CA2289751A1 - Gene encoding for systemic acquired resistance in arabidopsis - Google Patents

Abstract

The invention relates to genes which are related to the transmission of Systemic Acquired Resistance (SAR) in plants via the signal transduction pathway. A gene has been characterized as to its ability to encode for a protein which acts upstream from salicylic acid in the signal transduction pathway.

Description

GENE ENCODING FOR SYSTEMIC ACQUIRED
RESISTANCE IN ARABIDOPSIS
TECHNICAL FIELD
This invention relates to a gene specific to the signal transduction pathway for systemic acquired resistance in plants.
CROSS REFERENCE TO RELATED APPLICATION
This application claims the benefit of prior filed copending U. S. Provisional Application No. 60/046,475 filed May 14, 1997.

2 BACKGROUND OF THE INVENTION
The signal transduction pathway in plants that leads to Systemic Acquired Resistance (SAR) involves a number of stages. The first stage is the induction or immunization stage, where initial infection of a leaf by a pathogen to which the S plant is resistant results in a hypersensitive response (HR) leading to the formation of necrotic lesions. There are, however, some examples in which SAR occurs without necrosis. Cameron, et al., "Biologically induced systemic acquired resistance in Arabidopsis thaliana," Plant .I 5:715-725 ( 1994); Kelley, et al., "Physiological and molecular characteristics of elicitin-induced systemic acquired resistance in tobacco," Plant Physiol I 10:365-376 (1996). The expression in the inoculated leaf of a set of pathogenesis-related (PR) protein genes, commonly referred to as SAR genes, occurs during this induction stage in tobacco and cucumber (Kuc', J., Bioscience 32:854-856 (1982); Ward, et al., "Coordinate gene activity in response to agents that induce systemic acquired resistance,"
Plant Cell

3:49-59 (1991))and in Arabidopsis (Uknes, et al., "Acquired resistance in Arabidopsis," Plant Cell 4:645-656 ( 1992)); Uknes, et al., "Biological induction of systemic acquired resistance in Arabidopsis," Mol Plant Microbe Interact 6:692-698 ( 1993)). Accumulation of salicylic acid ( I 0- to 50-fold increase) also occurs during this stage. Yalpani, et al., "Salicylic acid is a systemic signal and an inducer of pathogenesis-related proteins in virus-infected tobacco," Plant Cell 3:809-(1991); Malamy, et al., "Salicylic acid: A likely endogenous signal in the resistance response of tobacco to viral infection," Science 250:1002-1004 (1990);
Metraux, et al., "Increase in salicylic acid at the onset of systemic acquired resistance in cucumber," Science 250:1004-1006 (1990); Uknes, et al., Plant Cell 4:645-656 (1992); Vernooij, et al., "Salicylic acid is not the translocated signal responsible for inducing systemic acquired resistance but is required in signal transduction,"
Plant Cel16:959-965 (1994).
It has been reported that a phloem-mobile signal may move from the necrotic leaf to the rest of the plant to establish SAR. Jennes, A. and Kuc', J., "Graft transmission of systemic resistance of cucumber to anthracnose induced by Colletotrichum lagenarium and tobacco necrosis virus," Phytopathology 69:753-756 ( 1979); Guedes, et al., "Induced systemic resistance to anthracnose in cucumber as influenced by the location of the inducer inoculation with Colletotrichum lagenarium and the onset of flowering and fruiting," Physiol Plant Pathol 17:229-233 (1980); Tuzun, S. and Kuc', J., "Movement of a factor in tobacco infected with Peronospora tabacina Adam which systemically protects against blue mold," Physiol Plant Pathol 26:321-330 (1985).
A study using '80-labeling of tobacco mosaic virus (TMV)-inoculated tobacco leaves revealed that a large percentage of the salicylic acid in the systemic leaves is synthesized in the initially infected leaf, consistent with salicylic acid itself being the mobile SAR signal. Shulaev, et al., "Is salicylic acid a translocated signal of systemic acquired resistance in tobacco," Plant Cell 7:1691-1701 (1995).
However, other studies using cucumber (Rasmussen, et al., "Systemic induction of salicylic acid accumulation in cucumber after inoculation with I'seudomonas syringae pv. syringae, " Plant Physiol 97:1342-1347 ( 1991 )), or grafting experiments with transgenic tobacco containing the bacterial NahG salicylic acid hydroxylase gene (and therefore exhibiting drastically reduced levels of salicylic acid), have led to the suggestion that salicylic acid is not the mobile signal. For example, NahG rootstocks, when inoculated with TMV, are capable of producing a phloem-mobile signal that can be perceived in the wild-type scion, which then becomes resistant to subsequent pathogen challenge. Gaffney, et al., "Requirement of salicylic acid for the induction of systemic acquired resistance," Science 261:754-756 (1993). These results suggest that salicylic acid is not required in the induction/immunization phase of SAR.
The second stage in the signal transduction pathway that leads to SAR is the establishment stage, which involves the perception of the mobile signal in systemic leaves. It is characterized by the expression of the same set of SAR
genes as induced around the primary necrotic lesion, as well as the accumulation of salicylic acid, although to lower levels than are induced in the primary leaf during the induction stage.
The final stage in the signal transduction pathway is the expression stage which occurs when the plant is challenged with a second, normally virulent, y W .: ~.u.~wv~ CA 02289751 1999-11-12

4 pathogen and responds to that pathogen as if it were an avirulent one. Kuc', J., Binsci~nce 32:854-8~6 (I982).
Recent studies using plants with very low levels of salicylic acid due to the e:cpression of the Nul7G salicylic acid hydroxylase gene product (Ga.ffney, et al., 2U .Science 261:754-7~6 (1993); Vernooij, et al., Plant Cell 6:99-965 ( 1994);
Delan,y, et al., Science 266:1247-1250 (1994)) have shown that salicylic acid is required not only in the establishment and expression stages of SAR, but also durinr~ incompatible HR interactions, and even during compatible interactions to limi: disease spread. The Arabidopsis SAR-defective mutants niml (Delanev, et 25 a!., Science 266:1247-1250 ( 19941) and nprl (Cao, et al., ''Characte:iration ef art Arabidopsis mutant that is nonresponsive to inducers of systemic acquired resistance.'' Planr Cell 6:1583-1592 (1994)) also show enhanced disease susceptibility to virulent pathogens, confirnins tile role of salicylic acid in general disease resistance. Mutant cprl plants (constitutive expressor of PR genes}
with 3U elevated levels of SAK gene expression and salicylic acid were shown to be resistant to normally vinzlent pathogens (,bowling, et al., ''A mutation in Arabidopsis that leads to constitutive expression of systemic acquired resistance,"
Plant C'el: 6:I84~-18~ 7 (1994)), pror~ding further genetic evidence in support of the importance of salicylic acid accu.~nulation in disease resistance.
3 S As outlined above, a number of Arabidopsis mutants affected in their ability to establish SAR have been isolated. However, these mutants also have a reduced general defense response, and therefore, the lesion in these plaints is not specific For S AR.
According to the present invention, Lipid Transfer Proteins (LTP'sj have 4U also been identif ed as irrpottant in the establishment of SAR in plants.
Pr eviously, L,TP's have been cloned from a number of plant species. and have been proposed to function in a range of processes including embryogenic development and epicuticulai w-ax depositior: (Pyee and Kolattukudy, "The gene for the major cuticular wax-associated protein and three homologous genes from broccoli , =~5 (Brassica oleracnu) and their expression patterns," Plant .lournal 7: 49-59 (1995); ' Toonen et al., "AtLTP 1 luciferase expression during carrot somatic ernbryogenesis," Plant ,Iournal 12: 1213-122 L (I997)}. In relation AMENDED SHEEj ,.. , -_ . ;., to the present invention, LTPs have already been implicated in plant defense responses. Thus, LTPs from two monocot species were shown to possess antimicrobial activity (Molina et al., "Lipid transfer proteins (nsLTPs) from barley and maize leaves are potent inhibitors of bacterial and fungal plant pathogens,"

5 FEBS'Letters 316: 119-122 (1993)), and transgenic tobacco and Arabidopsis genetically engineered to express a barley LTP were shown to exhibit enhanced tolerance against pathogens (Molina and Garcia-Olmedo, "Enhanced tolerance to bacterial pathogens caused by the transgenic expression of barley lipid transfer protein LTP2," Plant Journal 12: 669-675 ( 1997)). These lipid transfer proteins have not been shown to function in SAR signal transduction.
Previous screens for SAR mutants have bypassed the initial signal production steps involved in SAR by inducing SAR through the plant by spraying with 2,6-dichloroisonicotinic acid (INA), which has been shown to act at the same point as, or even downstream of, salicylic acid in the SAR signal transduction pathway.
Despite reports concerning the possible role of salicylic acid in SAR, and the reported correlation of SAR gene expression with SAR, there is virtually no information available on the molecular mechanisms that result in the induction, transmission and expression of SAR. Thus, there was a need for the isolation and characterization of the genes that are a part of the SAR signal transduction pathway, which encode for proteins which act upstream of the site of action of salicylic acid.
It has now been found that genes which code for proteins which act upstream from salicylic acid in the signal transduction pathway affect SAR in plants, and a method for isolating those genes is herein disclosed. In particular, the dir-1 gene, which encodes a novel lipid transfer protein which is involved in the signal transduction pathway, has now been isolated and characterized. The gene is useful as a reagent for determining the mechanisms involved in establishing SAR in plants, and for genetically engineering disease-resistance in plants. A method for genetic screening of SAR mutants has also been developed which allows for detection of SAR-defective mutants throughout the SAR signal transduction

6 pathway, and more specifically, for those SAR mutants which encode proteins which act upstream of where salicylic acid acts in the SAR signal transduction pathway. Proteins translated from the genes are useful for the production of antibodies to the proteins. The antibodies are useful as reagents in screening plants for examples of the SAR signal transduction genes.
SUMMARY OF THE INVENTION
The present invention is directed to a nucleic acid molecule encoding a protein capable of enhancing systemic acquired resistance in a plant. The protein acts upstream from where salicylic acid acts along the signal transduction pathway.
The invention is also directed to a vector nucleic acid comprising the dir-1 gene.
The invention further comprises plant cells transformed with a vector containing the dir-1 gene.
This invention further comprises a method for conferring systemic acquired resistance to a plant.
The invention also comprises a method for isolating SAR mutants which encode proteins which act upstream of salicylic acid in the signal transduction pathway.
These and other features, aspects and advantages of the present invention will become better understood with reference to the following description and appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 depicts the nucleotide sequence of the dir-1 gene.
Fig. 2 depicts the amino acid sequence of the dir-1 lipid transfer protein.
DETAILED DESCRIPTION
The present invention involves the isolation of genes involved in SAR
signal transduction which encode proteins which act upstream from where salicylic acid acts along the SAR signal transduction pathway. To isolate a gene involved in

7 SAR signal transduction which encodes a protein which acts upstream from where salicylic acid acts along the pathway, the following method is used: Using techniques as described in Example 1, wild type and T-DNA transformed plants are grown and observed for disease symptoms. SAR mutants are isolated, and SAR
competence tests are performed as described in Example 1. The SAR mutants are then tested to determine the position of the mutant gene in relation to the point of salicylic acid action in the pathway as described in Example 1. DNA is isolated from those plants which are homozygous for a SAR mutant gene, and where the gene is found to encode a protein which acts upstream from the site of salicylic acid action along the pathway, probed to isolate a cDNA, and sequenced using techniques as described in Example 2. The isolated genes, such as the dir-1 gene, can be used as vectors to transform plants for improved disease resistance by restoring or enhancing SAR. The plants to which the invention can be applied include the commercially important forage legumes such as but not limited to 1 S alfalfa, large-seeded legumes (grain legumes) such as but not limited to soybeans, beans, and peas, but not limited to solanaceous species such as tobacco, potato and tomato, and monocots such as but not limited to corn, wheat, and rice.
Transformation may be accomplished by any known means including Agrobacterium (Shargool, Peter D. and Ngo, That T., Biotechnological Applications ofPlant Cultures, 1994, pp. 61-76.), biolistic process (Shargool and Ngo at p. 38) or electroporation. Transformed plants may be regenerated by standard protocols well known to those with average skill in the art, such as organogenesis from leaf discs or somatic embryogenesis. The transformed plants may be propagated sexually, or by cell or tissue culture.
The invention includes the expression of the proteins translated from the isolated genes such as the dir-1 gene. These proteins, such as the dir-1 lipid transfer protein (dir-1 LTP), are useful in developing antibodies to the protein.
The antibodies are valuable reagents to be used in the process of screening plants for the expression of isolated genes such as the dir-1 gene. To isolate a protein from a SAR signal transduction pathway gene, the gene product of the isolated gene is expressed as described in Example 5. Antibodies to the protein are

8 produced as described in Example 6. The antibodies to the protein are used as reagents for screening of plants for expression of the isolated gene as described in Example 6.
This invention includes a method for screening SAR-defective mutants throughout the SAR signal transduction pathway, and, more specifically, for screening for those mutants of genes which encode proteins which act upstream from where salicylic acid acts along the pathway. After transforming plants and then observing those plants for disease symptoms as described in Example 1, SAR
mutants are isolated. The SAR mutants are then tested to determine the position of the mutant gene in relation to toe point of salicylic acid action in the pathway as described in Example 1.

Isolation and characterization of dir-1 mutant Plant and Bacterial Growth Conditions Arabidopsis thaliana ecotype Wassilewskija (Ws) and the Feldman T-DNA
transformed seed pools (Arabidopsis Biological Resource Center at Ohio State University) were sterilized, plated on Murashige minimal organics media, then transferred to soil after ten days as previously described. Cameron et al., Plant J
5 : 715-725 ( 1994). Plants were placed in growth chambers at 22 to 24 ° C at a light intensity of 150 ~cE m 2 sec' with a nine hour photoperiod, and grown for 4-5 weeks. Pseudomonas syringae pv tomato (Pst) strains DC3000[pLAFR3]
(virulent) and DC3000[pLAFR3+avrRpt2] (avirulent) (Whalen, et al., "Identification of Pseudomonas syringae pathogens of Arabidopsis and a bacterial locus determining avirulence on both Arabidopsis and soybean," Plant Cell 3:49-59 ( 1991 )) and Ps pv maculicola M4[pLAFR3+avrRpml] (avirulent) (Debener, et al., "Identification and molecular mapping of a single Arabidopsis thaliana locus determining resistance to a phytopathogenic Pseudomonas syringae isolate,"
Plant J 1:289-302 (1991)) were grown as previously described. Cameron et al., Plant.l 5:715-725 ( 1994).

9 Isolation of SAR Mutants Four to five week old T-DNA mutagenized plants (M 1 generation) were given an immunizing inoculation of avirulent Pst ( 10' cfu/ml) on one leaf per plant, then two days later, challenge inoculated on three other leaves with virulent Pst (5x105 cfu/ml). Plants were observed four to five days later and symptoms compared to control avirulent (SAR induced) and virulent treatments performed on ecotype Ws. Those plants showing disease symptoms were selected as potential SAR mutants and allowed to set seed. Between 30 and 40 progeny of the putative SAR mutants were rescreened in the M2 generation as described below.
Genetic Characterization of dir-1 A number of dir-1 M2 plants were backcrossed to wild-type Ws and the resulting F1, F2, and F3 progeny subjected to a SAR competence test (described below) in order to determine whether the dir-1 mutation was recessive or dominant to wild-type, and also if the dir-1 phenotype cosegregated with the T-DNA
(kanamycin resistance marker).
SAR Competence Test Both dir-1 and wild-type Ws plants were subjected to a number of infection treatments. The control treatment (M,M) consisted of a primary inoculation with

10 mM MgCl2 on one leaf per plant, followed two days later by another inoculation with 10 mM MgClz on four other leaves/plant. An avirulent treatment (M,A) consisted of a primary inoculation with 10 mM MgCl2 on one leaf per plant, followed two days later by an avirulent inoculum ( 106 cfu/ml) on four other leaves/plant. A virulent treatment (M,V) consisted of a primary inoculation with 10 mM MgClz on one leaf/plant, followed two days later by a virulent inoculum (106 cfu/ml) on four other leaves per plant. A SAR treatment (A,V) consisted of a primary inoculation with avirulent bacteria ( 106 cfu/ml) on one leaf/plant, followed two days later with a virulent inoculum ( 106 cfu/ml) on four other leaves/plant.

Disease symptoms or their absence were noted for each treatment, and results confirmed by collecting leaf discs from each treatment for determination of in planta bacterial growth.
Quantification of Bacterial Growth in plants Eight leaf discs (4.0 mm in diameter) were harvested from individual plants and macerated in 10 mM MgCIZ. Appropriate dilutions were made in 10 mM
MgCl2 and plated on King's B agar (King, et al., "Two simple media for demonstration of phycocyanin and fluorescein," JLab Clin Med 44:301-307 (1954)) containing 100 mg/1 rifampicin (Sigma Chemical Co., St. Louis, MO.) and 10 20 mg/1 tetracycline (Sigma) to prevent growth of bacteria other than those used as inoculum.
Analysis of Salicylic Acid Levels Salicylic acid was determined by grinding 0.5 g of Arabidopsis leaf tissue (20-30 leaves) in liquid Nz, then extracting in SO% ethanol plus 0.04% 2-mercapto-1 S ethanol, followed by two volumes of 100% ethanol plus 0.04% 2-mercaptoethanol.
The combined ethanol extracts were dried under N2, until approximately 4 ml of water remained, then extracted two times with one volume of ethyl acetate. The ethyl acetate phase was dried under NZ, and the dried material resuspended in ~l of 100% methanol. Salicylic acid was quantified by reverse phase HPLC.
Samples (20 ~.I) were injected onto a 4.6 x 250 mm 5 um C-18 silica column (J.T.
Baker, Inc., Phillipsburg, NJ), equilibrated at ambient temperature with 95%

mM sodium acetate, pH 5.0, 5% methanol, and run isocratically at a flow rate of I.S ml/min. Salicylic acid (retention time approximately 7 min) was detected using a scanning fluorescence detector (Model FP-920; JASCO, Easton, MD) with excitation at 315 nm and emission at 405 nm, as described by Yalpani, et al.
(1991). The identity of salicylic acid peaks from representative samples was confirmed by comparison of the emission spectra with that of an authentic salicylic acid standard. Salicylic acid peaks were also confirmed by digesting a number of previously determined salicylic acid samples with salicylic acid hydroxylase (Sigma)

11 and demonstrating that the salicylic acid peaks disappeared after this treatment.
Total salicylic acid {free salicylic acid + glucose conjugate) was measured by digesting 50 ~I of the methanol extracts with ~3-glucosidase. The loss of salicylic acid during the extraction procedure varied from 36 to 50% and was corrected for during the calculation of salicylic acid levels (ng/g fwt).
RNA Extraction and Analysis RNA was isolated from frozen leaf samples (3-4 leaves/sample) using the small scale procedure of Verwoerd, et al., "A small scale procedure for the rapid isolation of plant RNAs," Nucleic Acids Res 17:23 8 2 ( i 989). Samples of total RNA (5 fig) were separated by electrophoresis through formaldehyde-agarose gels and blotted to Hybond-N nylon membranes (Amersham Corp., Arlington Heights, IL) as described in Sambrook, et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, pp. 7.43-7.46 (1989). Ethidium bromide (40 ~g/ml) was included in the sample buffer in order to confirm equal sample loading under UV light after electrophoresis. Arabidopsis cDNA clones for PR-1, PR-2 and PR-5 (Uknes, et al., Plant Cel14:645-656 (1992)) were 32P-labeled by random priming using a Random Primer Kit (Amersham).
Hybridizations and washes were carried out according to Church, G.M. and Gilbert, W., "Genomic sequencing," Proc Natl Acad Sci USA 81:1991-1995 (1984). A rDNA probe was used to reconfirm that each lane was loaded with similar levels of total RNA.
Isolation and Genetic Characterization of the Arabidopsis dir-1 Mutant Of I 1,000 T-DNA transformed (MI) plant lines screened, 200 putative SAR mutants were recovered. After retesting in the M2 generation, the dir-1 gene proved to be a true SAR defective mutant. In two separate SAR tests of M2 dir-plants, the SAR defective phenotype segregated with a ratio of 3:1 (chi square values p>0.9 and p>0.8) for SAR defective to SAR competent (wild-type) plants.
These results indicate that the dir-1 mutation is dominant to the wild-type SAR
gene function.

12 Three different M2 dir-1 plants were backcrossed to the wild-type Ws background in order to confirm the dominant nature of the dir-1 mutation. The progeny (approximately ten plants) of each cross were tested again for SAR
competence (see Table 1 for data on bacterial growth in planta). All the progeny from each cross were SAR defective, confirming the dominant nature of the dir-mutation.
Table 1: Bacterial Growth in planta (cfu/leaf disc) for dir-1 x Ws F3 progeny' Treatmentz Genotype Control Avirulent Virulent SAR
(M,M) (M,A) (M,V) (A,V)3 (A,V)4 Ws 102 8x105 5x10' 2.2x106 2x106 dir-1 F2-a 2x102 6.4x105 4.6x10' 8.0x10' 3.7x10' dir-1 F2-b Sx lOZ 8.7x105 2.8x10' 3.2x10' -' Eight leaf discs (2 replicates) were collected from each treatment, the average of 2 replicates is presented.
z (M,M) - Primary inoculation - MgCl2; 2nd challenge inoculation -MgClz (M,A)- Primary inoculation - MgClz; 2nd inoculation avirulent Pst; collect inoculated or uninoculated leaves after 2nd challenge.
(M, V) - Primary inoculation - MgCl2; 2nd inoculation - virulent Pst; collect inoculated leaves after 2nd challenge.
(A,V) - Primary inoculation - avirulent Pst; 2nd inoculation - virulent Pst; collect inoculated leaves after 2nd challenge.
3 (A, V), A= PstavrRpt2.
" (A, V), A= PsmavrRpml.
The backcrossed dir-1 lines were allowed to set seed and were plated out (250-500 seeds) on kanamycin-containing media to determine if the SAR
defective phenotype segregated with the kanamycin resistance gene present on the T-DNA
to determine whether the dir-1 mutation was the result of insertion of the T-DNA

13 into the dir-1 gene. Seven backcrossed F2 lines were analyzed. Segregation ratios (kanamycin resistant to kanamycin sensitive) ranged from 7 to 3.3, indicating that there was at one time more than one T-DNA insertion per plant. If there were two functional copies of the T-DNA present in these lines, the ratio of kanamycin resistant to sensitive plants would be 15:1. These F2 Lines (kanamycin resistant and sensitive) were transferred to soil, grown for four weeks, then tested for SAR
competence by measurement of disease symptoms and bacterial growth levels.
Very few of the kanamycin sensitive plants grew when transferred to soil, but for the most part, these plants were SAR competent. Plants from the F2 lines which grew on kanamycin ranged from 100% SAR defective as expected to 80% SAR
defective, indicating that there was a functional kanamycin resistance gene in some of the progeny that was not linked to the dir-1 mutation.
DNA was isolated from both SAR defective, kanamycin resistant F2 plants and from SAR competent, kanamycin sensitive F2 plants. The DNA from each sample was digested with EcoRl and subjected to electrophoresis, blotting, and probing with the plasmid pBR322, which is contained within the T-DNA
construct.
Errampalli, et al., "Embryonic lethals and T-DNA insertional mutagenesis in Arabidopsis," Plant Cell 3:149-157 (1991). A number ofthe F2 SAR defective, kanamycin resistant plants contained bands corresponding to the T-DNA that were not found in the SAR competent, kanamycin sensitive plants. A number of these dir-1 F2 kanamycin resistant plants were backcrossed again to wild-type Ws in order to eliminate any non-linked T-DNA sequences.
A number of the F2 families of the dir-1 x Ws backcross segregated 3:1 for kanamycin resistance. A SAR competence test was performed on individuals from two families that grew on kanamycin-containing media; if the T-DNA
cosegregates with the dir-1 phenotype, all individuals are SAR defective. Each individual in both families (approximately 120) was SAR defective as measured by phenotype and bacterial quantification. These results confirm that the dir-1 mutant phenotype was segregating with kanamycin resistance and the T-DNA insertion. F3 seed from a number of F2 individuals was collected and was used to isolate dir-1 homozygous plants. All kanamycin sensitive F3 plants were homozygous wild-type

14 for dir-1, and expressed SAR. The genetic analysis therefore indicates that the dir-1 mutation is caused by the insertion of a T-DNA, facilitating the strategy of cloning the interrupted gene by polymerase chain reaction (PCR) techniques.
Rescue of the dir-1 Phenotype with INA
S Spraying Arabidopsis with 2,6-dichloroisonicotinic acid (INA), can induce SAR to virulent fungal and bacterial pathogens. Uknes, et al., Mol Plant Microbe Interact 6:692-698 (1993). INA is thought to act at the same point or downstream of salicylic acid in the SAR signal transduction pathway. (Vernooij et al., 1995).
Therefore, dir-1 M2 plants were sprayed with INA and then challenged four days later with 1 O6 cfu/ml of virulent bacteria; the plants were observed three days later and the symptoms recorded. The virulent control treatment (M,V) produced disease symptoms on both Ws and dir-1 plants, whereas the SAR treatment (A,V) produced disease in the dir-1 plants and resistance or SAR in the Ws plants.
Both wild-type Ws and dir-1 plants that were sprayed with INA then challenged with virulent bacteria remained symptomless, indicating that INA induced SAR in dir-plants. This demonstrates that the dir-1 mutation is upstream of INA in the SAR
signal transduction pathway. Substances such as, but not limited to, aspirin (acetylsalicylic acid), methysalicylate, and benzothiazoles which, like INA, mimic the effects of salicylic acid, may also be used.
SAR Gene Expression and Salicylic Acid Levels in the Arabidopsis dir-1 Mutant In preliminary studies of dir-1 x Ws F2 progeny (kanamycin resistant, SAR
defective) following inoculation with avirulent Pst (SAR treatment), expression of the SAR genes PR-l, PR-2 and PR-S was reduced in the expression stage of SAR
as compared to wild-type plants. However, salicylic acid accumulated to similar or higher levels than observed in wild-type plants (Table 2), consistent with earlier findings which show that salicylic acid accumulation is involved not only in SAR, but also in the hypersensitive response (HR) and general defense responses in Arabidopsis. Delaney, et al., Science 266:1247-1250 (1994); Cameron et al., unpublished). Moreover, SAR was not induced in the dir-1 mutant when a different avirulent bacterium (Psm M4 avrRpml) was used as the immunizing primary inoculum, further indicating that the lesion in the dir-1 mutant plants is specific for the SAR signal transduction pathway.
5 Table 2: Free Salicylic Acid (nglgfw) in Ws and dir-1 (F2 progeny) Genotype Control Induction Establishment Expression (M,M-inoc)' (M,A-inoc)2 (M,A-uninoc)3 (A,V-inoc)4 Ws 95 1750 105 1186 dir-I F2-a 93 2195 I76 2500 dir-1 F2-b - 1257 167 1017 10 ' (M,M-inoc) - Primary inoculation - MgClz; 2nd challenge inoculation -MgClz; salicylic acid levels were determined for leaves collected from the 2nd inoculation.
2,3 (M,A)- Primary inoculation - MgClz; 2nd inoculation - avirulent Pst; collect inoc or uninoc leaves after 2nd challenge.

15 4 (A,V) - Primary inoculation - avirulent Pst; 2nd inoculation -virulent Pst; collect inoculated leaves after 2nd challenge.

Isolation and Characterization of the dir-1 Gene Genomic DNA was isolated from leaves of a dir-I homozygous line by standard procedures (Sambrook, et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press (1989). Application of thermal assymetric interlaced (TAIL) PCR (Liu et al., "Efficient isolation and mapping of Arabadopsis thaliana T-DNA insert junctions by thermal assymetric interlaced PCR", Plant Journal 8: 457-463 (1995)) using primers specific for the right border (RB) of the T-DNA resulted in cloned sequences containing the RB but, instead of plant flanking DNA, inverted T-DNA sequences were found, indicating that a rearrangement had occurred within the T-DNA leading to an insert with two left borders (LBs). TAIL-PCR was then performed using primers designed to anneal to

16 the LB of the T-DNA. Two specific sequences were amplified, consisting of LB
and plant genomic flanking sequences.
A combination of PCR and Southern blot analysis with genomic DNA from wild-type and dir-1 Arabidopsis indicated that the two sequences amplified by TAIL-PCR were contiguous in the genome of wild-type Arabidopsis, and were interrupted by the single T-DNA insertion. This insertion was in the 3' untranslated region of a gene, the open reading frame of which had significant sequence identity to that of a non-specific lipid transfer protein (LTP) from Phaseolns vnlgaris.
Sequencing the flanking DNA beyond the T-DNA insertion (away and out from the 3'-end of the LTP) revealed no open reading frame in over 1.1 KB of sequence. Furthermore, the LTP transcript levels were strongly reduced in total RNA preparations from the dir-1 line, suggesting that the T-DNA insertion does indeed interfere with the expression of the dir-1 gene. Probes from the genomic LTP sequence were used to isolate a cDNA encoding the complete LTP using standard methods (Sambrook, et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press (1989)).
DNA Sequence Analysis DNA was sequenced by the Sanger dideoxy sequencing method. (Sanger, et al., Proc Natl Acad ,Sci USA 74:5463-5467 ( 1977)). A Tag Dideoxy Terminator Cycle Sequencing Kit (Applied Biosystems Inc., Foster City, CA) was used according to the manufacturer's protocol. The products were separated on a 6%
polyacrylamide gel and the data processed by an ABI 373A automated DNA
sequencer. All manipulations of raw data generated from the automated system were done on the PC Gene DNA analysis software (Intelligenetics, Mountain View, CA).
The nucleotide sequence is shown in SEQ ID NO: 1.
Evidence indicates that the Arabidopsis LTP encoded by dir-1 is a novel protein involved in signal transduction for establishing systemic plant immunity to disease. Firstly, the nature of the genetic screen targeted mutations in systemic rather than local responses, and this was borne out by the fact that the dir-1

17 mutation does not affect the local response to avirulent pathogens or the level of susceptibility to virulent pathogens in naive plants, as would occur if the dir-1 gene product was in itself antimicrobial. Secondly, inducers of SAR such as INA
rescue the dir-1 phenotype, indicating that dir-1 is upstream of other signal functions such as nprl, mutations which eliminate responsiveness to INA (Cao et al., "Characterization of an Arabidopsis mutant that is nonresponsive to inducers of systemic acquired resistance," Plant Cell 6: 1583-1592 (1994)). Thirdly, systemic induction of pathogenesis-related (PR) proteins and micro-HRs, molecular markers for the establishment of SAR in systemic leaves (Cao et al., "Characterization of an Arabidopsis mutant that is nonresponsive to inducers of systemic acquired resistance," Plant Cell6: 1583-1592 (1994); Alvarez et al., "Reactive oxygen intermediates mediate a systemic signal network in the establishment of plant immunity," Ce(1 92: 773-784 (1998)) is suppressed in dir-1.
The T-DNA insertion in the 3'-untranslated region of dir-1 leads to a 1 S reduction in corresponding transcript levels and therefore reduced levels of functional dir-1 LTP. This, in turn, blocks signal transduction from the primary infected leaf to systemic leaves of the plant. The identification of the dir-1 gene having dir-1 LTP as a translation product indicates that lipid intermediates act as systemic signals in SAR.
The amino acid sequence for the dir-1 LPT was derived using PC Gene software and is given in SEQ ID NO: 2.

Construction of a vector containing the dir-1 gene Fragments containing the full length Arabidopsis dir-1 cDNA, or a functional homolog of dir-I from another plant species, are cloned in a binary vector plasmid, for example in place of the GUS gene in the binary vector plasmid pBI121.1 (R. A. Jefferson, T. A. Kavanagh, M. W. Bevan, "GUS fusions: 13-glucuronidase as a sensitive and versatile gene fusion marker in higher plants," The EMBRO Journal 6: 3901-3907 (1987), under the control of the cauliflower mosaic virus 35S promoter (or any other constitutively expressed or inducible promoter

18 active in plant cells) and the nopaline synthase terminator (or any other transcriptional termination signal active in plant cells). Cloning and vector construction uses standard recombinant DNA techniques in E. coli strains HB 1 or DHSa, performed according to (Sambrook, J., Fritsch, E.F. & Maniatis, T.
( 1989) Molecular Cloning: A Laboratory Manual (2nd Ed), Cold Spring Harbor Laboratory Press, New York). Orientations of the insert are confirmed by restriction mapping, and the 5' ends of each construct confirmed by sequencing from an oligonucleotide primer from the 355 promoter or other promoters know to be used in the art.

Use of dir-1 gene vector in the transformation of plants and creation of transgenic plants Tobacco and alfalfa plants are transformed with A. tumefaciens strain LBA4404 harboring the dir-1 gene construct by leaf disc methods. Transgenic tobacco plants (Nicotiana tabacum cv. Xanthi NF) are generated as described before (Rogers, et al., "Gene transfer in plants: Production of transformed plants using Ti plasmid vectors," Methods Enzymol 118:627-640 ( 1986)), with regeneration under kanamycin selection. Transgenic alfalfa plants are generated from the transformation and regeneration of competent alfalfa cultivar Regen SY
(Bingham, E.T., "Registration of alfalfa hybrid Regen-SY germplasm for tissue culture and transformation research," Crop Sci 31: I 098 ( I 991 )), following a modified version of published procedures (Bingham, et al., "Breeding alfalfa which regenerates from callus tissue in culture," Crop Sci 15:719-721 ( 1975)).
Briefly, leaf discs from young trifoliate leaves are inoculated with a suspension of Agrobacterium harboring the binary construct and incubated on solid BSh plates (Brown, O.C.W. and Atanassov, A., "Role of genetic background in somatic embryogenesis inMedicago," Plant Cell Tiss Organ Cult 4:111-122 (1985)) for four days ( 16 hours light at 24 ° C). The explants are then washed twice with water to remove bacteria and incubated for four more days on new BSh plates.
Explants are then washed twice with water and transferred to selection plates (BSh plates

19 with 100 mg/L timentin (Smith-Kiine Beecham, Philadelphia, PA) and 25 mg/L
kanamycin (Sigma, St. Louis, MO)). Calli and occasional embryos appear after two weeks and are transferred to new selection plates, making sure the calli are spread out. Plates are incubated for another week to allow development of additional embryos. The calli and embryos are then transferred to BS plates (no hormones, but with antibiotics as before). After two weeks, the calli and embryos are transferred to fresh BS plates (with antibiotics). After one to two weeks, individual embryos are cultured on MS plates (Murashige, T., and Skoog, F., "A
revised medium for rapid growth and bioassays with tobacco tissue culture,"
Physiol Plant 15:473-497 (1962)) with antibiotics (50 mg/L timentin and 25 mg/L
kanamycin); plantlets are formed within one to three weeks, occasionally with roots. These are transferred to plastic boxes (Magenta Corp, Chicago, IL) with MS agar media and antibiotics. Plants are maintained on MS media with antibiotics and propagated by cutting. Plants are also transferred to soil in the greenhouse.

Isolation of the dir -1 LTP
The Arabidopsis dir-1 gene product is expressed in E. toll SB221 cells as described (BS Shorrosh and RA Dixon, "Molecular cloning of a putative plant endomembrane protein resembling vertebrate protein disulfide isomerase and phosphatidylinositol-specific phospholipase C," Proc Natl Acad of Sci USA 88:
10941-10945). The expressed protein is resolved by polyacrylamide gel electrophoresis {Laemmli, U.K., "Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227: 680-685 (1970)). The protein band encoded by dir-1 is localized by staining with Coomassie brilliant blue, and regions from 12 gel lanes excised, cut into small fragments, destained to completion with 50% (v/v) isopropanol/3% (w/v) SDS overnight, rinsed with water, vacuum dried, ground in liquid nitrogen, and finally resuspended in phosphate buffered saline.

Production and use of antibodies to the dir-1 LTP protein Antiserum is obtained by immunizing a female New Zealand White rabbit.
The primary immunization contains approximately 30 ~g of the dir-i LTP in 2.7 ml 5 of complete Freund's adjuvant, injected subcutaneously along the back at nine separate sites (300 ml per site). Booster injections containing dir-1 LTP from six excised gel lanes (approximately 15~g) in incomplete Freund's adjuvant are given at 4 and 6 weeks after the primary injections. The serum is stored at -

20°C.
The antibodies are used as a reagent to detect the dir-1 LTP in transgenic 10 plants in a Western blot analysis. Leaf and stem proteins are extracted in 0.2 M
borate buffer, pH 8.8 and subjected to denaturing polyacrylamide gel electrophoresis by standard procedures (Ausubel et al. 1994. Current Protocols in Molecular Biology, John Wiley and Sons, Inc., New York). Proteins are transferred to Immobilon-P membranes (Millipore, Milford, MA) in 25 mM Tris-15 HCI, pH 8.3, 192 mM glycine, and 20% methanol (Towbin et al. 1979.
"Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications," Proc Natl Acad Sci LISA 9:4350-4354).
Membranes are blocked and probed with primary (anti-dir-1) and secondary antibodies, e.g., goat anti-rabbit IgG linked to a detection system such as alkaline 20 phosphatase, in 3% BSA in TTBS (0.1% Tween-20 in Tris-buffered saline) (Ausubel et al., Current Protocols in Molecular Biology ( 1994)).
Although the present invention has been described in considerable detail with reference to certain preferred versions thereof, other versions are possible.
Therefore, the spirit and scope of the appended claims should not be limited to the description of the preferred versions contained herein.

21 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: The Samuel Roberts Noble Foundation, Inc.
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(A) NAME: Dixon, Richard A.
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(ii) TITLE OF INVENTION: Gene Encoding for Systemic Acquired Resistance in Arabidopsis (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS:
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22 (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
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(B) REGISTRATION NUMBER: 31,966 (C) REFERENCE/DOCKET NUMBER: 11137/03402 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 219-981-3315 (B) TELEFAX: 214-981-3400 (2) INFORMATION FOR SEQ ID N0:1:
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(A) LENGTH: 939 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 36..341 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:

GAAAAAGAGA ATG
GAGGAGGATA GCG
AGC
AAG
AAA
GCA

Met Ala Ser Lys Lys Ala AlaMetAla MetMetAla MetIle ValIleMetAla MetLeuVal Asp ThrSerVal AlaIleAsp LeuCys GlyMetSerGln AspGluLeu Asn GluCysLys ProAlaVal SerLys GluAsnProThr SerProSer Gln ProCysCys ThrAlaLeu GlnHis AlaAspPheAla CysLeuCys Gly TyrLysAsn SerProTrp LeuGly SerPheGlyVal AspProGlu Leu

23 Ala Ser Ala Leu Pro Lys Gln Cys Gly Leu Ala Asn Ala Pro Thr Cys (2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 102 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Ala Ser Lys Lys Ala Ala Met Ala Met Met Ala Met Ile Val Ile Met Ala Met Leu Val Asp Thr Ser Val Ala Ile Asp Leu Cys Gly Met Ser Gln Asp Glu Leu Asn Glu Cys Lys Pro Ala Val Ser Lys Glu Asn Pro Thr Ser Pro Ser Gln Pro Cys Cys Thr Ala Leu Gln His Ala Asp Phe Ala Cys Leu Cys Gly Tyr Lys Asn Ser Pro Trp Leu Gly Ser Phe Gly Val Asp Pro Glu Leu Ala Ser Ala Leu Pro Lys Gln Cys Gly Leu Ala Asn Ala Pro Thr Cys

Claims (20)

We claim:

1. A nucleic acid molecule which encodes a lipid transfer protein wherein the encoded lipid transfer protein exhibits SAR signal transducing property, said nucleic acid molecule obtainable by a process comprising the steps of:
a. growing wild type and T-DNA transformed plants and observing for disease symptoms;
b. isolating SAR mutants showing disease symptom;
c. performing SAR competence tests to validate the SAR mutation in said SAR mutants, wherein SAR is compromised in a SAR mutant plant upon first being treated on one leaf of said plant with an avirulent inoculum and later being treated on other leaves of said plant with a virulent inoculum as compared to a wild type plant which exhibits SAR upon first being treated on one leaf with an avirulent inoculum and later being treated on other leaves with a virulent inoculum;
d. testing said SAR mutants to determine the position of the mutant gene in relation to the point of salicylic acid action in the signal transduction pathway, wherein said SAR mutants which are first sprayed with substance that mimics salicylic acid and acts at about the same point as salicylic acid in the SAR
signal transduction pathway, challenged with a virulent inoculum, and then remain symptomless exhibit a SAR mutant gene upstream of the point of salicylic acid action in the signal transduction pathway;
e. isolating DNA from those plants which are homozygous for a SAR
mutant gene, and where the gene is found to encode a protein which acts upstream from the sits of salicylic acid action cloy the signal transduction pathway;
and f. probing said DNA to isolate cDNA.

2. The nucleic acid of claim 1, wherein said nucleic acid can function as a vector for plants, said nucleic acid encoding a lipid transfer protein which exhibits a SAR signal transducing property.

3. The nucleic acid molecule of claim 1 or 2, wherein said encoded lipid transfer protein is depicted in SEQ ID NO:2.

4. The nucleic acid molecule of claim 3, wherein said is purified cDNA.

5. The nucleic acid of claim 1 or 2, wherein said nucleic acid comprises nucleotides 1 to 439 as depicted in SEQ ID NO:1.

6. The nucleic acid of claim 5, wherein said nucleic acid is purified cDNA.

7. A vector comprising the nucleic acid molecule of claim 3.

8. A vector comprising the nucleic acid molecule of claim 5.

9. A transformed plant comprising the vector of claim 7.

10. A transformed plant comprising the vector of claim 8.

11. A plant cell and plants transformed with a recombinant nucleic acid molecule which encodes a lipid transfer protein, wherein said encoded lipid transfer protein exhibits SAR signal transducing property, wherein said recombinant nucleic acid molecule is obtainable by a process comprising the steps of:
a. growing wild type and T-DNA transformed plants and observing for disease symptoms;
b. isolating SAR mutants showing disease symptoms;
c. performing SAR competence tests to validate the SAR mutation in said SAR mutants, wherein SAR is compromised in a SAR mutant plant upon first being treated on one leaf of said plant with an avirulent inoculum and later being treated on other leaves of said plant with a virulent inoculum as compared to a wild type plant which exhibits SAR upon first being treated on one leaf with an avirulent inoculum and later being treated on other leaves with a virulent inoculum;
d. testing sand SAR mutants to determine the position of the mutant gene in relation to the point of salicylic acid action in the signal transduction pathway, wherein said SAR mutants which are first sprayed with substance that mimics salicylic acid and acts at about the same point as salicylic acid in the SAR
signal transduction pathway, challenged with a virulent inoculum. and then remain symptomless exhibit a SAR mutant gene upstream of the point of salicylic acid action in the signal transduction pathway;
e. isolating DNA from those plants which are homozygous or an SAR
mutant gene. and where the gene is found to encode a protein which acts upstream from the site of salicylic acid action alone the signal transduction pathway;
and f. probing said DNA to isolate cDNA.

12. The plant cell and plants according to claim 11, wherein said recombinant nucleic acid molecule is integrated into the plant's chromosomal DNA.

13. The recombinant nucleic acid molecule of claim 11 or 12, wherein said recombinant nucleic acid molecule comprising nucleotides 1 to 439 as depicted in SEQ ID NO:1.

14. A lipid transfer protein encoded by nucleotides 36 - 341 of SEQ 1D
NO:1.

15. A lipid transfer protein having an amino acid sequence as depicted in SEQ ID NO:2, wherein said protein exhibits STIR signal transducing property.

l6. An antibody which specifically birds the protein of Claim 14 or 15.

27

17. A method of conferring systemic acquired resistance to a plant comprising transforming said plant with a vector comprising a nucleic acid molecule encoding a lipid transfer protein exhibiting SAR signal transducing property, said nucleic acid molecule obtainable by a process comprising the steps of:
a. growing wild type and T-DNA transformed plants and observing for disease symptoms;
b. isolating SAR mutants showing disease symptoms;
c. performing SAR competence tests to validate the SAR mutation in said SAR mutants, wherein SAR is compromised in a SAR mutant plant upon first being treated on one leaf of said plant with an avirulent inoculum and later being treated on other leaves of said plant with a virulent inoculum as compared to a wild type plant which exhibits SAR upon first being treated on one leaf with an avirulent inoculum and later being treated on other leaves with a virulent inoculum:
d. testing said SAR mutants to determine the position of the mutant gene in relation to the point of salicylic acid action in the signal transduction pathway, wherein said SAR mutants which are first sprayed with substance that mimics salicylic acid and acts at about the same point as salicylic acid in the SAR
signal transduction pathway, challenged with a virulent inoculum, and then remain symptomless exhibit a SAR mutant gene upstream of the point of salicylic acid action in the signal transduction pathway;
e. isolating DNA from those plants which are homozygous for an SAR
mutant gene, and where the gene is found to encode a protein which acts upstream from the site of salicylic acid action along the signal transduction pathway;
and f. probing said DNA to isolate cDNA.

18. The method of Claim 17, wherein said nucleic acid region comprises nucleotides 1 to 439 of SEQ ID NO:1.

19. The method of Claim 18, wherein said protein has an amino acid sequence as depicted in SEQ ID NO:2.

20. A method of isolating SAR mutants which possess a nucleic acid region which encodes a lipid transfer protein exhibiting SAR signal transducing property, wherein said protein acts upstream from the point of salicylic acid action along the signal transduction pathway:
a. transforming plants with an insertional mutagen;
b. observing for disease symptoms;
c. isolating SAR mutants showing disease symptoms;
d. spraying said SAR mutants with a salicylic acid like substance;
e. observing the phenotypes of said SAR mutants; and f. selecting for said SAR mutants in which the wild type phenotype is restored as a result of said spraying, wherein said SAR mutants in which said wild type phenotype is restored have a nucleic acid region encoding a lipid transfer protein exhibiting SAR signal transducing property, wherein said protein acts upstream from the point of salicylic acid action along the signal transduction pathway.