WO2001007603A1 - Cloning of nps45: a gene encoding a putative translation initiation/stabilization factor - Google Patents

Abstract

An isolated clone of a gene NPS45 encoding a translational initiation factor is disclosed. The sequence of the clone is disclosed, as is the sequence of the resulting translational initiation factor. Since the clone is full length, it can be operable linked to a promoter for up or down regulation. Also disclosed is a method for expressing the clone, and a cell or plant including the clone.

Description

CLONING OF NPS45: A GENE ENCODING A PUTATIVE TRANSLATION INTIATION/STABILIZATION FACTOR

Background

Field of the Invention

The present invention relates to genetic translation initiation/stabilization factors in plants and methods for their regulation and use.

Description of the Related Art

Plants are constantly being challenged by a diverse variety of pathogens including bacteria, fungi, mycoplasma, nematodes, protozoa, parasites and viruses. In spite of this fact only a minority of plants are successfully colonized by pathogens. There are three basic reasons why a pathogen may fail to colonize a potential host plant: ( 1 ) the plant is unable to support the niche requirements of the pathogen, this is referred to as non-host resistance, (2) the plant has preformed chemical or structural barriers which inhibit pathogen ingress, and (3) the plant recognizes the pathogen as non-self and this leads to the induction of an array of defense responses. All of the above interactions result in resistance, and thus, are referred to as incompatible. A compatible interaction is one in which the host plant succumbs to the pathogen and disease ensues. Compatible interactions result when either preformed defenses are inappropriate, there is no recognition of the pathogen or when activated defense responses are ineffective.

Plants contain numerous genes that are involved in their defense against predators. These genes encode proteins that function in pathogen recognition, signal transduction and the activation of defense-associated responses. Genetically controlled disease resistance, or "heritable resistance", is governed by the presence of one, a few or many genes. Heritable resistance to plant pathogens may be classified as either horizontal (general) or vertical (race-specific). Horizontal resistance is controlled by many genes and is equally effective against all isolates of a pathogen. These genes appear to be responsible for controlling the numerous steps of the physiological processes in the plant that lead to the elaboration defense mechanisms. The general defense expressed in horizontal resistance does not necessarily prevent plants from becoming infected, but it does slow down the spread of disease. In contrast to horizontal resistance, vertical resistance is only effective against certain races of a pathogen. The term "race" refers to biotypes of pathogens that vary in their ability to cause disease on specific hosts. Vertical resistance is controlled by only one or a few genes known as disease resistance (R) genes. These genes are critical to host resistance because they control the recognition of elicitor molecules from pathogens and cause the activation of defense responses.

The gene-for-gene interaction is an example of vertical resistance. It was discovered over 80 years ago that genes at different loci could be responsible for resistance to different pathotypes. The importance of this observation was not, however, fully appreciated until the gene-for-gene theory was proposed. It was observed that in the Melampsora /zw^'-flax interaction, physiological variants of the pathogen, called races, produced different reactions in the host. Based on these observations the gene-for-gene theory was proposed, which states that for each gene that confers virulence to the pathogen, there is a corresponding gene in the host that confers resistance. These genes are referred to as avirulence (avr) and resistance (R) genes, respectively. Resistance occurs only when complimentary pairs of R-avr gene pairs are present. The absence or inactivity of either member of the gene pair results in susceptibility. The gene-for-gene theory, has been demonstrated for several host-pathogen interactions including those involving bacteria, fungi, nematodes and viruses.

The "elicitor-receptor" model perhaps best explains the molecular mechanisms underlying the gene-for-gene interaction. This model suggests that avr genes directly or indirectly encode elicitors that serve as ligands for receptors encoded by R genes. The interaction of R-avr genes triggers a signal transduction pathway that activates host defense responses. Characterization of some avr determinants has provided support for the elicitor-receptor model. Analysis of many R genes involved in gene-for-gene resistance has revealed that many encode features indicative of signal recognition and transduction.

To date more than 20 R genes with recognition specificity for defined avr genes have been isolated. These genes confer resistance to various bacterial, fungal and viral pathogens. One R gene, the Mi gene of tomato, has a dual function and is active against both nematodes and aphids. Surprisingly, these genes which protect against pathogens with very diverse lifestyles share many structural motifs. The similarities observed among the sequences of these genes suggest that disease resistance to diverse pathogens may operate through similar molecular pathways. Analysis of these common sequence patterns provides insight into how resistance genes function in pathogen recognition and subsequent defense reactions.

R genes have been divided into five classes based on their common structural motifs. These classes are as follows: (1) genes encoding detoxifying enzymes, (2) serine- threonine kinases, (3 a) intracellular leucine rich repeat (LRR) proteins with leucine zippers (LZ) and a nucleotide binding site (NBS), (3b) intracellular NBS-LRR proteins with a region with similarity to Toll and interleukin- 1 receptor (IL-1R) proteins, (4) intracellular LRR proteins with transmembrane domains (TM), and (5) extracellular LRR proteins with TM's.

The first plant resistance gene to be cloned was the Hml gene of maize. Hml confers resistance to race 1 isolates of the fungal pathogen Cochliobolus carbonum. Race 1 of C. carbonum produces a host-specific toxin called HC toxin. This toxin acts as a pathogenicity factor because race 1 cannot infect susceptible corn varieties if it is not present. Hml confers resistance because it encodes a reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent HC toxin reductase that inactivates HC toxin. The resistance mechanism employed by the Hml gene is unique in that the gene encodes a protein product that is directly active against the pathogenic factor. Thus, unlike the majority of R genes, Hml mediated resistance does not involve a typical R-avr gene interaction.

The Pto gene of tomato was the first resistance gene isolated which conforms to the classic gene-for-gene relationship. Pto confers resistance against Pseudomonas syringae pv. tomato expressing the avirulence gene avrPto. The Pto gene product encodes a serine-threonine protein kinase that shares homology with many mammalian kinases. In animal cells, protein kinases have been found to be key elements in signal transduction and amplification. The Pto kinase also has both autophosphorylating and substrate phosphorylating activity. This is of significance because many cellular processes, including some plant defense responses, are regulated by changes in protein phosphorylation. Additionally, it is believed that this kinase may also play a role in the signal transduction scheme leading to the hypersensitive response.

Other structural themes of interest are the LRR, LZ and NBS domains found in the RPS2 and RPM1 genes of Arabidopsis. RPS2 confers resistance against P. syringae pv. tomato strains with avirulence gene avrRpt2. Its counterpart, RPM1, protects against P. syringae pv. maculicola possessing either avrRpml or avrB genes. The LRR motif is a common feature found in all R genes except Hml and Pto. In yeast, Drosophila and humans, LRR's are mediators of protein-protein interactions. It is likely that plant LRR's may have a similar function in plants facilitating the interaction of R gene products with other proteins that are part of the defense arsenal. It is also significant that several peptide-binding transmembrane receptors possess extracellular LRR's. Thus, it is possible that the products of some LRR-containing R genes may act as a binding domains for ligands produced via avr gene activity. In addition, it has been demonstrated recently that, in some cases, LRR domains are essential for resistance gene function.

Although LRR's are proposed to play a role in the signal transduction pathway, there is no experimental evidence to support a direct interaction between LRR domains and avr proteins. The only evidence of a direct interaction between R and avr proteins has come from studies of the Pto-avrPto system. The direct interaction of Pto-avrPto is somewhat surprising since cytoplasmic kinases do not typically function as receptors. A possible explanation may lie in the finding that Pto requires a LRR protein, Prf, in order to function properly.

NBS domains, which are another prominent feature in R genes, are found in many protein families including the Ras group, ribosomal elongation factors, adenosine triphosphates (ATPases), heterotrimeric GTP binding proteins and adenylate kinases. These proteins are important for many eukaryotic cellular events such as cell growth, differentiation, cytoskeletal organization, vesicle transport and defense. The fact that NBS are found in R gene products suggests that these binding sites are necessary for R gene function. This idea is further supported by the fact that site-specific mutations that alter key residues in the NBS have been found to eliminate the HR-inducing function of RPS2. The class of genes which includes the tobacco N gene, the flax L6 and M genes and RPP5 of Arabidopsis are also characterized by LRR and ΝBS. In addition, they have a region that exhibits homology to cytoplasmic domains of the Drosophila developmental gene Toll and the mammalian immune response gene encoding interleukin-1 receptor (IL- 1R). The Toll receptor is responsible for the release and activation of Rel family transcription factor Dorsal. IL-1R is a receptor for cytokine IL-1 and causes the activation of the Rel-family transcription factor ΝF-_RB. This results in the synthesis of defense and signaling proteins that are involved in immune responses. Thus it is possible that plant resistance genes containing these motifs activate plant defenses by a mechanism similar to those used by IL-1R and Toll.

The Cf genes of tomato (Cf-2, Cf-4, Cf-5 and Cf-9) control resistance to different avr determinants of the leaf mold fungus Cladosporium fulvum. These genes encode proteins with putative transmembrane receptors with large extracytoplasmic LRR domains. Xa21, which confers resistance to the bacterium Xanthomonas oryzae pv. oryzae, encodes a putative transmembrane receptor with an extracellular LRR domain and an intracellular serine-threonine kinase. Both Cf genes and_¥_.27 are theorized to encode membrane bound receptors of kinase-like proteins.

Many of the structural features found in R genes support the elicitor-receptor model of host-pathogen gene interaction. Based on this model, avr products are presumed to be any extracellular molecule or surface feature produced by the pathogen. The host encoded receptor ( R gene) should possess a domain that enables detection of the avr signal. This receptor would likely be extracellular with a membrane-spanning domain that is connected to a protein domain involved in signal transduction. The LRR, ΝBS, kinase, Toll and IL-1R domains of R gene products are found in a number of eukaryotic proteins participating in protein-protein interactions and signal transduction. Thus, most resistance genes are likely to be involved in pathogen recognition and/or signal recognition.

One surprising finding that contradicts the elicitor-receptor model is that not all R-avr gene interactions take place outside of the cell. It was originally anticipated that R proteins would encode extracellular receptor-like proteins, a theory based largely on the fact that most plant pathogens live extracellular lives. While this appears to be the case for some R genes, the majority of R genes encode intracellular proteins. This suggests that the pathogen is detected only after cellular penetration or signal transduction. It has been demonstrated that avrRpt2 and avrB gene products interact with the Arabidopsis RPS2 protein which is localized in the plant cytoplasm. This leads to the conclusion that molecular recognition of the pathogen P. syringae must occur inside the cell in RPS2 and RPM1 mediated resistance.

Plants posses a very complex defense system that utilizes a combination of constitutive and induced defense mechanisms to protect themselves against phytopathogenic microbes. Constitutive defenses, those present prior to pathogen attack, include both structural features which inhibit pathogen ingress and toxic chemical compounds. These constitutive defenses are somewhat effective; however, most pathogens manage to overcome them and cause some degree of infection. Plants thus activate subsequent defense responses, which allow them to reinforce structural barriers and to mount a biochemical attack against predators. These induced defense responses include cell wall reinforcements, the oxidative burst, the hypersensitive response, synthesis of antimicrobial compounds and the production of pathogenesis-related (PR) proteins. With the exception of oxidative responses, all of these responses result from transcriptional activation of defense genes. The temporal and spatial patterns of defense gene activation may vary with some genes being activated rapidly at the initial site of invasion, whereas others are activated more slowly both locally and systemically throughout the plant. Immediate plant defense responses, which are believed to be involved in pathogen recognition and initial signaling events, include ion fluxes, changes in phosphorylation states, the activation of G proteins and protein kinases, the oxidative burst and the hypersensitive response.

One of the most rapid biochemical responses observed following pathogen attack is a dramatic increase in reactive oxygen species (ROS) known as the oxidative burst. The oxidative burst is considered to be the first line of chemical defense and has been observed to occur within minutes of pathogen invasion or elicitor treatment in many plant species. Increases in ROS may serve several functions in the resistance scheme. Elevated levels of ROS may act directly as a toxic substance to kill the pathogen or may activate cell wall cross-linking and lignifϊcation resulting in strengthening of cell walls and confinement of the pathogen to the initial infection site. Additionally, ROS may act in signal transduction as a messenger for the activation of defense genes.

Closely related to the oxidative burst is a programmed cell death event referred to as the hypersensitive response (HR). The HR is a rapid cell death which results in a layer or ring of dead cells forming at and around the site of infection which can often be visualized as a necrotic lesion. It is believed that this process produces a chemical and physical barrier that inhibits the growth and further spread of the pathogen. The HR is the most common response associated with active host resistance. It has been observed in plant-pathogen interactions involving several pathogen types including bacteria, fungi, nematodes and viruses. The molecular mechanisms responsible for HR are not yet fully understood. It has been proposed that the toxicity of active oxygen species may contribute to host cell death and it has been demonstrated that H₂0₂ may play a direct role in triggering the HR. In addition to impairing pathogen ingress, the HR may also act as a catalyst for subsequent defense reactions. Metabolic changes in the cells surrounding the necrotic lesion are believed to contribute to resistance by eliciting defense responses. Interestingly, HR is often associated with the onset of systemic acquired resistance. Thus, it is likely that the induction of defense genes associated with HR not only provides protection against the initial invader but also protects the plant from subsequent infection.

Occurring just after or concurrent with HR is the activation defense mechanisms responsible for the de novo synthesis of a diverse variety of proteins which either directly or indirectly inhibit the growth and development of invading pathogens. This plethora of proteins includes key enzymes of general phenylpropanoid metabolism, such as phenylalanine ammonia-lyase (PAL) and 4-coumarate: CoA ligase (4CL), the many proteins involved in the synthesis of phytoalexins, hydroxyproline rich glycoproteins (HRGP) and PR proteins.

The activation of the phenylpropanoid pathway has been documented in almost every plant-pathogen system analyzed. A diverse group of compounds that function in various aspects of plant defense are elaborated from this pathway. PAL, which is a key enzyme of the phenylpropanoid pathway, is involved in the synthesis of antimicrobial compounds called phytoalexins, the synthesis of materials for cell wall reinforcement such as lignin and the production of the signal molecule salicylic acid. Phytoalexins are a diverse group of low molecular weight, lipophilic, antimicrobial compounds that are synthesized in response to pathogen attack and in response to elicitors. The levels of these compounds increase greatly at the site of infection and accumulate to levels which are toxic to pathogens. Most phytoalexins are toxic to fungi but some are also toxic to bacteria and nematodes.

Lignification is a defense mechanism which renders cell walls more impermeable by making them more resistant to cell wall degrading enzymes and providing a physical barrier to pathogen growth. Lignin is an integral part of secondary cell walls of vascular plants and is resistant to breakdown by many microorganisms. Lignification of cell walls has been observed in a number of plant species following wounding, pathogen attack or elicitor treatment. Lignin deposition occurs earlier and is more pronounced in resistant plant varieties of several plant species including, potato, melon and cabbage when compared to their susceptible counterparts. Although lignin formation has been described primarily as a defense response in fungal pathogenesis, acting by forming a structural barrier that inhibits fungal hyphae penetration, evidence has been provided which suggests it can be active against bacterial pathogens also. Bacterial inhibition may occur as a result of toxic phenolic compounds or activated oxygen production, both of which are associated with lignification. Lignin precursors are phenolic compounds that exhibit anti-bacterial activity.

In studies involving wheat-Puccinia graminis interaction, inhibition of lignification by inhibiting the lignin precursors PAL and cinnamyl alcohol dehydrogenase resulted in the inhibition of the hypersensitive response. This suggests that lignin precursor production is somehow related to the development of the defense response. Perhaps loss of hypersensitive reaction is caused by the lack of lignin precursors or peroxidase generated free radicals, which under normal conditions, are toxic to plant cells.

The increased transcription of genes involved in the phenylpropanoid pathway is accompanied by the activation of an additional set of plant defense genes. Among these are the genes that encode pathogenesis-related proteins (PR proteins). The term PR protein was first used to describe numerous extracellular proteins that accumulated following Tobacco mosaic virus (TMV) infection of susceptible tobacco hosts. It has since been shown that several incompatible plant-pathogen interactions are associated with differential PR gene induction. PR proteins are also induced by several chemical elicitors including xylanase, ozone, ethylene, pectic polysaccharides and salicylic acid. The definition of PR proteins has therefore been broadened to include both intra- and extra- celluiarly localized proteins that accumulate following pathogen attack or elicitor treatment. Some PR proteins such as chitinase and β-l,3-glucanases are very inhibitory toward fungi while others such as lysozyme are active against bacteria. Examples of some PR proteins are PRl, β-1,3, glucanases, chitinases, lysozyme like proteins, osmotin like proteins, cysteine-rich proteins, glycine-rich proteins, proteinase inhibitors and peroxidases. PR proteins are an integral part of plant defense and the constitutive expression of some of these proteins, such as PRl in transgenic tobacco, has led to increased resistance to some fungal pathogens.

The plant defense responses discussed so far generally involve localized responses occurring in cells immediately surrounding the infection site. In addition to these very specific responses, some plants also exhibit a nonspecific "immunity" to subsequent pathogen infection. The phenomenon of induced resistance were first observed over 100 years ago, and in 1933, a review of several papers was published which detailed this phenomenon then known as "physiological acquired immunity." In 1961, sound evidence of induced resistance was provided. Plants were inoculated with TMV and these plants were resistant to subsequent infection by TMV as well as Tobacco Necrosis Virus and some bacteria. This inducible resistance is known as "systemic acquired resistance" (SAR).

Induced systemic resistance (ISR) is a generalized resistance that is developed in response to pathogen infection, elicitors from pathogens and non-pathogens, or treatment with certain chemicals. ISR is a broad-spectrum, long-lasting resistance in non-infected plant tissue that not only provides protection against the inducing agent but also protects against other pathogens including bacteria, fungi and viruses. ISR defense responses are activated in parts of the plant distal to the initial point of infection. It is believed that signals produced at the infection site are sent to distal parts of the plant where they cause activation of defense mechanisms. Initial investigations into the mechanisms of ISR focused primarily on markers that correlated with the occurrence of this event. One early observation was that the accumulation of PR proteins correlates with the onset of ISR. In addition a group of cDNAs were identified which were expressed during ISR. Several of the genes induced during ISR encode proteins with antimicrobial activity such as β-l,3-glucanase and chitinases. Another group of ISR genes encodes proteins related to the antifungal protein thaumatin. Expression of PRla genes in transgenic tobacco plants resulted in increased protection against the fungal pathogens Peronospora tabaci and Phytophthora parasitica. The fact that the products of these genes are antimicrobial in nature and that their over- expression leads to increased resistance indicate that these genes play an important role in the maintenance of ISR.

It is postulated that induced resistance is the result of the combined activation of several PR proteins. While this theory explains the occurrence of the ISR phenomenon, it does not explain how plant cells distal from the initial site of infection are alerted to activate these genes. Plants, unlike animals, do not have a circulatory system so they must rely on transmissible signal molecules that can activate defense mechanisms in cells distal from the point of infection. In order for a molecule to qualify as a systemic signal it must be produced by the plant, increase systemically following pathogen attack or elicitor treatment, move throughout the plant, induce the synthesis of defense-related proteins and chemicals and enhance resistance. Based on these criteria, three putative signal molecules, salicylic acid (SA), jasmonic acid (JA), and ethylene, have been identified.

Salicylic acid is a phenolic acid that accumulates prior to the onset of ISR. The first evidence of salicylic acid as the possible signal came as a result of a demonstration that salicylic acid could induce both resistance and PR protein accumulation in tobacco plants. Further evidence was provided by two additional findings. First, in cucumber and tobacco, salicylic acid accumulates in the phloem following pathogen infection and just prior to the onset of resistance. Second, the exogenous application of salicylic acid induces ISR gene expression and stimulates resistance to a variety of bacterial, fungal and viral pathogens. The most compelling evidence for the importance of salicylic acid in systemic resistance come from experiments using plants transformed with the bacterial nahG gene which encodes salicylate hydroxylase. This enzyme catalyzes the conversion of salicylic acid to an inactive compound catechol. Plants expressing the nahG gene were unable to accumulate salicylic acid in response to pathogen attack and were incapable of activating induced resistance or developing resistance to pathogens.

While it is quite clear that salicylic acid plays a role in resistance, it is not known if salicylic acid is the actual signal involved in ISR or just a precursor that activates the signal. Initial experiments indicated that salicylic acid might function as the long distance signal; however, other studies suggest that this may not be true. It has been demonstrated that removal of the inducer leaf prior to significant increase in salicylic acid levels did not inhibit ISR. Furthermore, transgenic rootstocks from nahG plants were able to induce resistance in non-transgenic scions. These results suggest that salicylic acid is not the translocated signal for SAR. Thus, although salicylic acid may function as a disease resistance signal, it may not be the translocated signal for SAR.

Another signal molecule that has been implicated in plant defense responses is jasmonic acid (JA) and its methyl ester methyl jasmonate (JAMe). The following lines of evidence suggest a role for these jasmonates in defense response: (1) Both jasmonic acid and JAMe induce genes which encode defense related compounds including, proteinase inhibitors, enzymes involved in flavanoid biosynthesis, the antifungal proteins thionin and osmotin and ribosome-inactivating proteins. (2) In several plant species, jasmonates induce the accumulation of a set of proteins known as jasmonate-induced proteins or JIPs. Many of these JIPs exhibit activities of expression patterns suggestive of a function in defense and stress responses. (3) In potato and tomato, jasmonic acid treatment results in local and systemic protection against the fungal pathogen Phytophthora infestans. (4) The Arabidopsis defensin gene PDF 1.2 (Penninckx et al, 1996)and thionin gene TM2.1 are both induced by pathogen invasion and methyl jasmonate. (5) Exogenous application of jasmonic acid induces the secondary metabolic pathways leading to phytoalexin production.

The most compelling evidence for the role of jasmonic acid and JAMe in intracellular signal transduction has come from wounding studies. In tomato plants the proteinase inhibitors, pin 1 and pin2, accumulate in leaves following herbivore attack. As a consequence of this accumulation, leaves containing these inhibitors are protected against the digestive serine proteinases of herbivorous insects. Application of jasmonic acid to tomato leaves induces the synthesis of pinl andpin2 in untreated leaves. In addition there is a rapid increase in endogenous jasmonic acid in response to wounding and elicitor treatment. Based on these findings, it was suggested that jasmonic acid acts as a component of the wound inducible signal transduction pathway that is responsible for the activation of proteinase inhibitor genes. It was later determined that although jasmonic acid plays a role in this pathway, it is not the primary signal. An 18 amino acid polypeptide called systemin is responsible for the wound induced systemic induction of proteinase inhibitors. Systemin, which is released at the wound site, stimulates the release of jasmonic acid in distal tissues. Specifically, the recognition of systemin leads to a rise in the concentration of linoleic acid, which is in turn processed into jasmonic acid via the octadecanoid pathway.

The gaseous plant hormone ethylene has also been proposed as a possible signal molecule. Ethylene regulates plant growth, development and a variety of stress responses. Ethylene production increases in response to pathogen challenge and the exogenous application of ethylene induces defense-related proteins. Enzymes involved in phytoalexin synthesis are also induced by ethylene. While it is quite clear that synthesis of ethylene increases in response to pathogen attack and that ethylene induces defense- related proteins, it has not been conclusively demonstrated that ethylene is essential for ISR. In fact, there is some evidence that suggests that ethylene is not required for ISR. Infected plants treated with inhibitors of ethylene biosynthesis are still capable of producing glucanases and chitinases. Furthermore, Arabidopsis mutants with ethylene insensitivity are still able to initiate defense responses suggesting ethylene is not required for the onset of ISR.

There is now evidence that suggests that ethylene acts in concert with jasmonic acid to induce the wound response in tomato. A rapid production of ethylene is observed upon wounding and the exogenous application of jasmonic acid induces ethylene biosynthesis. Inhibition of ethylene synthesis has a negative affect on the induction of the jasmonic acid pathway. It is proposed that ethylene functions in the wound response by regulating the level of JA. Thus, although ethylene may not be sufficient to induce systemic signaling, it is required for optimal functioning of the jasmonic acid pathway. Characterization of inducible defense responses has revealed a complex network of signaling pathways that function both independently and together to protect plants from predators. The salicylic acid pathway appears to function independently of jasmonic acid and ethylene while systemin, jasmonic acid, JAMe and ethylene act together to regulate wound responses. In addition to wounding, jasmonic acid and ethylene also act as signals in ISR which is elicited by treatment with various strains of root-colonizing biocontrol bacteria. PGPR (plant growth promoting rhizobacteria) mediated ISR functions independently of salicylic acid and PR gene expression, and thus, represents another unique signaling pathway involved in plant defense.

It must be noted that not all inducible defense responses are dependent on the known regulators, salicylic acid, jasmonic acid and ethylene, as evidenced by a recently identified wound- inducible defense gene whose expression is independent of salicylic acid, jasmonic acid and ethylene. There is also evidence that suggests that reactive oxygen intermediates generated during HR may act as signal molecules in systemic resistance. It is very likely that even more endogenous molecular signals will be identified in the future. The identification and characterization of these signals will help to elucidate the precise molecular mechanisms whereby these molecules activate the transcription of defense genes.

The last step in the signal transduction pathway is the transcriptional activation of defense genes. Transcription of eukaryotic cells is controlled by proteins that bind to specific DNA sequence motifs (cis regulatory sequences) located in the promoter and enhancer regions of genes. These proteins, known as transcriptional regulatory proteins or transcription factors, control gene expression by mediating the activity of RNA polymerase during the initiation phase of transcription. Identification of cis and trans acting elements involved in the regulation of defense genes is critical to our understanding of the resistance response.

Several putative elicitor-responsive s-elements have been identified in the promoters of various defense genes. Using in vivo foot-printing experiments with elicitor treated parsley cells, researchers were able to define putative cz^'_. -elements in the PAL-1 gene promoter. The PAL-1 gene possesses three CA-rich elements, designated as boxes P, A and L, that have been identified as sites of DNA-protein interactions (Logemann et al, 1995). It has not yet been conclusively demonstrated that these motifs function in defense gene induction; however, homologous sequences have been identified in the promoter regions of other stress-inducible genes and other genes involved in the synthesis of phenylpropanoid compounds.

The P box has also been identified as a putative cz^'s-acting element in the PAL genes of French bean, Arabidopsis, potato, tomato and rice. A DNA binding protein, BPF- 1 , which is induced in elicitor treated and infected parsley cells, binds to the P box indicating that it may be a transcription factor involved in defense gene regulation.

Another set of binding motifs, the G box (CACGTG) and H box [CCTACC(N)7CT] , are both found in the promoters of genes encoding enzymes involved in phenylpropanoid synthesis such as PAL, 4CL and CHS. The G box functions in the regulation of a diverse group of genes including those involved in responses to developmental signals, abscisic acid, UV radiation, wounding and pathogen attack. The G box is also known to be a general recognition site for the bZIP family of transcription factors.

Salicylic Acid-responsive cz^'s-elements have also been identified in defense genes. A barley β-l,3-glucanase gene contains a lObp SA-responsive element TCA (TCATCTTCTT) that has been observed in over 30 different stress-induced genes. Salicylic acid treatment increases the binding activity of the tobacco nuclear protein TCA-1. TCA-1 interacts with the TCA element; however, the function of TCA- 1 in defense gene action remains unknown. Another salicylic acid responsive element is the AS-1 cis element found in the Cauliflower mosaic virus (CaMV) 35S promoter. This element, which is also found in some plant genes, is recognized by the TGA family of bZIP transcription factors and responds to signals elaborated by both auxin and wounding.

A jasmonate inducible cw-acting promoter sequence of potato has been identified in thej_w_ 2 gene. A 'G box' sequence (CACGTGG) is essential for jasmonic acid response. Another jasmonate inducible sequence element (TGACG) has been identified in the lipoxygenase promoter. This element is a known binding site for bZIP trans-acting factors and is essential for jasmonate induced defense responses. In addition to identifying cis -elements involved in the regulation of defense responses, recent studies have also focused on factors that affect the functional properties of transcriptional regulatory proteins. One mechanism that plays a key role in regulating the activity of transcription factors is reversible phosphorylation. PR- la is a pathogenesis-related gene that is induced in potato following wounding, elicitor treatment or infection with P. infestans. PBF-1 is a nuclear factor that binds to an elicitor responsive element of the PR- 10a promoter. Both wounding and elicitor treatment induce the phosphorylation of PBF-1. Phosphorylation of PBF-1 increases its binding activity for the cis element of PR- 10a gene and this is believed to cause the transcriptional activation of PR- 10a.

The H box binding proteins KAP- 1 and KAP-2 also appear to be mediated by phosphorylation. Dephosphorylation of KAP- 1 and KAP-2 by alkaline phosphatase treatment changed the mobility of KAP-DNA complexes in gel retardation assays. This suggests that the formation of KAP-DNA complexes is mediated by phosphorylation.

G/HBF- 1 is a member of the bZIP transcription factor family that binds to the G box of the bean chsl5 promoter. Activation of chs 15 in response to pathogen attack does not involve increases in transcript or protein levels of G/HBF- 1 , but it does cause a rapid phosphorylation of G/HBF- 1 by a protein serine-kinase. As in other cases, this phosphorylation increases the binding activity of G/HBF- 1 to its cz^'s-acting elements.

For many years transcriptional regulation of genes was assumed to be the primary mechanism for controlling gene expression. Regulation at the level of translation, although acknowledged, was considered to be a novelty. By the mid 1980's, the majority of translation factors involved in protein synthesis had been identified and characterized for many systems and it was believed that there was little else to be learned about translation. It was not until the advent of molecular techniques, including the cloning of cDNA's encoding translation factors, that it was realized that many of these factors function as more than just general mediators of protein synthesis. The result has been a boom in translation research that has revealed a scheme of translational regulation that is far more vast and complicated than ever imagined. In spite of the tremendous progress made in this area, there is still much to be learned about how gene expression is regulated at the level of translation, particularly, in response to pathogenesis and other cellular stresses. There also exists a significant gap in our understanding of translational mechanisms in plant systems.

Eukaryotic cells employ a variety of mechanisms that enable them to mount an effective response to endogenous and exogenous stresses, including control of protein synthesis. Regulation of translation may allow cells to respond quickly to changes in physiological condition, thereby bypassing regulation at the level of transcription which may involve a considerable time lag between perception of the physiological stimulus and the de novo synthesis of response proteins. Translational control is defined as the modulation of efficiency of translation of messenger ribonucleic acid (mR A) and translation-coupled regulation of mRNA stability. Translational regulation may be global, affecting the overall rate of protein synthesis, or specific, translating only a portion of the mRNA in a cell or even a single one. Regulation of specific mRNA's typically occurs in response to developmental cues such as light, embryo development, wounding, heat shock and oxygen deprivation. The down regulation of protein synthesis may serve as a protective measure against the harmful effects of toxic agents and ensure the conservation of resources required for survival under adverse conditions. Likewise, it is possible that over expression of some factors may lead to the enhanced translation of stress response proteins.

The process of mRNA translation can be divided into three phases: initiation, elongation and termination. The goal of the initiation phase is to position the ribosome at the start of the coding region. Successful initiation requires the participation of many eukaryotic factors known as translation initiation/stabilization factors (elFs). The primary function of these proteins is to insure efficient assembly of the "translational apparatus". The initiation process involves the binding of the 40S and then 60S ribosomal subunit to the messenger RNA molecule via a series of molecular interactions that are catalyzed by translation initiation/stabilization factors. This process begins with the association of several initiation factors and the initiator tRNA with the 40S subunit to form the 43 S preinitiation complex. Specifically, 40S subunits are captured for initiation by binding with elFl, elFIA and eIF3. In a separate reaction, eIF2 binds with GTP and the Met- tRNA_f to form a tertiary complex. Factor eIF4C then assists in the binding of the tertiary complex to the 40S subunit, thus, forming the 43 S preinitiation complex. The 43 S preinitiation complex binds to the mRNA at the 5' end via the help of factors eIF4A, eIF4B, eIF4E and eIF4F, and then, migrates in the 5' - 3' direction (in a process called scanning) to the appropriate initiation codon, usually AUG. Once the AUG codon is reached, eIF2 catalyzes the hydrolysis of the bound GTP, an event triggered by eIF5, and the initiation factors are released from the 40S subunit. This release allows the joining of the 60S ribosomal subunit to form an 80S ribosome at the initiation codon. The "translational apparatus" is then ready to begin translation of the coding region. As a consequence of forming the 80S complex, eIF2 bound to GDP is released from the 40S subunit. Initiation factor eIF2B then facilitates the exchange of eIF2 bound GDP for GTP. This results in eIF2-GTP complex that is ready for another cycle of initiation.

Regulation of protein synthesis can occur at either the initiation or elongation stages through changes in either the phosphorylation state or cellular concentration of only a few initiation factors. Many initiation factors are phosphoproteins and it has been demonstrated that there is a direct correlation between the phosphorylation of these factors and the rate of protein synthesis. The initiation stage is the most commonly observed target of physiological control, particularly two steps in the initiation pathway, the binding of the Met-tRNA_f to the 40S ribosomal subunit mediated by eIF2 and binding of the 43 S preinitiation complex to the 5' end of mRNA mediated by eIF4E. Although it is likely that there may be many factors that act in a regulatory manner, to date, most experimental evidence supports the use of eIF2 and eIF4E in translational regulation. The primary regulational mechanism observed for these two factors, thus far, is phosphorylation.

As previously mentioned, eIF2B acts as a recycling factor by catalyzing the guanine-nucleotide exchange on eIF2 to regenerate active GTP bound eIF2. The phosphorylation of eIF2α causes eIF2 to bind eIF2B more tightly, thereby blocking the guanine-nucleotide exchange. The lack of eIF2-GTP-Met-tRNAf complex impairs the initiation process and protein synthesis is inhibited. Three eIF2 kinases respond to cellular stress by phosphorylating the eIF2 subunit eIF2 .

The first eIF2α kinase, a heme-controlled repressor, HCR, was identified in rabbit reticulocytes. This kinase is activated in response to heme deprivation, heavy metals and heat shock proteins. HCR inhibits protein synthesis in erythroid cells when heme levels are insufficient. In the presence of heme, HCR loses its kinase activity towards eIF2α and protein synthesis proceeds unimpaired.

The second eIF2 kinase, PKR, is activated by double-stranded RNA species. PKR plays an important role in the defense of mammalian cells against viral invasion. Double stranded RNA produced during virus replication activates PKR which then phosphorylates eIF2α. This results in a shut down of translation in virus-infected cells, thereby blocking viral propagation (Dever, 1999).

GCN2 is the third eIF2α kinase identified, and it has been extensively characterized in the yeast Saccharomyces cerevisiae. GCN2 is activated in response to amino acid deprivation in the cell. GCN2 phosphorylates eIF2α and this enhances the translation of mRNA encoding the protein GCN4. GCN4 is a transcription factor that activates the expression of genes encoding enzymes involved in the de novo synthesis of amino acids. Thus, the phosphorylation of eIF2α by GCN2 results in the activation of mechanisms which allow the cell to compensate for its nutritional deficiency.

It is not currently known if eIF2 in plants is regulated in the same manner as its mammalian and yeast counterparts. Both mammalian kinases and a wheat germ casein kinase phosphorylate the wheat eIF2α homologue p42. However, it has not yet been demonstrated that phosphorylation by these two kinases has any effect on protein synthesis activity. A plant kinase similar to mammalian PKR has been identified in barley leaves and it phosphorylates the p42 subunit of wheat germ and inhibits protein synthesis. This is the first real evidence that p42 may be regulated in the same manner as other eIF2α factors. Although an equivalent to the recycling factor eIF2B has not yet been identified in plants, there is some evidence that Arabidopsis and rice may both have functional equivalents of this protein.

Another step in the initiation pathway that is important in regulatory control is the binding of the 43S preinitiation complex to the 5' end of mRNA. eIF4E binds the 5' cap of eukaryotic mRNA's and brings the mRNA into a complex with other initiation factors and ribosomes. It is essential for the translation of capped mRNA's. The activity of eIF4E is regulated by phosphorylation and through inhibitory binding proteins (4E-BP's) which mediate its availability for initiation complex assembly. Stresses such as arsenite and anisomycin and the cytokines tumor necrosis factor-alpha and interleukin- 1 beta cause increased phosphorylation of eIF4E by a mitogen and stress activated kinase, MNK1. Phosphorylation of this factor increases its affinity for capped mRNA thereby enhancing protein synthesis. In contrast stresses such as heat shock, sorbitol and H₂0₂ increase the binding of eIF4E to its inhibitor 4E-BP1. This blocks the phosphorylation of eIF4E by MNK1 and decreases the activity of eIF4E. Thus, the activity of eIF4E may be up or down regulated in response to different cellular stress.

In addition to phosphorylation, cellular concentration of translation initiation/stabilization factors may also affect translational regulation. Several studies have examined the effect of initiation factor concentration on translation using overexpression studies. Overexpression of initiation factors eIF2α, eIF4A and eIF5A had no effects on translation in transiently transfected COS cells. Similar results where obtained when elFIA, eIF2β, and eIF2γ were over-expressed. These results suggest that these factors are not rate limiting. In contrast, increased expression of eIF4B was found to inhibit protein synthesis.

One surprising finding was that eIF4E acts as proto-oncogene. Over-expression of this factor leads to changes in cell morphology, loss of growth control and induces tumor formation in mice. Increases in the level of eIF4E mRNA and protein have also been observed in a variety of transformed cell lines and tumors. It is believed that overexpression of eIF4E increases the expression of growth control gene products that are normally translationally repressed. It was observed that there is a more efficient translation of mRNA's encoding growth promoting proteins cyclin Dl and orinithine decarboxylase in cells that overexpress eIF4E. These results suggest that increased levels of eIF4E may be an important component in the development of cancer.

Recently another translation initiation/stabilization factor has been implicated as a possible regulator of translation. elF 1 , which has recently been described as " the Cinderella factor", was originally considered to be a non-essential factor. It was not until the importance of the yeast homologue SUI1 was realized, that elFl was given serious study. The SUIl gene, which was first identified in the yeast Saccharomyces cerevisiae, is a component of eIF3 that is essential for its function. In yeast, SUIl has been shown to act along with eIF2 to allow the Met-tRNA to recognize the initiator codon. In addition to being a component of eIF3, SUIl has also been shown to function singly in the same manner as elFl, and is thus considered to be the yeast equivalent of elFl .

Recent research suggests that SUIl may also act as a general monitor of translational accuracy during both the initiation and elongation stages of translation. A novel allele of SUIl called moft-1 was recently identified. Yeast strains possessing the mofl-1 allele exhibit increases in programmed -1 ribosomal frameshifting. Ribosomal frameshifting is a mechanism used by many viruses in order to form fusion proteins that enhance the viral propagation. Addition of the Mof2/Suil protein reduced ribosomal frameshifting efficiencies of moβ-1 cells to wild type levels. In addition, expression of human elF 1 protein also reduced frameshifting efficiencies indicating that the function of this protein is highly conserved. It was concluded that Mof2/Suil protein functions as a general monitor of translational accuracy.

The up-regulation of elF 1 in response to stress may be a defensive response that allows cells to control the translation of mRNA's whose products might prove harmful to the cell. Over-expression of elFl inhibits the colony forming efficiency of human cancer cells. In addition, it has also been shown that the hepatitis B virus (HBV) X antigen down-regulates the expression of elFl . Tissue analyses of HBV infected patients with hepatocellular carcinoma revealed an absence of elFl gene expression in tumor tissue. This lack of expression was not observed in normal hepatic tissues. These findings are consistent with findings that many RNA viruses can use a single transcript to synthesize multiple proteins via ribosomal frameshifting. It is possible that certain viruses down- regulate the expression of elF 1 , which under normal circumstances, prevents negative frameshifting, as a survival mechanism.

SUI 1 has also been demonstrated to be involved in genotoxic stress responses. A geno toxic stress-inducible transcript, A 121, exhibits high homology to the yeast translation initiation/stabilization factor SUI 1. The genotoxic agents, UV and UV mimicking agents, induce the expression of A121 in hamster cells. The expression of A121 is agent-specific and stresses such as heat shock and serum starvation do not induce this gene. These findings are significant because this is the first time that stress-mediated regulation of elF's has been shown to occur at the mRNA level. Furthermore, they provide support for the role of translational regulation in cellular stress.

The bacterial pathogen Xanthomonas campestris pv. campestris (XCC), causes black rot disease in a wide variety of crucifers. Several members of the cabbage (Brassica oleracea) family including, broccoli, brussel sprouts, cauliflower, collards, rutabaga and turnip are susceptible to this pathogen. XCC was first discovered on in the United States in 1898. The bacteria usually enter the host via the hydathodes but may also gain entry via wounds created by insect injury or other mechanical damage. The XCC-cabbage interaction is quite unique in that XCC does not colonize the living cells of the cabbage leaf, but is instead, confined to the non-living vessel members of the xylem. In natural cabbage infections, XCC typically enters hydathodes located at the leaf tips. In compatible interactions involving susceptible cabbage plants, the pathogen spreads systemically throughout the xylem vessels without any difficulty. Colonization of the vascular system restricts water flow causing a blackening of the veins. The tissue delimited by these veins becomes chlorotic and desiccates, producing a characteristic v- shaped lesion. In contrast, in incompatible interactions, XCC multiplies very actively initially but eventually both lesion development and bacterial multiplication are inhibited. Symptoms in these plants appear as localized necrotic lesions delimited by a dark rim or a small black area at the site of the infected hydathode.

There is very little information on the genetic and molecular mechanisms involved in the B. oleracea-XCC interaction. There is some evidence that the hydathodes are the primary locus of resistance. The growth of the pathogen is restricted to the hydathode region in resistant plants. Furthermore, wound inoculations bypass early host defenses in incompatible interactions. It has been observed that when resistant plants suffer injury, as due to insects or hail, susceptible type lesions are able to develop but do not progress as extensively as those observed in susceptible interactions. The fact that bacteria entering at wound sites are able to establish an initial infection lend support to the belief that the primary mechanism for black rot resistance in cabbage may be located at the hydathodes. It is postulated that in wound inoculations, the bacteria are delivered directly into the xylem vessels, and thus, by-pass any resistance mechanisms at the hydathodes. Although there is a defense response that occurs in resistant plants not naturally infected, the response is delayed. It is not known whether the defense reaction that occurs at the hydathode is the same or different from that occurring at remote infection sites. It is possible that in wound inoculations a signal is produced that triggers hydathodal defense responses. It is also possible that this type of inoculation triggers more general defense reactions that may work in conjunction with those elaborated at the hydathodes.

It is interesting to note that microscopic observations have shown that in compatible reactions the bacteria have little or no contact with the parenchymatous cells of the hydathodal chamber. However, in incompatible reactions there is extensive contact with the parenchymatous cells. It is hypothesized that contact with the living cells, such as xylem and phloem parenchyma and mesophyll cells, somehow elicits the defense response. Perhaps the excitation of these cells initiates the production of lytic substances active against the bacteria such as lysozyme, which hydrolyzes peptidoglycan in bacterial cell walls. Lysozyme activity has been observed in Brassica spp. including turnip and cauliflower. Additionally, some plant chitinases have been found to have lysozyme activity. Chitinase/lysozyme (CHL) activity has been described in bean, pea, turnip and cabbage. In turnip, two isozymes of CHL were reported to accumulate following infection with X. campestris pv. vitans. Research in our laboratory has also shown that CHL is constitutively expressed in resistant cabbage cultivars and expression is upregulated after bacterial colonization. CHL is detected in susceptible cultivars only during the late stages of infection. Lysozyme activity corresponded to CHL accumulation with significantly greater lysozyme activity occurring in both control and inoculated tissues of resistant plants.

Brief Description of the Figures

Figure 1 is a nucleotide sequence of the clone NPS45.

Figure 2 is a nucleotide and predicted amino acid sequence of the open reading frame of NPS45.

Figure 3 shows a southern blot analysis of genomic DNA from varieties of cabbage broccoli hybrids. Resistant varieties LC122 and LC121 are shown in A and B respectively, and susceptible varieties LC67 and LC52 are shown in C and D. Five micrograms DNA was cut with Bam I (lane 1), EcoRl (lane 2), Kpnl (lane 3), anάXhol (lane 4), electrophoresed on a 0.8% agarose gel, transferred to Hybond -N+ membrane and probed with NPS45.

Figure 4 shows a northern blot analysis of the expression patterns of NPS45 in response to the bacterial pathogen Xanthomonas campestris pv. Campestris versus expression when treated with water.

Figure 5 shows a northern blot analysis of the expression patterns of NPS45 in response to treatment with jasmonic acid of a disease resistant and a susceptible cabbage plant.

Figure 6 shows a northern blot analysis of the expression of NPS45 at different stages of plant life in disease resistant and susceptible cabbage plants.

Figure 7 shows a northern blot analysis of the expression of NPS45 in response to heat shock.

Figure 8 shows a northern blot analysis of the expression of NPS45 in response to cold shock.

Summary of the Invention

An isolated clone of a gene NPS45 encoding a translational initiation factor is disclosed. The sequence of the clone is set forth, as is the sequence of the resulting translational initiation/stabilization factor. Since the clone is full length, it can be operably linked to a promoter for up or down regulation. Also disclosed is a method for expressing the clone, and a cell or plant including the clone.

Detailed Description of the Invention

The Brassica oleracea-Xanthomonas campestris pv. campestris (XCC) model system was used to investigate the physiological and molecular aspects of defense gene regulation. Of particular interest was the identification and characterization of transcription and translation factors involved in the differential regulation of defense response genes. As a result, the clone NPS45 has now been isolated. NPS45 hybridizes under high stringency conditions to putative translation initiation/stabilization factors of the elFl/SUIl gene family. As previously stated, elFl/SUIl is one of the least characterized translation initiation/stabilization factors. We have also characterized the expression of the putative elFl/SUIl gene, NPS45, in response to the bacterial pathogen XCC.

NPS45 as shown in Figure 1 is a 560 base pair clone that hybridizes under high stringency conditions to SUIl genes of Arabidopsis, Japanese Willow, corn and rice. Analysis of genomic DNA from B. oleracea varieties revealed that NPS45 is most likely encoded by a small multigene family. This finding is consistent with findings that a multigene family encodes the sbSUIl gene of Japanese willow. It contrasts that of the rice SUIl homolog GOS2 which is encoded by a single gene.

Currently only five putative elFHSUIl genes have been identified in plant species. Three of these genes, those from Arabidopsis, Japanese Willow and rice, are constitutively expressed in various plant tissues. There are no published reports on the specific function of these genes. The primary function of the yeast and human counterparts of the elFl/SUIl genes is to position the translational apparatus at the initiation codon and to dissociate aberrant complexes from the mRNA. Expression of the human elFl protein in yeast strains exhibiting increased -1 ribosomal frame shifting was found to reduce frame shifting efficiencies to wild type levels in the same manner as the yeast Suil protein. This demonstrates that the function of the Suil/elFl protein is highly conserved in nature. The elFl protein has not yet been purified from any plant species. The hybridization of NPS45 to the elFl gene family under high stringency conditions indicated that the NPS45 gene should play a similar role. As it turns out, this is correct. NPS45 participates in the interaction between mRNA and genes activated for expression.

NPS45 was first identified as part of the reaction of plants to stress. Specifically, bacteria pathogen stress was first studied. Isolated clones back-hybridized, and bacterial stress was used to compare the reaction of disease resistant and disease susceptible cabbage. Upon stressing the plants, expression of genes involved in the stress response is initiated. The plants under stress were studied to determine the active genes through study of the mRNA present during stress. Subtraction of the early infected tissue of disease susceptible cabbage mRNA from mRNA of uninfected tissue of the same leaf yielded several clones. Examination of these clones identified a partial clone of NPS45 that led to identification of the full-length gene. More detailed explanations follow.

In order to determine if expression of NPS45 was in any way associated with resistance of 5. oleracea to the bacterial pathogen XCC, the expression patterns of NPS45 in resistant and susceptible varieties of 5. oleracea inoculated with XCC were examined. In the resistant variety HC, NPS45 expression was observed in both control plants and in all XCC inoculated plants. In PB (susceptible cabbage), however, NPS45 was expressed only in the symptomatic tissue, but there was decreased expression at days 1, 2, and 3 in plants inoculated with XCC. The constitutive expression of NPS45 in the resistant variety and its non-constitutive and late-induced expression in the susceptible variety upon XCC infection are suggestive of a role in resistance. Thus controlling the expression of this gene may allow regulation of resistance to different stresses on the plant. The NPS45 gene was also found to participate in the regulation of translation when resistant and susceptible plants were treated with jasmonate. This may indicate a role for jasmonate in defense responses.

NPS45 is a full-length complete gene. As such, it may be operably linked to a promoter by techniques well known to those skilled in the art. Once accomplished, the resulting expression vector can be introduced into a cell, either in vitro or in an organism. The organism is preferably a plant, although the expression vector could also be introduced into other types of organisms. The gene can then be expressed by inducing the promoter in the transfected or transformed host cell. The promoter of this gene can also be used to regulate other genes in the same fashion.

The change in expression of the NPS45 gene in plants inoculated with XCC indicates the participation of NPS45 in bacterial pathogen stress. It was also determined that NPS45 participates in heat and cold stress but not in wound stress.

In heat shocked and heat stressed B. oleracea plants, a general pattern of decreasing NPS45 expression was observed in all varieties assayed. NPS45 expression was affected more during prolonged periods of heat stress than during brief exposure to high temperatures. The pattern of expression observed in our study is similar to that observed for other organisms with the exception being that the response to heat shock occurred more rapidly than that observed in the B. oleracea varieties we tested. In both Drosophila and mammalian cells, the down regulation of eIF4E occurs within minutes of heat shock treatment. The decreased expression observed may be due to the specific down-regulation of NPS45 or, as in other cases, it may result from the shut down of the translational machinery.

There was an interesting pattern of NPS45 expression observed in cold stressed plants. NPS45 levels decrease during early hours of cold stress then increase at later times. In LC 122 (a cabbage/broccoli hybrid from the University of Wisconsin), there was a great decrease in expression at 4, 6 or 18 hours and in LC 67 (another similar hybrid) at 4 and 6 hours. In both varieties the expression observed at later times, 3, 7 and 12 days, is greater that seen in the control. The observed decrease in NPS45 expression may correlate with the stress-induced inhibition of protein synthesis. If this is true, then the increased expression observed at later times may be due to increased translation of cold-response messages. Regardless, it is clear that NPS45 plays a role in the regulation of the cold and heat stress responses.

Our results suggest that NPS45 does not play a role in the wound response. Overall, the expression of NPS45 did not increase or decrease in response to wounding. Currently, there is no information available on the role of other translation initiation/stabilization factors in wounding. However, the level of elongation factor EF- lα has been shown to increase in correlation with increased protein synthesis. It is thus possible that the expression of other factors involved in protein synthesis may also increase in a similar manner.

The participation of NPS45 in bacterial pathogen, heat shock, and cold shock stresses, but not in wound response is consistent with NPS45's role in translation of genes. The production of NPS45 would increase efficiency of translation, conserving plant resources in times of hot or cold stress. Accordingly, regulating NPS45 would enable the control of the heat, cold, and disease susceptibility of the plant. The present invention enables the user to regulate these factors and activate the defense mechanisms of the plant. By facilitating the binding of the mRNA and ribosome, NPS45 promotes the efficient translation of activated genes. The allows fast response of the plant to external stresses by allowing a more efficient, but more importantly a faster translation of genes activated by the plant in response to stresses. The downside risk of having NPS45 expressed constitutively is unknown at present. It may be that a plant that expresses NPS45 constitutively would become overly sensitive, producing an exaggerated response to a transient stress. However, the full implications of this are unknown at present. It has been found that certain chemicals can activate the regulatory region of NPS 45. Chief among these chemicals is jasmonic acid (JA or jasmonate).

EXAMPLES

Isolation of NPS45

The cabbage plants from disease-susceptible cabbage variety (Perfect Ball) were inoculated via petiole injections with a strongly pathogenic strain of Xanthomonas campestris pv. campestris (XCC), genetically modified to bioluminescence (using Vibrio fisherii Lux cassette). In plant location of this XCC strain was precisely monitored using computer assisted charge-coupled-device (CCD) camera to detect light emitted by bioluminescent bacteria. Samples were taken from infected leaf tissue and non-infected tissue of the same plant (No bacteria was detected). Total RNA was isolated from cabbage leaves inoculated by XCC, and from uninfected parts of the leaves. Polyadenylated mRNA was taken from both sources. The first cDNA strands were synthesized using standard procedures. Subtractive hybridization was carried out according to the protocol of the Subtracter Kit (Invitrogen, Carlsbad, CA). Messenger RNA expressed only in the diseased plant tissue was obtained by the subtraction. The subtraction was repeated several times and final material was amplified and stored at 4°C. The clones were grouped based on hybridization patterns, and groups were named as NPS10, 15, 20, 25, 30, 35,40, 45, 50 etc. Some of these clones were randomly selected for northern blotting under high stringency conditions (65 °C) and using Church's solution and a standard protocol for northern blotting. Based on the results of northern blots, some clones were sequenced using a 373 A automated DNA sequencer (Perkin-Elmer), and further studied by hybridization etc. NPS45 (a 340 base pair partial length clone) hybridizes strongly, under highly stringent conditions (hybridization and washing using Church's solution at 65°C), only to mRNA obtained from XCC-infected Brassica tissue. This clone was used in subsequent library screening to obtain the full-length clone. Specifically, the library was plated at 40,000-45,000 pfu/plate by adding an aliquot of library stock to 600 μl of XL 1 -Blue host cells (OD₆oo = 0.5 host cells/plate) and then incubating the mixture for 15 minutes at 37°C. An aliquot of the infected bacteria was added to 6.5 ml of top agar (0.5% NaCl, 0.2% MgS0₄.7H₂0, 0.5% yeast extract, 1% NZ amine (casein hydro lysate), 0.7% agarose, pH 7.5) and then plated on large 150 mm NZY (0.5% NaCl, 0.2% MgS0₄, 0.5% yeast extract, 1% NZ amine (casein hydrolysate), 1.5% agar; pH 7.5) plates. Plates were incubated for approximately 8 hours at 37°C and then chilled for 2 hours at 4°C. Plaques were transferred to Hybond-NX nylon membranes (Amersham, Arlington Heights, IL). The membrane-bound DNA was denatured in 1.5M NaCl, 0.5M NaOH, neutralized in 1.5 M NaCl, 0.5M Tris-HCl pH 8.0 and rinsed in 0.2 M Tris-HCl pH 7.5, 2X SSC. DNA was crosslinked to the membrane using a UV crosslinker at a setting of 120,000 μj of energy. Membranes were stored at 4°C.

Probe Synthesis and Hybridizations

Probes were radiolabeled by incoφorating [α³² P] dCTP using random hexanucleotides, Klenow fragment and the partial length NPS45 cDNA template as described by the Prime-a-Gene labeling system protocol (Promega Corporation, Madison, WI). Following labeling, reactions were passed over a Sephadex G-50 column to remove unincorporated nucleotides. Library membranes were hybridized in 6XSSC, 0.5% SDS, 5X Denhardts solution and lOOμg/ml salmon sperm DNA for 16 hours at 65°C. Washing was done in IX SSC, 0.1% SDS for 15 minutes at room temperature followed by 3 washes in 0.2X SSC, 1% SDS for 30 minutes at 65°C. Membranes were exposed overnight on a phosphorimager screen (Molecular Imaging System, Bio-Rad, Hercules, CA). Blots were analyzed using the Molecular Analyst Software program (Bio-Rad, Hercules, CA).

Secondary Screening and Isolation of Full Length Clone

Positive plaques were selected for secondary screening. Plaques were placed in 1 ml of SM buffer (0.58% NaCl, 0.2% MgSO₄.7H₂0, 50 mM Tris-HCl, pH 7.5, 10% gelatin) supplemented with 20 μl of chloroform and vortexed. An aliquot of this suspension was added to 200 μl of XL-1 Blue host cells and plated on a small 100 mm NZY plate (50-450 plaques per plate). Secondary screening was carried out as previously described. Following this screening plaques of interest were transferred to microcentrifuge tubes containing 500 μl of SM buffer and 20 μl of chloroform. The phage stocks were stored overnight at 4°C. One hundred microliters of each phage stock was added to 200 μl of XL 1 -Blue cells (OD600 = 1.0) and lμl of ExAssist helper phage (> lx 10⁶ pfu/ml). These mixtures were incubated at 37°C for 15 minutes and then 3 ml of 2X YT media (1.6% tryptone, 1% yeast extract, 0.5% NaCl) was added and incubated for 2.5 hours at 37°C with shaking. The tube was heated at 70°C for 20 minutes and then centrifuged at 4000g for 15 minutes at room temperature. The supernatant was placed in a sterile tube. Two hundred microliters of SOLR cells (OD₆oo ⁼ 1-0) was added to two microcentrifuge tubes and 1 μl or 50 μl of phage stock was added to each of these tubes, respectively, and the tubes were incubated for 15 minutes at 37°C. Following incubation, 100 μl of the mixture from each tube was plated on LB plates (1% NaCl, 1% bacto- tryptone, 0.5% yeast extract, 1.5% agar) supplemented with ampicillin (50μg/ml) and incubated overnight at 37°C.

Preparation of NPS45 cDNA

Single colonies were removed from each LB/ampicillin plate and placed in tubes containing 5ml of LB supplemented with 50μg/ml of ampicillin. Cultures were incubated overnight at 37°C with vigorous shaking. A mini-preparation of the plasmid DNA was performed using the alkaline lysis method as described by Maniatis (1982). Specifically, 1.5 ml of each bacterial culture was placed in eppendorf tubes and the tubes were spun at 12,000g for 30 seconds at 4°C. The supernatant was removed and the previous step repeated. The supernatant was again removed and the pellets were resuspended in an ice cold solution of 50 mM glucose, 10 mM EDTA and 25 mM Tris-CL pH. 8.0. Tubes were vortexed vigorously and then stored for 5 minutes at room temperature. Two hundred microliters of a freshly prepared solution of 0.2% NaOH, 1% SDS was then added to the tubes and the contents were mixed by inverting the tubes. Tubes were incubated on ice for 5 minutes and then 150 μl of ice cold potassium acetate (60 ml 5M potassium acetate, 11.5 ml glacial acetic acid, 28.5 ml H₂0) was added. Tubes were vortexed in an inverted position for 10 seconds and then placed on ice for 5 minutes. Following centrifugation at 12,000 g for 5 minutes at 4°C, the supernatant was transferred to a fresh tube and an equal volume of phenol/chloroform was added. Tubes were vortexed and then centrifuged at 12,000 g for 2 minutes at 4°C. The supernatant was transferred to a fresh tube and the DNA was precipitated with 2 volumes of 100% ice cold ethanol. Tubes were placed at - 20°C for 30 minutes and then centrifuged at 12,000 g for 5 minutes at 4°C. The supernatant was removed and pellets were rinsed in ice cold 70%> ethanol, dried and resuspended in 50 μl of TE buffer (10 mM Tris-Cl pH 8.0, 1 mM EDTA pH 8.0). An aliquot of this DNA was restricted with EcoRl and _Y7zoI and restriction reactions were electrophoresed on a 1% agarose gel. Southern blot analysis was performed in order to confirm that the isolated clones cross-hybridized with the 340 base pair NPS45. The gel was blotted onto Hybond N+ membrane (Amersham, Arlington Heights, IL ) and probes were prepared as described previously. Blots were hybridized in 6XSSC, 0.5%) SDS, 5X Denhardts solution and lOOμg/ml salmon sperm DNA for 16 hours at 65°C. Washing was done in IX SSC, 0.1% SDS for 15 minutes at room temperature followed by 3 washes in 0.2X SSC, 1% SDS for 30 minutes at 65°C. Blots were exposed and analyzed as described previously. A clone of 560 base pairs, which cross hybridized with the original 340 base pair NPS45 clone, was chosen for sequencing. The plasmid DNA that yielded this clone was restricted and the restriction digest was electrophoresed on a 1 % agarose gel. The 560 base pair cDNA fragment was extracted from the gel according to the protocol of the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). The cDNA was quantified using a fluorometer and then stored at -20°C for future use in hybridizations.

Plant Materials

Resistant (Hancock) and susceptible (Perfect Ball) cabbage varieties (Shamrock Seed Company, Salinas, CA) were chosen based on symptomology. In addition to this resistant (LC 122, LC 121) and susceptible (LC 67, LC 52) cabbage/broccoli hybrids (obtained from the University of Wisconsin) were also used. These varieties were derived from a cross between a black rot resistant cabbage line Badger Inbred- 16 (BI-16; Brassica oleracea subsp. capitata) and a susceptible inbred broccoli line OSU Cr-7 (Brassica oleracea subsp. italica). The LC 122, 121, 67 and 52 plants are members of the F₃ family generated from this cross. They vary in resistance to XCC with LC 122 being more resistant than LC 121 and LC 67 being more susceptible than LC 52. All plants were grown under greenhouse conditions (18-24°C, 14 hours of light per day) in Jiffy Mix Plus (Jiffy Products of America, Chicago, IL). Plants were fertilized every 7 days with Miracle Grow 15-30-15 according to company's recommendations (Scotts Miracle- Gro Products Inc., USA).

Genomic DNA was isolated from Brassica leaves using the Dneasy Plant Maxi Kit (Qiagen, Valencia, CA). Five micrograms of DNA were cut with EcoRI, BamHl, Kpnl and Xhol (Promega Corporation, Madison, WI), separated on a 0.8% agarose gel and transferred to Hybond N+ membrane (Amersham, Arlington Heights, IL) as described by Sambrook et al. (1989). The membrane was prehybridized for 5 minutes at 65°C in Perfecthyb hybridization buffer (Sigma Chemical Co., St. Louis, MO). The full- length 560 base pair NPS45 template cDNA was prepared as previously described. Probes were radiolabeled by incoφorating [α³² P] dCTP using random hexanucleotides, Klenow fragment and the full length NPS45 cDNA template as described by the Prime-a- Gene labeling system protocol (Promega Coφoration, Madison, WI). After labeling, unincoφorated nucleotides were removed from the reactions using the QIAquick Nucleotide Removal Kit (Qiagen, Valencia, CA). Probes were added to the prehybridization/hybridization buffer and membranes were hybridized for 18 hours at 65°C. Washing was carried out in IX SSC, 0.1%> SDS for five minutes at room temperature and two times in 0.2X SSC, 1% SDS for 20 minutes at 65°C. Blots were exposed and analyzed as previously described.

Bacterial Inoculations

A highly aggressive strain of XCC (FD91L) was used for this study. The inoculum was prepared by growing the bacteria in Media 210 (0.5%> sucrose, 0.8%> casein hydrolysate, 0.4% yeast extract, 0.2% K₂HP0₄ anhydrous, 0.03%> MgS0₄) supplemented with tetracycline at a concentration of 12.5 μg/ml at 28°C for 48 hours. Bacterial concentration was determined by spectrophotometer readings at 600 nm. Plants were petiole inoculated using a syringe and a 23 gauge needle (Becton Dickinson and Company, Franklin Lakes, New Jersey) as described by Shaw and Kado (1988). Resistant (Hancock, LC 122, LC 121) and susceptible (Perfect Ball, LC 67, LC 52) Brassica plants were inoculated at the 6-8 leaf stage with 3 x 10⁸ cfu/ml of XCC strain FD91L. Plants used as a control treatment were inoculated with sterile water. One plant was inoculated for each time period. Four leaves on each plant were inoculated. Samples were collected 1, 2, 3 and 7 days after inoculation. RNA Extraction and Northern Blot Analysis

Total RNA was extracted from Brassica leaves according to the Extract- A-Plant RNA isolation kit protocol (Clonetech, Palo Alto, Ca.). Two grams of leaf tissue were ground in liquid nitrogen and then placed in a solution containing 5 ml of extraction buffer (100 mM LiCl, 100 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS) and 5 ml of phenol preheated to 80°C. The buffer was supplemented with Vi volume of chloroform/isoamyl alcohol (24: 1 v/v) and following centrifugation, the RNA was precipitated in 4 M LiCl at -80°C for one hour. The RNA was pelleted by centrifugation, rinsed with 70% ethanol, dried and resuspended in DEPC (Diethyl Pyrocarbonate, Sigma Chemical Co., St. Louis, MO) treated water. RNA samples were quantified using a spectrophotometer. Fifteen microgram samples of RNA were denatured and electrophoresed on a 1.2% formaldehyde gel in IX MOPS (0.2M 3-N-Moφholino Propane Sulfonic Acid, 0.08 M NaC₂H₃0₂, 10 mM EDTA pH 8.0) buffer pH 7.0. The RNA was transferred to a Hybond N+ membrane (Amersham, Arlington Heights, IL). Probes were radiolabeled by incoφorating [ ³² P] dCTP using random hexanucleotides, Klenow fragment and NPS45 cDNA template as described by the Prime-a-Gene labeling system protocol (Promega Coφoration, Madison, WI). After labeling, unincoφorated nucleotides were removed from the reactions using the QIAquick nucleotide removal kit (Qiagen, Valencia, CA). Blots were hybridized at 65°C for 18 hours in Perfecthyb hybridization buffer (Sigma Chemical Co., St. Louis, MO). Washing was done in 2X SSC, 0.1% SDS for 5 minutes at room temperature followed by two washes in 0.5X SSC, 0.1% SDS for 20 minutes at 65°C and two washes in 0.1X SSC, 0.1% SDS for 20 minutes at 65°C. Membranes were exposed to a phosphorimager screen overnight (Molecular Imager System, Bio-Rad, Hercules, CA). RNA integrity and even loading were determined by visualization of ethidium bromide stained formaldehyde gel.

Analysis of clone NPS45

The full-length 560 base pair clone NPS45 (Genebank Accession No. AF098672) was isolated from a subtracted cDNA library constructed from induced mRNA of Perfect ball variety cabbage (Figure 1). The full-length cDNA contained an open reading frame of 113 amino acids that encodes a protein of predicted 12.5 kDa in weight (Figure 2). The clone NPS45 was sequenced. NPS45 hybridized under high stringency conditions to other genes belonging to SUI 1 translation initiation/stabilization factor family, including from Arabidopsis, Japanese Willow, rice and corn. Based on the high degree of hybridization patterns between NPS45 and SUIl genes it is clear that this gene encodes a translation initiation/stabilization factor of Brassica.

Southern Analysis

Southern analysis was performed to confirm that NPS45 is present in the B. oleracea genome and to determine if it is encoded for by one or more genes. The results are shown in Figure 3. Genomic DNA from Hancock (HC) and Perfect Ball (PB) cabbage varieties was restricted with BamHl and EcoRI. DNA cut with BamHl had one distinctive band while that restricted with EcoRI had four bands. Restriction of DNA from resistant LC varieties (LC 122 and LC 121) with BamHl, Kpnl and Xhol resulted in a pattern of two distinctive bands. In comparison, DNA cut with EcoRI had four and three bands for LC 122 and LC 121, respectively. LC 67 and LC 52 varieties exhibited the same banding pattern with the following exceptions: LC 67 DNA restricted with EcoRI exhibited 2 bands and LC 52 restricted with BamHl had 4 bands. These results suggest that NPS45 is encoded by a multigene family.

Northern Analysis

Northern analysis was performed to assess the expression patterns of NPS45 in response to the bacterial pathogen XCC. In HC, there was an increase in NPS45 expression in XCC treated tissue collected 1, 2, 3 and 7 days after inoculation when compared to water inoculated controls. The results are shown in Figure 4. In PB, expression of NPS45 was not detected until the late stages of the infection. Comparison of XCC treatments for these two varieties revealed that in HC, NPS45 is expressed in all XCC treated plants, whereas in PB, expression was observed only at day 7

Salicylic Acid (SA) and Jasmonic Acid (JA) Treatments Plants were sprayed with a 100 μM solution of salicylic acid (Sigma Chemical Co., St. Louis, MO) at 4 weeks of age. Samples were collected 2, 4, 6, 24 and 48 hours post-treatment. For jasmonic acid treatments, a 100 μM solution (Sigma Chemical Co., St. Louis, MO) was prepared in 100%> ethanol. This solution was applied to 4 week old plants until drip. Plants in the control treatment were sprayed with 100%> ethanol only. Treatment with 100% ethanol did not cause any visible damage to plants. As with other treatments, samples were collected at 2, 4, 6, 24 and 48 hours post-treatment. The results of these tests are shown in Figure 5.

Plant Development and Stress Treatments

In order to determine the expression of NPS45 at different stages of plant growth, plants were grown as described above and samples were taken from 3, 5 and 7 leaf stage plants. Plants at the 6 to 8 leaf stage, approximately 5 weeks of age, were used for all stress treatments. A northern blot analysis of the expression of NPS45 in Hancock and Perfect Ball varieties at 4.5 and 6 weeks post-planting is shown in Figure 6. Leaves were wounded by piercing the leaf panel with a cork borer. Four leaves were chosen and six punctures were made on each side of the leaf panel. Samples were collected at 1, 2, 4, 6, 8, 24 and 48 hours after wounding. Samples were also collected from an unwounded control plant. One plant was used for each time period and all four leaves were collected and pooled together as one sample. For heat shock treatments, plants were placed at 45°C in the dark for 15, 30 or 90 minutes. The results are shown in Figure 7. To determine the affect of prolonged exposure to high temperatures, plants were heat stressed for 3, 6 or 8 hours. Again, one plant was for each time period and samples were collected as previously described. Cold stress treatments were carried out by placing plants at 4°C for varying lengths of time. Samples, four leaves for each plant, were collected at time intervals of 1, 4, 6, 18 and 24 hours and also 3, 7 and 12 days. The results are shown in Figure 8.

Plant Development

To assess whether the expression of NPS45 varied at different stages of plant growth, total RNA was isolated from the leaf tissue of plants at the 3, 5 and 7 leaf stage. Expression of NPS45 was greater in 5 and 7 leaf stage plants in HC, PB, LC 122 and LC 67. In HC there was a decrease in NPS45 expression in 7 leaf stage plants but expression was still greater than that observed in 3-leaf stage plants. Interestingly, NPS45 levels were greater in 5 and 7 leaf stage plants of LC 122 when compared to LC 67.

Stress Treatments

In wounded LC 122 plants, 1, 24 and 48-hour samples had a slight decrease in expression. The decrease observed at the later sampling times (24 and 48 hours) was also observed in LC 52 plants. In general, however, the expression of NPS45 in wounded plants did not differ from that observed in controls.

B. oleracea varieties were incubated at 45°C for varying lengths of time to determine the affect of heat shock and heat stress on NPS45 expression. In both HC and PB plants there was very little difference in the level of mRNA transcripts in heat shocked tissue when compared with the control. There was, however, a slight decrease in NPS45 expression in HC and PB plants exposed to 45°C temperature for prolonged periods of time; 3-8 hours. This decrease was observed earlier in PB, at 3 hours, than in HC, at 6 hours. A similar pattern of expression was observed in LC 122 and LC 67 plants with the decrease in NPS45 expression in heat stressed plants being more pronounced than that observed in HC. In both LC 122 and LC 67 plants there was a great decrease in the expression of NPS45 following 6 or 8 hours of heat stress. The decreased expression of NPS45 appeared to occur earlier in LC 67 where decreased expression was observed following 15 minutes of heat shock.

To determine the affect of low temperatures on NPS45 expression, B. oleracea varieties were placed at 4°C for varying lengths of time. In LC 122 and LC 67 plants expression was first decreased and then increased in cold treated plants. In LC 122 plants, there was very little expression observed following 4, 6 and 18 hours of cold treatment . Likewise in LC 67, there was also a great decrease in expression 4 and 6 hours post- treatment. Expression of NPS45 increased at 24 and 18 hours for LC 122 and LC 67, respectively. Additionally, the expression of this gene at these later times is greater than that observed in the controls.

Our results suggest that NPS45 does not play a role in the wound response. Overall, the expression of NPS45 did not increase or decrease in response to wounding. Currently, there is no information available on the role of translation initiation/stabilization factors in wounding. However, the level of elongation factor EF- lα has been shown to increase in correlation with increased protein synthesis. It is thus possible that the expression of other factors involved in protein synthesis may also increase in a similar manner.

Claims

Claims

1. An isolated DNA sequence that comprises the nucleotide sequence of figure 1.

2. An isolated DNA sequence that comprises a nucleotide sequence which encodes the polypeptide of figure 2.

3. The DNA of claim 2 comprising the nucleotide sequence from base 28 to 369 of the nucleotide sequence of figure 1.

4. An isolated and purified polypeptide encoded by the DNA of claim 2.

5. An isolated and purified polypeptide encoded by the DNA of claim 3 and comprising the amino acid sequence of figure 2.

6. An isolated DNA sequence comprising one of:

(a) nucleic acid molecules which hybridize under highly stringent conditions to a nucleic acid molecule consisting of the nucleotide sequence of figure 1 , and which encode a naturally occurring polypeptide comprising the amino acid sequence of figure 2;

(b) nucleic acid molecules that differ from the nucleic acid molecules of (a) in codon sequence due to the degeneracy of the genetic code, and;

(c) complements of (a) or (b).

7. The isolated DNA sequence of claim 6, wherein the isolated nucleic acid molecule encodes a polypeptide comprising the amino acid sequence of figure 2.

8. An expression vector comprising the isolated DNA sequence of claim 6 operably linked to a promoter.

9. A host cell transformed or transfected with the expression vector of claim

10. A method for expressing nucleic acid molecules comprising operably joining the isolated DNA sequence of claim 6 to a promoter, introducing the joined isolated DNA sequence into a host cell, culturing the host cell under conditions which allow expression of the isolated DNA sequence.

11. A plant including the transformed or transfected host cell of claim 9.

12. The method of claim 10 wherein said cell is contained within a plant.

13. The method of claim 10, wherein, the isolated DNA sequence of claim 6 encodes a polypeptide comprising the amino acid sequence as set forth in figure 2.

14. The method of claim 10, wherein the isolated DNA sequence of claim 6 comprises the DNA sequence of figure 1.

15. A method for expressing a polypeptide comprising: operably joining the isolated DNA sequence of claim 1 to a promoter, wherein the isolated nucleic acid molecule encodes a polypeptide; introducing the isolated DNA sequence into a host cell; and culturing the host cell under conditions which allow expression of the polypeptide.

16. The method of claim 15, further comprising isolating the encoded polypeptide.

17. The method of claim 16, wherein the encoded polypeptide comprises the amino acid sequence as set forth in figure 2.

18. The method of claim 16, wherein said polypeptide consists of the amino acid sequence as set forth in figure 2.