of pathogenicity factors. Slater, H., A. Alvarez-Morales, C. E. Barber, M. J. Daniels, and J. M. Dow. 2000. A two-component system involving an HD-GYP domain protein links cell-cell signaling to pathogenicity gene expression in Xanthomonas campestris. Molecular Microbiology 38:986-1003. It has been found that several other bacterial species can both positively and negatively interact with the DSF-mediated cell-cell signaling in Xf, but the manner in which the interaction occurred nor whether such strains had the potential to affect the virulence of Xf in grape was not previously known. In this study, the role of DSF-production by Xf on its behavior within plants and insects as well as the manner in which other bacterial strains affect such cell signaling has been extensively investigated, and the extent to which other endophytes could modulate density-dependent behaviors and virulence in Xf by interfering with cell-cell signaling has been determined.
Results
A strain of Xf Temecula was constructed in which the rpfF gene, which is required for production of the signal in Xcc, is knocked out. This mutant was constructed using exchange of the wild-type allele for a deleted copy carrying an antibiotic resistance gene on a suicide plasmid. The rpfF mutant of Xf does not make DSF as determined using previously constructed “signal-sensing” strains of Xcc to determine DSF production by Xf and other bacterial strains. rpfF mutant strains were tested for their ability to infect and move within host plants and to cause Pierce's disease symptoms. The rpfF gene appears to play a role in modulating disease progress because the timing and severity of symptom development are greatly exacerbated in grapevines infected with rpfF mutants when compared to the wild type. The mechanism behind these differences has been investigated. No detectable difference in populations or movement between the wild type and rpfF mutants has been found, although the sampling methods used would not be able to detect small increases in colonization if they existed. As noted previously, it is hypothesized that rpfF mutants may be causing increased vessel blockage in the grapevine, leading to increased symptom expression. A green fluorescent rpfF mutant was made to investigate the pattern of colonization by the mutant and compare it to that of the wild type. Importantly, when rpfF was over-expressed in Xf under the control of a high and constitutive promoter, the severity of disease in plants was greatly reduced, as illustrated in FIGS. 7 and 8. The Xf strain that overproduced DSF caused disease symptoms in grape, but only at the site of inoculation. The mutant cells did not move within the plant as did wild-type strains. These results all support a model, illustrated in FIG. 9, that DSF regulates genes required for movement of Xf from colonized vessels. Such results suggest that elevating DSF levels in plants should reduce movement of Xf in the plant.
The transmissibility of the rpfF mutant strain by an insect vector was also tested. The rpfF mutant was virtually untransmissible. This defect in transmissibility by the signaling-deficient mutant reveals the importance of cell-cell signaling in insect transmission. Leafhoppers fed on rpfF mutant-infected plants ingested rpfF cells but were able to rapidly clear themselves whereas the wild type is never cleared as illustrated in FIG. 10.
In addition, a variety of bacteria were isolated from grapevines from vineyards affected by Pierce's disease as well as tomato and cruciferous crop plants infected with the signal-producing pathogens Xanthomonas campestris pv. vessicatoria and Xcc, respectively and tested for their ability to interfere with cell-cell signaling in Xf in an assay using the signal-sensing strain described above. Several strains that negatively affected signaling in Xcc were found while several strains were found to produce DSF. By adding purified DSF to either cell-free extracts of the strains with a negative influence on signaling or to whole cells it was found that at least two mechanisms of interference with signaling could be observed. Some strains such as strains C, E, G, H, and J are able to degrade DSF while other inhibitor strains did not do so, and apparently have another means of interfering with DSF perception by Xcc. The several strains that produced DSF were all identified as Xanthomonas species. The 16S rRNA gene from these strains was sequenced to determine their species identity (noted in the Table 1, below):










TABLE 1





Strain
Genus
Origin
Mechanism of DSF Interference







A
Paenibacillus
Grape
Unknown inhibition


B
Paenibacillus
Grape
Unknown inhibition


C
Pseudomonas
Cabbage
Enzymatic digestion


D
Staphylococcus
Grape
Unknown inhibition


E
Bacillus
Broccoli
Enzymatic digestion


G
Pseudomonas
Cabbage
Enzymatic digestion


H
Pseudomonas
Cabbage
Enzymatic digestion


J
Pseudomonas
Tomato
Enzymatic digestion


L
Staphylococcus
Grape
Unknown inhibition


I
Xanthomonas
Tomato
DSF production


U
Xanthomonas
Broccoli
DSF production


V
Xanthomonas
Broccoli
DSF production


W
Xanthomonas
Broccoli
DSF production


X
Xanthomonas
Broccoli
DSF production


Y
Xanthomonas
Tomato
DSF production


Z
Xanthomonas
Grape
DSF production









To verify that disease control is due to DSF interference, mutants of bacterial strains that disrupt the ability of these strains to produce or degrade DSF were constructed and it has been shown that these mutants are deficient in Pierce's disease (PD) control. Both mutants unable to produce DSF as well as mutants deficient in degradation of DSF exhibited less ability to control PD when coinoculated with Xf, suggesting that altering DSF abundance within the plant was a major factor contributing to disease control by these DSF-interfering strains.
Given that DSF overabundance appears to mediate an attenuation of virulence in Xf grape has been transformed with the rpfF gene of Xf to enable DSF production in planta. Preliminary results indicate that transient expression of rpfF in Nicotiana benthamiana following infiltration with appropriate Agrobacterium tumefaciens strains resulted in high levels of DSF production, suggesting that grape cells will produce DSF when transformed with the bacterial rpfF gene. The bacterial genes required for DSF degradation have been cloned and identified in antagonist strain G, enabling their exploitation for disease control by both over-expression in various bacterial endophytes of grape as well as by expression within plants themselves. Non-endophytic bacterial species were also established in high numbers inside grape leaves and petioles following spray application to plants with a high concentration of a silicon-based surfactant with a low surface tension, suggesting the possibility of producing protective compounds such as DSF in plants using a variety of bacteria.
As noted above, to test the ability of bacteria that alter Xf signaling to alter the process of disease in plants, grapevines with Xf and strains that either inhibit or activate cell-cell signaling were co-inoculated in greenhouse studies. The incidence of Pierce's disease was greatly reduced by all of the signaling interfering strains that were tested, as illustrated in FIGS. 4 and 11. DSF-producing strains generally reduced disease severity more than did strains that interfered with signaling in Xf (FIG. 3). These results were highly repeatable and indicate that alteration of signal molecules within plants can have a profound effect on the disease process.
Disease control by topical inoculation has also been demonstrated. The results of inoculation of DSF-producing bacteria, such as DSF-producing strains X and 8004, to the foliage of plants where they colonize and presumably produce DSF as well as by pre-treatment of plants by injection of these antagonists before inoculation with Xf, are illustrated in FIG. 12.
It has also been found that it is possible to establish large populations of bacteria within grape leaves, stems and petioles by simple topical applications of bacterial suspensions to plants in solutions of organosilicone surfactants having very low surface tensions. Non-endophytic bacterial species were also established in high numbers inside grape leaves and petioles following spray application to plants with a high concentration (e.g., about 0.2 to 1.0% V/V, for example about 0.5% to 1.0%) of a silicon-based surfactant with a low surface tension (e.g., less than 25 dynes/cm), such as the product Break-Thru®, available from Goldschmidt Chemical Corporation, suggesting that it may be possible to produce protective compounds such as DSF in plants by a variety of bacteria. Bacteria were found to colonize grape for at very high population sizes (e.g., greater than 106 cells/petiole) for extended periods of time following topical application (FIG. 13). While these bacteria apparently do not spread throughout the plant after inoculation as does Xf, by introducing it into the intercellular spaces and perhaps even the xylem of the plant by use of the surfactants that stimulate spontaneous infiltration of the plant, the bacteria can be inoculated into all sites within the plant. Such non-pathogenic bacteria then increased in population size after inoculation, apparently by growing locally where they had been inoculated. This finding suggests exploiting a variety of DSF-interfering bacteria for practical control of PD. Such bacteria could produce large amounts of DSF after introduction into the plant. The observation that large numbers of bacteria can be introduced into grape plants by simple topical applications of cell suspensions in a penetrating surfactant provides an additional disease control strategy that should prove practical for commercial use.
To establish a rigorous connection between DSF production and disease control, mutant strains have been constructed of those DSF-producing bacteria that perform best in the disease control assays described above that no longer could produce DSF. These mutants were then compared to their parent strains in the disease control assays. Mutants of DSF-degrading strains that no longer could degrade DSF were also made. As illustrated in FIGS. 14-16, all mutants unable to produce DSF were diminished in ability to reduce PD when co-inoculated with Xf compared to their DSF-producing wild-type strain, many dramatically so.
Likewise, as illustrated in FIG. 17, mutant strain G741, a mutant of DSF-degrading parental strain G that no longer could degrade DSF also was greatly reduced in ability to control PD when co-inoculated with Xf compared to its parental strain. These results suggest strongly that it is the production of, or degradation of DSF in plants by these antagonistic bacteria that makes a large contribution to their ability to reduce PD. The results thus strongly suggest that any method that either increases or decreases DSF abundance in Xf-infected plants will have a large effect on the incidence and/or severity of Pierce's disease.
To increase the usefulness of any interfering agents identified in this screen, genes conferring the DSF-interference phenotypes can be molecularly identified. This gene can then be introduced into other organisms, such as plants.
Interfering strain G, typical of strains that apparently degrade DSF, was subjected to transposon mutational analysis of the interfering activity. Several insertional mutations that block degradation of DSF were identified. The genes have been inactivated individually by random Tn5 mutagensis and cloned the disrupted loci. A library of mutants of strain G was obtained by mating strains with pRL27, a suicide vector carrying a Tn5-derivative transposon. Larsen, R. A., Wilson, M. M., Guss, A. M. & Metcalf, W. W. Genetic analysis of pigment biosynthesis in Xanthobacter autotrophicus Py2 using a new, highly efficient transposon mutagenesis system that is functional in a wide variety of bacteria. Arch Microbiol 178, 193-201 (2002). Transposon insertion events conferred kanamycin resistance to the host cells, allowing efficient selection of mutated cells. The resulting mutant collections were screened for a loss of the ability to interfere with DSF signaling using the DSF biosensor-based screen described above. Mutations of the carAB genes, encoding carbamoyl-phosphate synthetase activity, in antagonist G abolish DSF degradation. Multiple mutants of these two genes have been found to disrupt DSF production:
Genbank Accession No. NC—007005 (gi:66043271) (SEQ ID NO:5 and SEQ ID NO:6, respectively)








carA:



translation = “MTKPAILALADGSIFRGEAIGADGQTVGEVVFNTA


 


MTGYQEILTDPSYAQQIVTLTYPHIGNTGTTPEDAESDRVWSAGLVIRDL


 


PLVASNWRNKLSLGDYLKANNVVAIAGIDTRRLTRILREKGAQNGCILAG


 


DNITEEAAIAAARGFPGLKGMDLAKEVCTKDTYEWRSSVWDLKTDSHPEI


 


AASELPYHVVAYDYGVKVNILRMLVERGCRVTVVPAQTPASEVLAYKPDG


 


VFLSNGPGDPEPCDYAIKAIREVLETDIPVFGICLGHQLLALAAGAKTVK


 


MGHGHHGANHPVQDLDTGVVMITSQNHGFAVDEATLPSNVRAIHKSLFDG


 


TLQGIELTDKSAFSFQGHPEASPGPNDVAPLFDRFIEAMAKRR”


 


carB:


translation = “MPKRTDIKSILILGAGPIVIGQACEFDYSGAQACK


 


ALREEGYRVILVNSNPATIMTDPAMADATYIEPIKWQTVAKIIEKERPDA


 


LLPTMGGQTALNCALDLEREGVLEKFGVEMIGANADTIDKAEDRSRFDKA


 


MKSIGLACPRSGIAHSMEEANAVLEKLGFPCIIRPSFTMGGTGGGIAYNR


 


EEFEEICARGLDLSPTKELLIDESLIGWKEYEMEVVRDKKDNCIIVCSIE


 


NFDPMGVHTGDSITVAPAQTLTDKEYQILRNASLAVLREIGVETGGSNVQ


 


FGICPNTGRMVVIEMNPRVSRSSALASKATGFPIARVAAKLAVGYTLDEL


 


SNEITGGKTPASFEPSIDYVVTKLPRFAFEKFAKADARLTTQMKSVGEVM


 


AIGRTFQESLQKALRGLEVGVCGLDPKLDLSHPESMSTLKRELTVPGAER


 


IWYVADAFRAGMTVEEIFAMNMIDPWFLVQIEDLIKDEEKIKTLGLSAID


 


RDVMFRLKRKGFSDARLAKLLGVTEKNLRTHRHKLEVFPVYKRVDTCAAE


 


FATDTAYLYSTYEEECEANPSTRDKIMILGGGPNRIGQGIEFDYCCVHAA


 


LALREDGYETIMVNCNPETVSTDYDTSDRLYFEPVTLEDVLEIVRVEKPK


 


GVIVQYGGQTPLKLARALEAAGVPIIGTSPDAIDRAEDRERFQHMVERLN


 


LRQPPNATVRSEDEAIRAAAKIGYPLVVRPSYVLGGRAMEIVYQEDELKR


 


YLREAVQVSNDSPVLLDHFLNCAIEMDVDAVCDGTDVVIGAIMQHIEQAG


 


VHSGDSACSLPPYSLPAHIQDEMREQVKKMALELGVVGLMNVQLALQGED


 


IYVIEVNPRASRTVPFVSKCIGVSLAMIAARVMAGKTLKELNFTKEIIPN


 


FYSVKEAVFPFAKFPGVDPILGPEMKSTGEVMGVGDTFGEAFAKAQMGAS


 


EVLPTGGTAFISVRDDDKPLVEAVARDLINLGFEIVATAGTAKLIEAAGL


 


KVRRVNKVTEGRPHVVDMIKNDEVTLIINTTEGRQSIADSYSIRRNALQH


 


KIYCTTTIAAGEAICEALKFGPEKTVRRLQDLHAGLKA”






The enzyme's mechanism of DSF degradation can be investigated by over-expressing it. The genetics of DSF degradation in this and other strains can be explored using this mutagenesis strategy or by constructing cosmid libraries in pLAFR3 (Huynh, T. V., Dahlbeck, D. & Staskawicz, B. J. Bacterial Blight of Soybean Regulation of a Pathogen Gene Determining Host Cultivar Specificity. Science 245, 1374-1376 (1989)) and screening for the ability to degrade DSF or interfere with signaling when introduced into E. coli, or if necessary Xcc, using the DSF biosensor strategy described above with reference to FIGS. 1 and 2. The location of genes can be determined by standard subcloning and insertional mutagenesis approaches, and the identity of the genes revealed by sequencing of subcloned fragments.
Disease control by DSF-interfering strains can be optimized if they are good colonists of grapevine. To maximize disease control the various genes conferring DSF interference can be expressed in effective non-pathogenic endophytic colonists of grapevine such as Alcaligenes xylosoxidans denitrificans (Axd) and Agrobacterium vitis (Av). This strategy should deliver the disease control agent directly to the site of the pathogen and result in highly effective control. Since the rpfF gene of Xf is sufficient to confer expression of DSF in other bacteria, it is being introduced into these two species. For Axd, technology developed in the laboratory of Dr. David Lampe, which employs a transposon-containing plasmid, pSP14, to deliver genes directly into the chromosome of Axd can be used (Bextine, B. R., Lauzon, C. R., Potter, S. E., Lampe, D. & Miller, T. E. Delivery of a genetically marked Alcaligenes sp. to the glassy-winged sharpshooter for use in a paratransgenic control strategy. Current Microbiology 48, 327-31 (2004). Both DSF-activating and DSF-inhibiting genes under the control of the constitutive Kan promoter can be transferred from Tn903 into Axd. The DSF degradation genes can introduced into Av on the stable vector pVSP61.
DSF degradation reduces pathogenicity of Xcc and virulence of Xf. Black rot symptoms in mustard are reduced when strain G is co-inoculated with the pathogen, however this reduction in symptom severity is not seen when strain G741, a Tn5 mutant that can no longer degrade DSF, is co-inoculated (FIG. 18). Pierce's disease occurrence in grapevines is reduced in frequency when strain G is co-inoculated with the pathogen (FIG. 19). The gene mutated in strain G741 as well as the other four mutants was the carAB locus, cited above. This indicates that the carAB locus encodes a DSF-degradation activity. Therefore, the carAB-encoded enzyme is an example of a DSF-degrading enzyme that can be used to degrade DSF.
Discussion of Example Results
Substantial data show that cell-cell signaling plays a major role in the epidemiology and virulence of Xf and that disruption of cell signaling is a promising means of controlling Pierce's disease and other DSF-mediated plant diseases. Strikingly, Xf strains that cannot signal are also not transmissible by nor colonize an efficient insect vector. This result reveals an important and previously unappreciated connection between cell-cell signaling and transmission as well as the requirement for biofilm formation for transmission. These findings provide a basis for a transmission-mediated disease control strategy.
The findings that mutants unable to signal are hypervirulent, but that strains of Xf that overproduce DSF have low virulence and do not move within grape indicated that value of the elucidation of Xf's colonization strategies rather than traits predicted to contribute to virulence based on studies of other plant pathogens. Bacterial strains have been identified that can interfere with Xf signaling. These strains proved very effective as protective agents for grapevines when co-inoculated with Xf. Both positive and negative interference with DSF signaling reduced disease in grape suggesting that signaling is normally finely balanced in the disease process; such a finely balanced process might be readily disrupted. Since in bacteria rpfF is sufficient to encode a synthase capable of DSF production, expression of DSF directly in plants is a attractive approach for disease control. Preliminary results are very encouraging that DSF can be made in plants. Alternatively, the use of various bacteria to express DSF in plants may prove equally effective in altering Xf behavior and hence disease control. And, the observation that large numbers of bacteria can be introduced into grape plants by simple topical applications of cell suspensions in a penetrating surfactant provides an additional disease control strategy that should prove practical for commercial use.
CONCLUSION
Agricultural diseases caused by DSF-producing pathogens can be controlled by blocking cell-cell signaling in organisms susceptible to these pathogens. Blocking cell-cell DSF signaling reduces virulence (in the case of the Xanthomonads) or spread (in the case of Xylella) of the pathogen.
The control may be by several potential methods: 1) by using a DSF-interference (inhibitor or activator) strain as a biocontrol agent; 2) by expressing a DSF-interference gene (degrader, analog, or synthase) in an effective endophytic or epiphytic colonist of the plant; or, 3) by expressing a DSF-interference gene in the plant or rootstock.
While the invention has been primarily described herein with reference to bacterial diseases or plants, some of these methods could also be useful for control of fungi, since some fungal pheromones are structurally related to DSF. In addition, several human pathogens express DSF and may use this signal to control pathogenicity. Therefore, the DSF-interference-based control strategies described herein may have application for control of non-bacterial and/or non-plant pathogens.
Although the foregoing invention has been described in some detail for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims. It should be noted that there are many alternative ways of implementing both the process and compositions of the present invention. Accordingly, the present embodiments are to be considered as illustrative and not restrictive, and the invention is not to be limited to the details given herein.
All references cited herein are incorporated by reference in their entirety for all purposes.

## Claims

1. A transgenic plant cell, the cell comprising:
a promoter active in plant cells, wherein the promoter is operatively linked to a coding sequence of a bacterial rpf gene that encodes a protein that participates in the production of microbial α,β unsaturated fatty acid signal molecules, wherein the rpf gene is from a bacterium of the genus Xanthomonas or Xylella, and wherein the rpf gene encodes RpfB protein or RpfF protein.

2. The transgenic plant cell of claim 1, wherein the plant cell is selected from the group consisting of a genus Brassicaceae cell, a genus Solanaceae cell, a rice cell and a grape cell.

3. The transgenic plant cell of claim 1, wherein the rpf gene is from a bacterium of the genus Xanthomonas.

4. The transgenic plant cell of claim 3, wherein the bacterium is X. campestris pathovar campestris (Xcc), and the plant cell is a Brassicacea cell.

5. The transgenic plant cell of claim 3, wherein the bacterium is X. campestris pathovar vesicatoria (Xcv), and the plant cell is a Solanaceae cell.

6. The transgenic plant cell of claim 3, wherein the bacterium is X. oryzae pathovar oryzae (Xoo), and the plant cell is a rice cell.

7. The transgenic plant cell of claim 1, wherein the rpf gene is from a bacterium of the genus Xylella.

8. The transgenic plant cell of claim 7, wherein the bacterium is X. fastidiosa (XJ), and the plant cell is a grape cell.

9. The transgenic plant cell of claim 1, wherein the rpf gene encodes RpfB protein.

10. The transgenic plant cell of claim 1, wherein the rpf gene encodes RpfF protein.

11. The transgenic plant cell of claim 10, wherein the rpf gene encodes Xf RpfF.

12. The transgenic plant cell of claim 11, wherein the rpf gene encodes SEQ ID NO:1.

13. The transgenic plant cell of claim 10, wherein the rpf gene encodes Xcc RpfF.

14. The transgenic plant cell of claim 13, wherein the rpf gene encodes SEQ ID NO:2.

15. The transgenic plant cell of claim 1, wherein the promoter comprises a constitutive plant promoter.

16. The transgenic plant cell of claim 1 wherein the promoter is selected from the group consisting of the cauliflower mosaic virus 35S promoter and the ubiquitin promoter.

17. A transgenic plant comprising cells according to claim 1.

18. A transgenic plant cell, the cell comprising:
a promoter active in plant cells, wherein the promoter is operatively linked to a coding sequence of a bacterial carA gene or carB gene.

19. The transgenic plant cell of claim 18, wherein the promoter is operatively linked to a coding sequence of a bacterial carA gene.

20. The transgenic plant cell of claim 19, where the carA gene encodes SEQ ID NO:5.

21. The transgenic plant cell of claim 18, wherein the promoter is operatively linked to a coding sequence of a bacterial carB gene.

22. The transgenic plant cell of claim 21, where the carB gene encodes SEQ ID NO:6.

23. A transgenic plant comprising cells according to claim 18.