DNA binding proteins that interact with NPR1

The present invention provides nucleic acids that encode bZIP polypeptides that are capable of interacting with NPR1. The present invention also provides for bZIP polypeptides that are capable of interacting with NPR1 as well as transgenic plants comprising a nucleic acids that encode bZIP polypeptides that are capable of interacting with NPR1. Also provided by the present invention is a method for enhancing resistance of plants to pathogens by introducing a recombinant expression cassette comprising a plant promoter operably linked to a polynucleotide sequence encoding a bZIP polypeptide that interacts with NPR1. The present invention also provides for a method of identifying additional bZIP polypeptides that interact with NPR1.

BACKGROUND OF THE INVENTION 
Plant pathogens cause hundreds of millions of dollars in damage to crops in 
the United States annually and cause significantly more damage worldwide. 
Traditional plant breeding techniques have developed some plants that 
resist specific pathogens, but these techniques are limited to genetic 
transfer within breeding species and can be plagued with the difficulty of 
introducing non-agronomic traits that are linked to pathogen resistance. 
Furthermore, traditional breeding has focused on resistance to specific 
pathogens rather than general, or systemic, resistance to a wide spectrum 
of pathogens. Therefore, an important goal in agriculture is to identify 
genetic components that enable plants to resist pathogens, thereby 
allowing for the development of systemically resistant plants through 
biotechnology. 
Systemic acquired resistance (SAR) is a general plant resistance response 
that can be induced during a local infection by an avirulent pathogen. 
While early studies of SAR were conducted using tobacco mosaic virus (TMV) 
and its Solanaceous hosts (see, e.g., Ross, A. F. Virology 14: 340-358 
(1961)), SAR has been demonstrated in many plant species and shown to be 
effective against not only viruses, but also bacterial and fungal 
pathogens (see, e.g., Kuc, J. Bioscience 32:854-860 (1982) and Ryals, et 
al., Plant Cell 8:1809-1819 (1996)). A necessary signal for SAR induction 
is salicylic acid (SA); plants that fail to accumulate SA due to the 
expression of an SA-oxidizing enzyme salicylate hydroxylase are impaired 
in SAR (Gaffney, T., et al. Science 261:754-756 (1993)). Conversely, an 
elevation in the endogenous level of SA or exogenous application of SA or 
its synthetic analogs, such as 2,6-dichloroisonicotinic acid (INA), not 
only results in an enhanced, broad-spectrum resistance but also stimulates 
concerted expression of a battery of genes known as pathogenesis-related 
(PR) genes (see, e.g., Malamy, J., et al. Science 250:1002-1004 (1990); 
Metraux, J.-P., et al. Science 250:1004-1006 (1990); Rasmussen, J. B., et 
al. Plant Physiol 97:1342-1347 (1991); Yalpani, N., et al. Plant Cell 
3:809-818 (1991); White, R. F. Virology 99:410-412 (1979); Metraux, J.-P., 
et al. (1991) In Advances in Molecular Genetics of Plant-Microbe 
Interactions, eds. Hennecke, H. & Verma, D. P. S. (Kluwer Academic, 
Dordrechet, The Netherlands), Vol. 1, pp. 432-439; Ward et al. Plant Cell 
3:1085-1094 (1991); and Uknes et al. Plant Cell 4:645-656 (1992)). PR 
genes may play direct roles in conferring resistance because their 
expression coincides with the onset of SAR and some of the PR genes encode 
enzymes with antimicrobial activities (see, e.g., Ward et al. Plant Cell 
3:1085-1094 (1991); and Uknes et al. Plant Cell 4:645-656 (1992)). 
Therefore, understanding the regulation of PR gene expression has been a 
focal point of research in plant disease resistance. 
Two classes of A. thaliana mutants with altered PR gene expression have 
been identified. One class constitutively expresses PR genes while the 
other class is impaired in the SA- or INA-induced PR gene expression 
(Lawton, K., et al. (1993) in Mechanisms of Defense Responses in Plants, 
eds. Fritig, B. & Legrand, M. (Kluwer Academic, Dordrecht, The 
Netherlands), pp. 422-432; Bowling, S. A., et al. Plant Cell 6:1845-1857 
(1994); Bowling, S. A., et al. Plant Cell 9:1573-1584 (1997); Clarke, J. 
D., et al. Plant Cell 10:57-569 (1998); Cao, H., et al. Plant Cell 
6:583-1592 (1994); Delaney, T. P., et al. Proc. Natl. Acad. Sci. USA 
92:602-6606 (1995); Glazebrook, J., et al., Genetics 143, 973-982 (1996); 
Shah, J., et al. Mol. Plant-Microbe. Interact. 10:69-78 (1997)). 
Interestingly, from the second class of mutants only one genetic locus, 
NPR1 (also known as NIMI), has been identified. NPR1 has been shown to be 
a key component of the SA-regulated PR gene expression and disease 
resistance because nprl mutants fail to express PR1, PR2, and PR5 and 
display enhanced susceptibility to infection even after treatment with SA 
or INA. Furthermore, transgenic plants overexpressing NPR1 display a more 
dramatic induction of PR genes during an infection and show complete 
resistance to Pseudomonas syringae pv. maculicola 4326 and Peronospora 
parasitica Noco, two very different pathogens that are virulent on 
wild-type A. thaliana plants (Cao, H., et al. Proc. Natl. Acad. Sci. USA 
95:6531-6536 (1998)). 
Sequence analysis of NPR1 does not reveal any obvious homology to known 
transcription factors (see, e.g., Cao, H., et al. Cell 88:57-63 (1997) and 
Ryals, J. A., et al. Plant Cell 9:425-439 (1997)). Therefore, it is 
unlikely that NPR1 is directly involved in transactivating the promoters 
of PR genes. However, NPR1 contains at least four ankyrin repeats, which 
are found in proteins with very diverse biological functions and are 
involved in protein-protein interactions (Bork, P. (1993) Proteins: 
Structure, Function, and Genetics 17, 363-374. Michaely, P., and Bennet, 
V. (1992) Trends in Cell Biology 2:127-129.). The functional importance of 
the ankyrin repeat domain has been demonstrated by mutations found in the 
npr1-1 and the nim1-2 alleles where the highly conserved histidine 
residues in the third and the second ankyrn repeats, respectively, are 
changed to a tyrosine. Because these conserved histidine residues are 
involved in the formation of hydrogen bonds which are crucial in 
stabilizing the three dimensional structure of the ankyrin-repeat domain 
(Gorina, S. & Pavletich, N. P. Science 274, 1001-1005 (1996)), npr1-1 and 
nim1-2 mutations may cause disruption in the local structure within the 
ankyrin-repeat domain and abolish its ability to interact with other 
proteins. These data suggest that NPR1 probably exerts its regulatory 
function by interacting with other proteins. 
SA-responsive promoter elements such as the as-1 element in the 35S 
promoter of cauliflower mosaic virus (CaMV) and the ocs and nos elements 
in opine synthase promoters of Agrobacterium have previously been 
identified and characterized (Lam, E., et al. Proc. Natl. Acad. Sci. USA 
86, 7890-7894 (1989); Qin, X-F., et al. Plant Cell 6, 863-874 (1994) ; and 
Ellis, J. G., et al. Plant J. 4, 433-443 (1993)). The as-1 element has 
been shown to bind to a tobacco transcription factor, SARP (salicylic acid 
response protein), which is immunologically related to the tobacco protein 
TGA1a, a bZIP transcription factor (Jupin, I. & Chua N-H. (1996) EMBO J. 
15:5679-5689). In A. thaliana, there are at least six bZIP genes 
identified that have homology to the tobacco TGA transcription factor 
(Kawata, T., et al. Nucleic Acids Res. 20, 1141 (1992); Xiang, C., et al. 
Plant Mol. Biol. 34, 403-415 (1997); Zhang, B., et al. Plant J. 4,711-716 
(1993 ); Schindler, U., et al., A. R. Plant Cell 4, 1309-1319 (1992); 
Miao, Z. H., et al. Plant Mol. Biol. 25, 1-11 (1994); and Lam, E. & Lam, 
Y. K.-P. Nucleic Acids Res. 23, 3778-3785 (1995)). These TAG transcription 
factors have been shown to have different affinities for the as-1 element 
in in vitro binding assays (Lam, E. & Lam, Y. K.-P. Nucleic Acids Res. 23, 
3778-3785 (1995)). While strong, binding of AHBP-1b requires two tandem 
copies of the TGACG motif present in the as-1 element, binding of TGA6 
appears to be unaffected by the number of motifs because a single copy 
seems to be sufficient. Other bZIP genes have been identified in wheat 
(see, e.g., Foley et al., Plant J. 3(5):669-79 (1993) and tobacco (see, 
e.g., Fromm, et al, Mol. Gen. Genet. 229:181-88 (1991) and Katagiri et 
al., Nature 340:727-30 (1989)). Although functions have been postulated 
for some of the above-described bZIP gene products, little is known about 
the regulation of bZIP gene products and there are no reports of their 
interaction with any other proteins associated with plant disease 
resistance. 
Recently, the promoter of the A. thaliana PR-1 gene has been thoroughly 
analyzed using deletion and linker scanning mutagenesis performed in 
transgenic plants as well as in vivo footprinting analysis (Lebel, E., et 
al. Plant J. 16, 223-234 (1998)). Through these analyses, two 
INA-responsive elements have been defined. One element at -610 is similar 
to a recognition sequence for the transcription factor NF-.kappa.B, while 
the other promoter element around residue -640 contains a CGTCA motif (the 
complementary sequence is TGACG) which is present in the as-1 element. The 
CGTCA motif was shown by linker-scanning mutagenesis to be essential for 
both SA and INA induction of PR-1 gene expression. 
In spite of the recent progress in understanding the genetic control of 
plant resistance to pathogens, little progress has been reported in the 
identification and analysis of genes interacting with key regulators of 
pathogen resistance such as NPR1. Characterization of such genes would 
allow for the genetic engineering of plants with a variety of desirable 
traits. The present invention addresses these and other needs. 
SUMMARY OF THE INVENTION 
This invention relates to bZIP polynucleotides and polypeptides that bind 
to NPR1, as well as the use of such polynucleotides and polypeptides to 
generate transgenic plants that have enhanced resistance to plant 
pathogens. For example, the invention provides molecular strategies for 
enhancing resistance to pathogens by modulating bZIP expression and 
activity using bZIP gene constructs. Thus, by regulating bZIP expression, 
transgenic plants with increased or decreased pathogen resistance can be 
produced. 
The present invention provides for isolated nucleic acids comprising a 
polynucleotide that is at least 95% identical over at least 500 base pairs 
to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9. The 
isolated nucleic acids can be derived, for instance, from rice or tomato. 
In preferred embodiments, the polynucleotide encodes SEQ ID NO:2, SEQ ID 
NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10. 
The invention also provides for transgenic plants comprising a recombinant 
expression cassette comprising a plant promoter operably linked to a 
polynucleotide that is at least 95% identical over at least 500 base pairs 
to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9 and 
that encodes a polypeptide capable of interacting with NPR1. 
A promoter can be operably linked to the polynucleotide sequence. The plant 
promoters used in the invention are not critical to the invention. The 
promoter can be constitutive, inducible or specific for an organ, tissue, 
or cell. 
The present invention also provides for methods of enhancing plant 
resistance to pathogens by introducing into a plant a recombinant 
expression cassette with a plant promoter operably linked to a bZIP 
polynucleotide sequence and selecting for a plant with enhanced 
resistance. In one embodiment, the method is performed on rice or tomato 
plants. In another embodiment, plant resistance is determined by measuring 
for increased expression from a defense-related promoter. In preferred 
embodiments of this method, the bZIP polynucleotide encodes SEQ ID NO:2, 
SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10. In other preferred 
embodiments, the bZIP polynucleotide of the method are SEQ ID NO:1, SEQ ID 
NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9. 
The invention also provides a method of identifying other polypeptides that 
are involved in plant disease resistance. The method comprises identifying 
a polypeptide that binds to NPR1 and determining whether the identified 
polypeptide modulates disease resistance. In some embodiments, the 
determining step comprises determining whether the identified polypeptide 
modulates expression of at least one defense-related gene. Preferably the 
defense related gene encodes a pathogenesis-related protein. In another 
preferred embodiment, the determining step comprises introducing into a 
plant a polynucleotide that encodes the identified polypeptide. In yet 
other embodiments, the polypeptide used in the method is derived from 
tomato or rice. 
Definitions 
The phrase "nucleic acid sequence" refers to a single or double-stranded 
polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to 
the 3' end. It includes chromosomal DNA, self-replicating plasmids, 
infectious polymers of DNA or RNA and DNA or RNA that performs a primarily 
structural role. 
The term "promoter" refers to regions or sequence located upstream and/or 
downstream from the start of transcription and which are involved in 
recognition and binding of RNA polymerase and other proteins to initiate 
transcription. A "plant promoter" is a promoter capable of initiating 
transcription in plant cells. 
The term "plant" includes whole plants, shoot vegetative organs/structures 
(e.g. leaves, stems and tubers), roots, flowers and floral 
organs/structures (e.g. bracts, sepals, petals, stamens, carpels, anthers 
and ovules), seed (including embryo, endosperm, and seed coat) and fruit 
(the mature ovary), plant tissue (e.g. vascular tissue, ground tissue, and 
the like) and cells (e.g. guard cells, egg cells, trichornes and the 
like), and progeny of same. The class of plants that can be used in the 
method of the invention is generally as broad as the class of higher and 
lower plants amenable to transformation techniques, including angiosperms 
(monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and 
multicellular algae. It includes plants of a variety of ploidy levels, 
including aneuploid, polyploid, diploid, haploid and hemizygous. 
"Increased or enhanced bZIP activity or expression of the bZIP gene" refers 
to an augmented change in bZIP activity. Examples of such increased 
activity or expression include the following. BZIP activity or expression 
of the bZIP gene is increased above the level of that in wild-type, 
non-transgenic control plants (i.e. the quantity of bZIP activity or 
expression of the bZIP gene is increased). BZIP activity or expression of 
the bZIP gene is in an organ, tissue or cell where it is not normally 
detected in wild-type, non-transgenic control plants (i.e. spatial 
distribution of bZIP activity or expression of the bZIP gene is 
increased). BZIP activity or expression is increased when bZIP activity or 
expression of the bZIP gene is present in an organ, tissue or cell for a 
longer period than in a wild-type, non-transgenic controls (i.e. duration 
of bZIP activity or expression of the bZIP gene is increased). 
A polynucleotide sequence is "heterologous to" an organism or a second 
polynucleotide sequence if it originates from a foreign species, or, if 
from the same species, is modified from its original form. For example, a 
promoter operably linked to a heterologous coding sequence refers to a 
coding sequence from a species different from that from which the promoter 
was derived, or, if from the same species, a coding sequence which is not 
naturally associated with the promoter (e.g. a genetically engineered 
coding sequence or an allele from a different ecotype or variety). 
"Recombinant" refers to a human manipulated polynucleotide or a copy or 
complement of a human manipulated polynucleotide. For instance, a 
recombinant expression cassette comprising a promoter operably linked to a 
second polynucleotide may include a promoter that is heterologous to the 
second polynucleotide as the result of human manipulation (e.g., by 
methods described in Sambrook et al., Molecular Cloning-A Laboratory 
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 
(1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & 
Sons, Inc. (1994-1998)) of an isolated nucleic acid comprising the 
expression cassette. In another example, a recombinant expression cassette 
may comprise polynucleotides combined in such a way that the 
polynucleotides are extremely unlikely to be found in nature. For 
instance, human manipulated restriction sites or plasmid vector sequences 
may flank or separate the promoter from the second polynucleotide. One of 
skill will recognize that polynucleotides can be manipulated in many ways 
and are not limited to the examples above. 
A polynucleotide "exogenous to" an individual plant is a polynucleotide 
which is introduced into the plant by any means other than by a sexual 
cross. Examples of means by which this can be accomplished are described 
below, and include Agrobacterium-mediated transformation, biolistic 
methods, electroporation, and the like. Such a plant containing the 
exogenous nucleic acid is referred to here as a T.sub.1 (e.g. in 
Arabidopsis by vacuum infiltration) or R.sub.0 (for plants regenerated 
from transformed cells in vitro) generation transgenic plant. Transgenic 
plants that arise from sexual cross or by selfing are descendants of such 
a plant. 
A "bZIP nucleic acid" or "bZIP polynucleotide sequence" of the invention is 
a subsequence or full length polynucleotide sequence (e.g., SEQ ID NO:1, 
SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9) which, encodes a 
polypeptide (e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ 
ID NO:10) or its complement. BZIP polypeptides of the invention are 
characterized by the presence of a leucine zipper domain and a basic 
domain (see, e.g., Baranger, Curr Opin. Chem. Biol. 2(1):18-23 (1998); 
Xiang, et al., Plant Mol. Biol. 34:403-15 (1997); and Ramachandran et al., 
Curr. Opin. Genet. Dev. 4:642-46 (1994)). The leucine zipper domain can 
function to dimerize with other proteins (see, e.g., Vinson et al., 
Science 246:911-16 (1989)). BZIP proteins can therefore form hetero- and 
homodimers, allowing for different DNA or protein specificities. The basic 
domain of a bZIP protein is the region of the polypeptide that binds to 
DNA. BZIP polynucleotides of the invention are preferably at least 95% 
identical, more preferably at least 97% identical and most preferably at 
least 99% identical over at least 500 base pairs to SEQ ID NO:1, SEQ ID 
NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9. 
BZIP polypeptides of the invention interact with NPR1. This interaction can 
be by direct protein-protein interaction. Alternatively, the interaction 
may be indirect. For instance, a third polypeptide may bind to both the 
bZIP polypeptide and NPR1, thereby keeping all three polypeptides in 
proximity to one another. Protein interactions can be measured by a number 
of different methods that are known to those of ordinary skill in the art. 
Examples of systems to measure such interaction include, inter alia, the 
yeast two-hybrid system (see, e.g., Fields, Nature 340(6230):245-6 (1989) 
and Finley, R. L. JR & Brent R. (1996) in DNA Cloning--Expression Systems: 
A Practical Approach, eds. Glover D. & Hames B. D (Oxford University 
Press, Oxford, England), pp. 169-203), immunoprecipitation (see, e.g., 
Current Protocols in Molecular Biology Volumes 2, .sctn.10.16, John Wiley 
& Sons, Inc. (1994-1998)), or the use of various sequence tags (e.g., TAG, 
His, etc.) that allow for the isolation of a polypeptide under 
nondenaturing conditions (see, e.g., Chen & Hai Gene 139(1):73-5 (1994); 
and Current Protocols in Molecular Biology Volumes 2, .sctn..sctn.10.1A-B, 
10.15, John Wiley & Sons, Inc. (1994-1998)). These methods can therefore 
be used to identify proteins that interact with NPR1. One of ordinary 
skill in the art will recognize that protein-protein interactions can be 
measured by any number of methods and are not limited to those described 
above. 
In the case of both expression of transgenes and inhibition of endogenous 
genes (e.g., by antisense, or co-suppression) one of skill will recognize 
that the inserted polynucleotide sequence need not be identical, but may 
be only "substantially identical" to a sequence of the gene from which it 
was derived. As explained below, these substantially identical variants 
are specifically covered by the term bZIP nucleic acid. 
In the case where the inserted polynucleotide sequence is transcribed and 
translated to produce a functional polypeptide, one of skill will 
recognize that because of codon degeneracy a number of polynucleotide 
sequences will encode the same polypeptide. These variants are 
specifically covered by the terms "bZIP nucleic acid", "bZIP 
polynucleotide" and their equivalents. In addition, the terms specifically 
include those full length sequences substantially identical (determined as 
described below) with an bZIP polynucleotide sequence and that encode 
proteins that retain the function of the bZIP polypeptide (e.g., resulting 
from conservative substitutions of amino acids in the bZIP polypeptide). 
An "NPR1 polynucleotide sequence" of the invention is a subsequence or full 
length polynucleotide sequence which, encodes a polypeptide or its 
complement, as described, for instance, by Cao, H., et al. Cell 
88(1):57-63 (1997) and Ryals, J. A., et al. Plant Cell 9:425-439 (1997) 
(see, also, GenBank Accession Nos. U76707 and U87794). One of ordinary 
skill in the art will recognize that NPR1 also encompasses any orthologs 
of NPR1 from different plant species. For instance, as described below, 
the tomato NPR1 gene is an NPR1 ortholog and therefore is an "NPR1 
polynucleotide" of the invention. 
A "salicylic acid (SA) responsive element" is a cis-acting DNA sequence 
that modulates a particular gene's expression in response to exposure of a 
plant or part of a plant to salicylic acid, salicylate, or any chemical 
capable of inducing systemic acquired resistance in plants e.g., 
acetyl-salicylic acid or 2,6 dichloroisonicotinic acid (see, e.g., Ward, 
et al. Plant Cell 3:1085-1094 (1991)). Known SA-responsive promoter 
elements include, for example, the as-1 element in the 35S promoter of 
cauliflower mosaic virus (CaMV) and the ocs-1 and nos elements in opine 
synthase promoters of Agrobacterium (see, e.g., Lam, E., et al. Proc. 
Natl. Acad. Sci. USA 86:7890-7894 (1989); Qin, X-F., et al. Plant Cell 
6:863-874 (1994); and Ellis, J. G., et al. Plant J. 4:433-443 (1993)). A 
motif that arises in many SA responsive elements is TGACG (or its 
complement CGTCA). For example, the CaMV 35S promoter contains the as-1 
element, identified as a 21 bp DNA sequence that comprises the sequence 
TGACG (see, e.g., Qin, et al., Plant Cell 6:863-74 (1994); Xiang, C., et 
al. Plant Mol. Biol. 34:403-415 (1997)). The hex element, another SA 
responsive element, also comprises the TGACG sequence (Xiang, C., et al. 
Plant Mol. Biol. 34:403-415 (1997); Katagiri et al., Nature 340:727-30 
(1989)). Similarly, the nos-1 element of the CaMV 35S promoter also 
comprises the TGACG motif and plays a role in controlling gene expression 
in response to exposure of a plant to salicylic acid (see, e.g., Lam, et 
al. J Biol Chem 265(17):9909-13 (1990) and Kim et al. Plant Mol Biol 
124(1):105-17 (1994)). Another SA responsive element is the ocs element of 
the CaMV 35S promoter. The ocs consensus element is TGACGTAAGCGCTTAGTCA 
(SEQ ID NO:17) (see, e.g., Zhang, B., et al. Plant J. 4, 711-716 (1993)) 
and represents a family of ocs elements found in higher plants (see, e.g., 
Bouchez, et al., EMBO J. 8:4197-204 (1989)). One potential binding site of 
bZIP proteins within the ocs sequence comprises the motif(A)CGTCA (see, 
e.g., Lebel etal., Plant J. 16(2):223-233 (1998) and Rushton et al., EMBO 
J. 15:5690-5700 (1996)). Additional examples of SA-responsive elements are 
discussed, inter alia, in Stange, et al. Plant J. 11(6): 1315-24 (1997); 
Horvath et al. Mol Plant Microbe Interact. 11(9):895-905 (1998); and 
Ulmasov, et al. Plant Mol Biol. 26(4):1055-64 (1994). 
A "defense-related" gene refers to a plant nucleic acid whose expression 
increases when a plant is contacted with, or infected by, a pathogen. One 
of ordinary skill in the art will recognize that defense-related genes 
encode polypeptides with diverse predicted functions. Typically, 
defense-related genes encode polypeptides that may inhibit or destroy an 
invading pathogen or pathogen product. For instance, several 
defense-related genes are predicted to encode chitinases that can destroy 
the cell wall of invading fungal pathogens. The expression of many defense 
related genes is also induced or increased upon exposure to salicylic acid 
(SA) or SA analogs such as 2,6-dichloroisonicotinic acid (INA). Examples 
of defense-related genes include genes that encode pathogenesis-related 
proteins (PR) (see, e.g., Ward, et al. Plant Cell 3:1085-1094 (1991); 
Reuber et al. Plant J. 16(4):473-85 (1998); Heitz T, et al. Mol Gen Genet 
245(2):246-54 (1994); and Stintzi et al. Biochimie 75(8):687-706 (1993)). 
Pathogenesis proteins include several proteins with homology to proteins 
with functions including .beta.-1,3-glucanase and chitinases. Not all PR 
proteins have predicted functions (e.g., PR-1). Other examples of defense 
related genes include those encoding phytoalexins, phenylalanine ammonia 
lyase (), proteinases, peroxidases, glutathoine-S transferases, 
lipoxygenases, as well as genes such as the rice Pir7b gene (see, e.g., 
Waspi, et al., Eur. J. Biochem. 254(1):32-7 (1998)), and SRG1 and SRG2 
from alfalfa (see, e.g., Truesdell & Dickman, Plant Mol Biol. 33(4):737-43 
(1997)), which were identified by the characteristic of induction upon 
pathogen infection. See, e.g., Hunt, et al. Gene 179(1):89-95 (1996); 
Fluhr, et al. Biochem Soc Symp 60:131-41 (1994); Bowles, et al. Annu Rev 
Biochem 59:873-907 (1990); Glazebrook, et al. Annu Rev Genet 31:547-69 
(1997); Dixon, R., et al., Adv Genet. 28:165-234 (1990); Ward, E., et al., 
Plant Cell 3:1085-1094 (1991); Lawton, et al., Plant J. 10:71-82 (1996); 
and Friedrich, L., et al., Plant J. 10:61-70 (1996) for additional 
examples and reviews of defense-related genes. 
"Pathogens" include, but are not limited to, viruses, bacteria, nematodes, 
fungi or insects (see, e.g., Agrios, Plant Pathology (Academic Press, San 
Diego, Calif.) 1988). 
Two nucleic acid sequences or polypeptides are said to be "identical" if 
the sequence of nucleotides or amino acid residues, respectively, in the 
two sequences is the same when aligned for maximum correspondence as 
described below. The terms "identical" or percent "identity," in the 
context of two or more nucleic acids or polypeptide sequences, refer to 
two or more sequences or subsequences that are the same or have a 
specified percentage of amino acid residues or nucleotides that are the 
same, when compared and aligned for maximum correspondence over a 
comparison window, as measured using one of the following sequence 
comparison algorithms or by manual alignment and visual inspection. When 
percentage of sequence identity is used in reference to proteins or 
peptides, it is recognized that residue positions that are not identical 
often differ by conservative amino acid substitutions, where amino acids 
residues are substituted for other amino acid residues with similar 
chemical properties (e.g., charge or hydrophobicity) and therefore do not 
change the functional properties of the molecule. Where sequences differ 
in conservative substitutions, the percent sequence identity may be 
adjusted upwards to correct for the conservative nature of the 
substitution. Means for making this adjustment are well known to those of 
skill in the art. Typically this involves scoring a conservative 
substitution as a partial rather than a full mismatch, thereby increasing 
the percentage sequence identity. Thus, for example, where an identical 
amino acid is given a score of 1 and a non-conservative substitution is 
given a score of zero, a conservative substitution is given a score 
between zero and 1. The scoring of conservative substitutions is 
calculated according to, e.g., the algorithm of Meyers & Miller, Computer 
Applic. Biol. Sci. 4:11-17 (1988) e.g., as implemented in the program 
PC/GENE (Intelligenetics, Mountain View, Calif., U.S.A.). 
The phrase "substantially identical," in the context of two nucleic acids 
or polypeptides, refers to sequences or subsequences that have at least 
60%, preferably 80%, most preferably 90-95% nucleotide or amino acid 
residue identity when aligned for maximum correspondence over a comparison 
window as measured using one of the following sequence comparison 
algorithms or by manual alignment and visual inspection. This definition 
also refers to the complement of a test sequence, which has substantial 
sequence or subsequence complementarity when the test sequence has 
substantial identity to a reference sequence. 
One of skill in the art will recognize that two polypeptides can also be 
"substantially identical" if the two polypeptides are immunologically 
similar. Thus, overall protein structure may be similar while the primary 
structure of the two polypeptides display significant variation. Therefore 
a method to measure whether two polypeptides are substantially identical 
involves measuring the binding of monoclonal or polyclonal antibodies to 
each polypeptide. Two polypeptides are substantially identical if the 
antibodies specific for a first polypeptide bind to a second polypeptide 
with an affinity of at least one third of the affinity for the first 
polypeptide. 
For sequence comparison, typically one sequence acts as a reference 
sequence, to which test sequences are compared. When using a sequence 
comparison algorithm, test and reference sequences are entered into a 
computer, subsequence coordinates are designated, if necessary, and 
sequence algorithm program parameters are designated. Default program 
parameters can be used, or alternative parameters can be designated. The 
sequence comparison algorithm then calculates the percent sequence 
identities for the test sequences relative to the reference sequence, 
based on the program parameters. 
A "comparison window", as used herein, includes reference to a segment of 
any one of the number of contiguous positions selected from the group 
consisting of from 20 to 600, usually about 50 to about 200, more usually 
about 100 to about 150 in which a sequence may be compared to a reference 
sequence of the same number of contiguous positions after the two 
sequences are optimally aligned. Methods of alignment of sequences for 
comparison are well-known in the art. Optimal alignment of sequences for 
comparison can be conducted, e.g., by the local homology algorithm of 
Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment 
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the 
search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. 
USA 85:2444 (1988), by computerized implementations of these algorithms 
(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software 
Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by 
manual alignment and visual inspection. 
One example of a useful algorithm is PILEUP. PILEUP creates a multiple 
sequence alignment from a group of related sequences using progressive, 
pairwise alignments to show relationship and percent sequence identity. It 
also plots a tree or dendogram showing the clustering relationships used 
to create the alignment. PILEUP uses a simplification of the progressive 
alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987). The 
method used is similar to the method described by Higgins &.Sharp, CABIOS 
5:151-153 (1989). The program can align up to 300 sequences, each of a 
maximum length of 5,000 nucleotides or amino acids. The multiple alignment 
procedure begins with the pairwise alignment of the two most similar 
sequences, producing a cluster of two aligned sequences. This cluster is 
then aligned to the next most related sequence or cluster of aligned 
sequences. Two clusters of sequences are aligned by a simple extension of 
the pairwise alignment of two individual sequences. The final alignment is 
achieved by a series of progressive, painvise alignments. The program is 
run by designating specific sequences and their amino acid or nucleotide 
coordinates for regions of sequence comparison and by designating the 
program parameters. For example, a reference sequence can be compared to 
other test sequences to determine the percent sequence identity 
relationship using the following parameters: default gap weight (3.00), 
default gap length weight (0.10), and weighted end gaps. 
Another example of algorithm that is suitable for determining percent 
sequence identity and sequence similarity is the BLAST algorithm, which is 
described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software 
for performing BLAST analyses is publicly available through the National 
Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This 
algorithm involves first identifying high scoring sequence pairs (HSPs) by 
identifying short words of length W in the query sequence, which either 
match or satisfy some positive-valued threshold score T when aligned with 
a word of the same length in a database sequence. T is referred to as the 
neighborhood word score threshold (Altschul et al, supra). These initial 
neighborhood word hits act as seeds for initiating searches to find longer 
HSPs containing them. The word hits are extended in both directions along 
each sequence for as far as the cumulative alignment score can be 
increased. Extension of the word hits in each direction are halted when: 
the cumulative alignment score falls off by the quantity X from its 
maximum achieved value; the cumulative score goes to zero or below, due to 
the accumulation of one or more negative-scoring residue alignments; or 
the end of either sequence is reached. The BLAST algorithm parameters W, 
T, and X determine the sensitivity and speed of the alignment. The BLAST 
program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring 
matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 
(1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a 
comparison of both strands. 
The BLAST algorithm also performs a statistical analysis of the similarity 
between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. 
Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the 
BLAST algorithm is the smallest sum probability (P(N)), which provides an 
indication of the probability by which a match between two nucleotide or 
amino acid sequences would occur by chance. For example, a nucleic acid is 
considered similar to a reference sequence if the smallest sum probability 
in a comparison of the test nucleic acid to the reference nucleic acid is 
less than about 0.2, more preferably less than about 0.01, and most 
preferably less than about 0.001. 
"Conservatively modified variants" applies to both amino acid and nucleic 
acid sequences. With respect to particular nucleic acid sequences, 
conservatively modified variants refers to those nucleic acids which 
encode identical or essentially identical amino acid sequences, or where 
the nucleic acid does not encode an amino acid sequence, to essentially 
identical sequences. Because of the degeneracy of the genetic code, a 
large number of functionally identical nucleic acids encode any given 
protein. For instance, the codons GCA, GCC, GCG and GCU all encode the 
amino acid alanine. Thus, at every position where an alanine is specified 
by a codon, the codon can be altered to any of the corresponding codons 
described without altering the encoded polypeptide. Such nucleic acid 
variations are "silent variations," which are one species of 
conservatively modified variations. Every nucleic acid sequence herein 
which encodes a polypeptide also describes every possible silent variation 
of the nucleic acid. One of skill will recognize that each codon in a 
nucleic acid (except AUG, which is ordinarily the only codon for 
methionine) can be modified to yield a functionally identical molecule. 
Accordingly, each silent variation of a nucleic acid which encodes a 
polypeptide is implicit in each described sequence. 
As to amino acid sequences, one of skill will recognize that individual 
substitutions, in a nucleic acid, peptide, polypeptide, or protein 
sequence which alters a single amino acid or a small percentage of amino 
acids in the encoded sequence is a "conservatively modified variant" where 
the alteration results in the substitution of an amino acid with a 
chemically similar amino acid. Conservative substitution tables providing 
functionally similar amino acids are well known in the art. 
The following six groups each contain amino acids that are conservative 
substitutions for one another: 
1) Alanine (A), Serine (S), Threonine (T); 
2) Aspartic acid (D), Glutamic acid (E); 
3) Asparagine (N), Glutamine (Q); 
4) Arginine (R), Lysine (K); 
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (see, e.g., Creighton, 
Proteins (1984)). 
An indication that two nucleic acid sequences or polypeptides are 
substantially identical is that the polypeptide encoded by the first 
nucleic acid is immunologically cross reactive with the antibodies raised 
against the polypeptide encoded by the second nucleic acid. Thus, a 
polypeptide is typically substantially identical to a second polypeptide, 
for example, where the two peptides differ only by conservative 
substitutions. Another indication that two nucleic acid sequences are 
substantially identical is that the two molecules or their complements 
hybridize to each other under stringent conditions, as described below. 
The phrase "selectively (or specifically) hybridizes to" refers to the 
binding, duplexing, or hybridizing of a molecule only to a particular 
nucleotide sequence under stringent hybridization conditions when that 
sequence is present in a complex mixture (e.g., total cellular or library 
DNA or RNA). 
The phrase "stringent hybridization conditions" refers to conditions under 
which a probe will hybridize to its target subsequence, typically in a 
complex mixture of nucleic acid, but to no other sequences. Stringent 
conditions are sequence-dependent and will be different in different 
circumstances. Longer sequences hybridize specifically at higher 
temperatures. An extensive guide to the hybridization of nucleic acids is 
found in Tijssen, Techniques in Biochemistry and Molecular 
Biology--Hybridization with Nucleic Probes, "Overview of principles of 
hybridization and the strategy of nucleic acid assays" (1993). Generally, 
highly stringent conditions are selected to be about 5-10.degree. C. lower 
than the thermal melting point (T.sub.m) for the specific sequence at a 
defined ionic strength pH. Low stringency conditions are generally 
selected to be about 15-30.degree. C. below the T.sub.m. The T.sub.m is 
the temperature (under defined ionic strength, pH, and nucleic 
concentration) at which 50% of the probes complementary to the target 
hybridize to the target sequence at equilibrium (as the target sequences 
are present in excess, at T.sub.m, 50% of the probes are occupied at 
equilibrium). Stringent conditions will be those in which the salt 
concentration is less than about 1.0 M sodium ion, typically about 0.01 to 
1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the 
temperature is at least about 30.degree. C. for short probes (e.g., 10 to 
50 nucleotides) and at least about 60.degree. C. for long probes (e.g., 
greater than 50 nucleotides). Stringent conditions may also be achieved 
with the addition of destabilizing agents such as formamide. For selective 
or specific hybridization, a positive signal is at least two times 
background, preferably 10 time background hybridization. 
Nucleic acids that do not hybridize to each other under stringent 
conditions are still substantially identical if the polypeptides which 
they encode are substantially identical. This occurs, for example, when a 
copy of a nucleic acid is created using the maximum codon degeneracy 
permitted by the genetic code. In such cases, the nucleic acids typically 
hybridize under moderately stringent hybridization conditions. 
In the present invention, genomic DNA or cDNA comprising ANT nucleic acids 
of the invention can be identified in standard Southern blots under 
stringent conditions using the nucleic acid sequences disclosed here. For 
the purposes of this disclosure, suitable stringent conditions for such 
hybridizations are those which include a hybridization in a buffer of 40% 
formamide, 1 M NaCl, 1% SDS at 37.degree. C., and at least one wash in 
0.2.times. SSC at a temperature of at least about 50.degree. C., usually 
about 55.degree. C. to about 60.degree. C., for 20 minutes, or equivalent 
conditions. A positive hybridization is at least twice background. Those 
of ordinary skill will readily recognize that alternative hybridization 
and wash conditions can be utilized to provide conditions of similar 
stringency. 
Nucleic acids that do not hybridize to each other under stringent 
conditions are still substantially identical if the polypeptides that they 
encode are substantially identical. This occurs, for example, when a copy 
of a nucleic acid is created using the maximum codon degeneracy permitted 
by the genetic code. In such cased, the nucleic acids typically hybridize 
under moderately stringent hybridization conditions. Exemplary "moderately 
stringent hybridization conditions" include a hybridization in a buffer of 
40% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in 1.times. 
SSC at 45.degree. C. A positive hybridization is at least twice 
background. Those of ordinary skill will readily recognize that 
alternative hybridization and wash conditions can be utilized to provide 
conditions of similar stringency. 
A further indication that two polynucleotides are substantially identical 
is if the reference sequence, amplified by a pair of oligonucleotide 
primers, can then be used as a probe under stringent hybridization 
conditions to isolate the test sequence from a cDNA or genomic library, or 
to identify the test sequence in, e.g., an RNA gel or DNA gel blot 
hybridization analysis. 
DESCRIPTION OF THE SPECIFIC EMBODIMENTS 
The present invention provides the first definitive evidence that bZIP 
genes and their gene products interacts with, and are regulated by, NPR1, 
a key component in plant pathogen resistance. The present invention also 
provides for the first time rice and tomato bZIP polynucleotides encoding 
polypeptides that interact with NPR 1. 
Because the bZIP gene product most likely functions as a transcription 
factor that binds to salicylic acid-responsive DNA elements (see, e.g. the 
postulated bZIP binding site in the PR-1 promoter: Lebel, E., et al., 
Plant J. 16, 223-234 (1998)), one of skill will recognize that desired 
phenotypes associated with altered bZIP activity can be obtained by 
modulating the expression or activity of bZIP-regulated genes. Any of the 
known methods described for increasing or decreasing expression or protein 
activity can be used for this invention. 
Increasing BZIP Activity or BZIP Gene Expression 
Any of a number of means well known in the art can be used to increase bZIP 
activity in plants. Enhanced expression is useful, for example, to enhance 
systemic resistance to pathogens. Any organ can be targeted, such as shoot 
vegetative organs/structures (e.g. leaves, stems and tubers), roots, 
flowers and floral organs/structures (e.g. bracts, sepals, petals, 
stamens, carpels, anthers and ovules), seed (including embryo, endosperm, 
and seed coat) and fruit. Alternatively, one or several bZIP genes can be 
expressed constitutively (e.g., using the CaMV 35S promoter). 
Increased bZIP activity or bZIP expression can also be used to enhance 
resistance of plants to specific pathogens. Thus, for instance bZIP 
expression can be targeted to induce defense-related genes harmful to 
specific pathogens. 
Increasing bZIP Gene Expression 
Isolated sequences prepared as described herein can be used to introduce 
expression of a particular bZIP nucleic acid to increase gene expression 
using methods well known to those of skill in the art. Preparation of 
suitable constructs and means for introducing them into plants are 
described below. 
One of skill will recognize that the polypeptides encoded by the genes of 
the invention, like other proteins, have different domains that perform 
different functions. Thus, the gene sequences need not be full length, so 
long as the desired functional domain of the protein is expressed. The 
distinguishing features of bZIP polypeptides, including the leucine zipper 
and basic domain, are discussed in Foley et al. Plant J. 3:669-79 (1993) 
and Singh et al. Plant Cell 2:891-903 (1990). The bZIP polypeptides of the 
invention interact with NPR1. 
Modified protein chains can also be readily designed utilizing various 
recombinant DNA techniques well known to those skilled in the art and 
described in detail below. For example, the chains can vary from the 
naturally occurring sequence at the primary structure level by amino acid 
substitutions, additions, deletions, and the like. These modifications can 
be used in a number of combinations to produce the final modified protein 
chain. 
Modification of Eendogenous bZIP Genes 
Methods for introducing genetic mutations into plant genes and selecting 
plants with desired traits are well known. For instance, seeds or other 
plant material can be treated with a mutagenic chemical substance, 
according to standard techniques. Such chemical substances include, but 
are not limited to, the following: diethyl sulfate, ethylene imine, ethyl 
methanesulfonate and N-nitroso-N-ethylurea. Alternatively, ionizing 
radiation from sources such as, X-rays or gamma rays can be used. 
Alternatively, homologous recombination can be used to induce targeted gene 
modifications by specifically targeting the bZIP gene in vivo (see, 
generally, Grewal and Klar, Genetics 146: 1221-1238 (1997) and Xu et al., 
Genes Dev. 10: 2411-2422 (1996)). Homologous recombination has been 
demonstrated in plants (Puchta et al., Experientia 50: 277-284 (1994), 
Swoboda et al., EMBO J. 13: 484-489 (1994); Offringa et al., Proc. Natl. 
Acad. Sci. USA 90: 7346-7350 (1993); and Kempin et al. Nature 389:802-803 
(1997)). 
In applying homologous recombination technology to the genes of the 
invention, mutations in selected portions of an bZIP gene sequences 
(including 5' upstream, 3' downstream, and intragenic regions) such as 
those disclosed here are made in vitro and then introduced into the 
desired plant using standard techniques. Since the efficiency of 
homologous recombination is known to be dependent on the vectors used, use 
of dicistronic gene targeting vectors as described by Mountford et al., 
Proc. Natl. Acad. Sci. USA 91: 4303-4307 (1994); and Vaulont et al., 
Transgenic Res. 4: 247-255 (1995) are conveniently used to increase the 
efficiency of selecting for altered bZIP gene expression in transgenic 
plants. The mutated gene will interact with the target wild-type gene in 
such a way that homologous recombination and targeted replacement of the 
wild-type gene will occur in transgenic plant cells, resulting in 
suppression of bZIP activity. 
Alternatively, oligonucleotides composed of a contiguous stretch of RNA and 
DNA residues in a duplex conformation with double hairpin caps on the ends 
can be used. The RNA/DNA sequence is designed to align with the sequence 
of the target bZIP gene and to contain the desired nucleotide change. 
Introduction of the chimeric oligonucleotide on an extrachromosomal T-DNA 
plasmid results in efficient and specific bZIP gene conversion directed by 
chimeric molecules in a small number of transformed plant cells. This 
method is described in Cole-Strauss et al., Science 273:1386-1389 (1996) 
and Yoon et al. Proc. Natl. Acad. Sci. USA 93: 2071-2076 (1996). 
Other Means for Increasing bZIP Activity 
One method to increase bZIP expression is to use "activation mutagenesis" 
(see, e.g. Hiyashi et al. Science 258:1350-1353 (1992)). In this method an 
endogenous bZIP gene can be modified to be expressed constitutively, 
ectopically, or excessively by insertion of T-DNA sequences that contain 
strong/constitutive promoters upstream of the endogenous bZIP gene. As 
explained below, preparation of transgenic plants overexpressing bZIP can 
also be used to increase bZIP expression. Activation mutagenesis of the 
endogenous bZIP gene will give the same effect as overexpression of the 
transgenic bZIP nucleic acid in transgenic plants. Alternatively, an 
endogenous gene encoding an enhancer of bZIP activity or expression of the 
endogenous bZIP gene can be modified to be expressed by insertion of T-DNA 
sequences in a similar manner and bZIP activity can be increased. 
Another strategy to increase bZIP expression can be the use of dominant 
hyperactive mutants of bZIP by expressing modified bZIP transgenes. For 
example expression of modified bZIP with a defective domain that is 
important for interaction with a negative regulator of bZIP activity can 
be used to generate dominant hyperactive bZIP proteins. Alternatively, 
expression of truncated bZIP proteins which have only a domain that 
interacts with a negative regulator can titrate the negative regulator and 
thereby increase endogenous bZIP activity. Use of dominant mutants to 
hyperactivate target genes is described in Mizukami et al. Plant Cell 
8:831-845 (1996). 
Inhibition of bZIP Activity or Gene Expression 
As explained above, bZIP activity is important in controlling the 
expression of a number of defense-related genes through interaction with 
the gene's promoters as well as with other proteins (e.g., RNA 
polymerase). Inhibition of bZIP gene expression activity can be used, for 
instance, to reduce pathogen resistance in plants. In particular, targeted 
expression of bZIP nucleic acids that inhibit endogenous gene expression 
(e.g., antisense or co-suppression) can be used to reduce pathogen 
resistance. 
Inhibition of bZIP Gene Expression 
The nucleic acid sequences disclosed here can be used to design nucleic 
acids useful in a number of methods to inhibit bZIP or related gene 
expression in plants. For instance, antisense technology can be 
conveniently used. To accomplish this, a nucleic acid segment from the 
desired gene is cloned and operably linked to a promoter such that the 
antisense strand of RNA will be transcribed. The construct is then 
transformed into plants and the antisense strand of RNA is produced. In 
plant cells, it has been suggested that antisense suppression can act at 
all levels of gene regulation including suppression of RNA translation 
(see, Bourque Plant Sci. (Limerick) 105: 125-149 (1995); Pantopoulos In 
Progress in Nucleic Acid Research and Molecular Biology, Vol. 48. Cohn, W. 
E. and K. Moldave (Ed.). Academic Press, Inc.: San Diego, Calif., U.S.A.; 
London, England, UK. p. 181-238; Heiser et al. Plant Sci. (Shannon) 127: 
61-69 (1997)) and by preventing the accumulation of mRNA which encodes the 
protein of interest, (see, Baulcombe Plant Mol. Bio. 32:79-88 (1996); 
Prins and Goldbach Arch. Virol. 141: 2259-2276 (1996); Metzlaffet al. Cell 
88: 845-854 (1997), Sheehy et al., Proc. Nat. Acad. Sci. USA, 85:8805-8809 
(1988), and Hiatt et al., U.S. Pat. No. 4,801,340). 
The nucleic acid segment to be introduced generally will be substantially 
identical to at least a portion of the endogenous bZIP gene or genes to be 
repressed. The sequence, however, need not be perfectly identical to 
inhibit expression. The vectors of the present invention can be designed 
such that the inhibitory effect applies to other genes within a family of 
genes exhibiting identity or substantial identity to the target gene. 
For antisense suppression, the introduced sequence also need not be full 
length relative to either the primary transcription product or fully 
processed mRNA. Generally, higher identity can be used to compensate for 
the use of a shorter sequence. Furthermore, the introduced sequence need 
not have the same intron or exon pattern, and identity of non-coding 
segments may be equally effective. Normally, a sequence of between about 
30 or 40 nucleotides and about full length nucleotides should be used, 
though a sequence of at least about 100 nucleotides is preferred, a 
sequence of at least about 200 nucleotides is more preferred, and a 
sequence of about 500 to about 3500 nucleotides is especially preferred. 
A number of gene regions can be targeted to suppress bZIP gene expression. 
The targets can include, for instance, the coding regions, introns, 
sequences from exon/intron junctions, 5' or 3' untranslated regions, and 
the like. 
Another well-known method of suppression is sense co-suppression. 
Introduction of nucleic acid configured in the sense orientation has been 
recently shown to be an effective means by which to block the 
transcription of target genes. For an example of the use of this method to 
modulate expression of endogenous genes (see, Assaad et al. Plant Mol. 
Bio. 22: 1067-1085 (1993); Flavell Proc. Natl. Acad. Sci. USA 91: 
3490-3496 (1994); Stam et al. Annals Bot. 79: 3-12 (1997); Napoli et al., 
The Plant Cell 2:279-289 (1990); and U.S. Pat. Nos. 5,034,323, 5,231,020, 
and 5,283,184). 
The suppressive effect may occur where the introduced sequence contains no 
coding sequence per se, but only intron or untranslated sequences 
homologous to sequences present in the primary transcript of the 
endogenous sequence. The introduced sequence generally will be 
substantially identical to the endogenous sequence intended to be 
repressed. This minimal identity will typically be greater than about 65%, 
but a higher identity might exert a more effective repression of 
expression of the endogenous sequences. Substantially greater identity of 
more than about 80% is preferred, though about 95% to absolute identity 
would be most preferred. As with antisense regulation, the effect should 
apply to any other proteins within a similar family of genes exhibiting 
identity or substantial identity. 
For co-suppression, the introduced sequence, needing less than absolute 
identity, also need not be full length, relative to either the primary 
transcription product or fully processed mRNA. This may be preferred to 
avoid concurrent production of some plants that over-express the 
introduced sequence. A higher identity in a sequence shorter than 
full-length compensates for a longer, less identical sequence. 
Furthermore, the introduced sequence need not have the same intron or exon 
pattern, and identity of non-coding segments will be equally effective. 
Normally, a sequence of the size ranges noted above for antisense 
regulation is used. In addition, the same gene regions noted for antisense 
regulation can be targeted using co-suppression technologies. 
Oligonucleotide-based triple-helix formation can also be used to disrupt 
bZIP gene expression. Triplex DNA can inhibit DNA transcription and 
replication, generate site-specific mutations, cleave DNA, and induce 
homologous recombination (see, e.g., Havre and Glazer J. Virology 
67:7324-7331 (1993); Scanlon et al. FASEB J. 9:1288-1296 (1995); 
Giovannangeli et al. Biochemistry 35:10539-10548 (1996); Chan and Glazer 
J. Mol. Medicine (Berlin) 75: 267-282 (1997)). Triple helix DNAs can be 
used to target the same sequences identified for antisense regulation. 
Catalytic RNA molecules or ribozymes can also be used to inhibit expression 
of bZIP genes. It is possible to design ribozymes that specifically pair 
with virtually any target RNA and cleave the phosphodiester backbone at a 
specific location, thereby functionally inactivating the target RNA. In 
carrying out this cleavage, the ribozyme is not itself altered, and is 
thus capable of recycling and cleaving other molecules, making it a true 
enzyme. The inclusion of ribozyme sequences within antisense RNAs confers 
RNA-cleaving activity upon them, thereby increasing the activity of the 
constructs. Thus, ribozymes can be used to target the same sequences 
identified for antisense regulation. 
A number of classes of ribozymes have been identified. One class of 
ribozymes is derived from a number of small circular RNAs that are capable 
of self-cleavage and replication in plants. The RNAs replicate either 
alone (viroid RNAs) or with a helper virus (satellite RNAs). Examples 
include RNAs from avocado sunblotch viroid and the satellite RNAs from 
tobacco ringspot virus, lucerne transient streak virus, velvet tobacco 
mottle virus, solanum nodiflorum mottle virus and subterranean clover 
mottle virus. The design and use of target RNA-specific ribozymes is 
described in Zhao and Pick, Nature 365:448-451 (1993); Eastham and 
Ahlering, J. Urology 156:1186-1188 (1996); Sokol and Murray, Transgenic 
Res. 5:363-371 (1996); Sun et al., Mol. Biotechnology 7:241-251 (1997); 
and Haseloff et al., Nature, 334:585-591 (1988). 
Modification of Endogenous BZIP Genes 
Methods for introducing genetic mutations described above can also be used 
to select for plants with decreased bZIP expression. 
Other means for Inhibiting bZIP Activity 
BZIP activity may be modulated by eliminating the proteins that are 
required for bZIP cell-specific gene expression. Thus, expression of 
regulatory proteins and/or the sequences that control bZIP gene expression 
can be modulated using the methods described here. 
Another strategy is to inhibit the ability of a bZIP protein to interact 
with itself or with other proteins. This can be achieved, for instance, 
using antibodies specific to bZIP. In this method cell-specific expression 
of bZIP-specific antibodies is used to inactivate functional domains 
through antibody:antigen recognition (see, Hupp et al., Cell 83:237-245 
(1995)). Interference of activity of a bZIP interacting protein(s) can be 
applied in a similar fashion. Alternatively, dominant negative mutants of 
bZIP can be prepared by expressing a transgene that encodes a truncated 
bZIP protein. Use of dominant negative mutants to inactivate target genes 
in transgenic plants is described in Mizukami et al., Plant Cell 8:831-845 
(1996). 
Purification of bZIP Polypeptides 
Either naturally occurring or recombinant bZIP polypeptides can be purified 
for use in functional assays. Naturally occurring bZIP polypeptides can be 
purified, e.g., from plant tissue and any other source of a bZIP homolog. 
Recombinant bZIP polypep ides can be purified from any suitable expression 
system. 
The bZIP polypeptides may be purified to substantial purity by standard 
techniques, including selective precipitation with such substances as 
ammonium sulfate; column chromatography, immunopurification methods, and 
others (see, e.g., Scopes, Protein Purification: Principles and Practice 
(1982); U.S. Pat. No. 4,673,64; Ausubel et al., supra, and Sambrook et 
al., supra). 
A number of procedures can be employed when recombinant bZIP polypeptides 
are being purified. For example, proteins having established molecular 
adhesion properties can be reversible fused to the bZIP polypeptides. With 
the appropriate ligand, the bZIP polypeptides can be selectively adsorbed 
to a purification column and then freed from the column in a relatively 
pure form. The fused protein is then removed by enzymatic activity. 
Finally the bZIP polypeptides could be purified using immunoaffinity 
columns. 
Isolation of bZIP Nucleic Acids 
Generally, the nomenclature and the laboratory procedures in recombinant 
DNA technology described below are those well known and commonly employed 
in the art. Standard techniques are used for cloning, DNA and RNA 
isolation, amplification and purification. Generally enzymatic reactions 
involving DNA ligase, DNA polymerase, restriction endonucleases and the 
like are performed according to the manufacturer's specifications. These 
techniques and various other techniques are generally performed according 
to Sambrook et al., Molecular Cloning-A Laboratory Manual, Cold Spring 
Harbor Laboratory, Cold Spring Harbor, New York, (1989) or Current 
Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. 
(1994-1998). 
The isolation of bZIP nucleic acids may be accomplished by a number of 
techniques. For instance, oligonucleotide probes based on the sequences 
disclosed here can be used to identify the desired gene in a cDNA or 
genomic DNA library. To construct genomic libraries, large segments of 
genomic DNA are generated by random fragmentation, e.g. using restriction 
endonucleases, and are ligated with vector DNA to form concatemers that 
can be packaged into the appropriate vector. To prepare a cDNA library, 
mRNA is isolated from the desired organ, such as leaves, and a cDNA 
library which contains a bZIP gene transcript is prepared from the mRNA. 
Alternatively, cDNA may be prepared from mRNA extracted from other tissues 
in which bZIP genes or homologs are expressed. 
The cDNA or genomic library can then be screened using a probe based upon 
the sequence of a cloned bZIP gene disclosed here. Probes may be used to 
hybridize with genomic DNA or cDNA sequences to isolate homologous genes 
in the same or different plant species. Alternatively, antibodies raised 
against a bZIP polypeptide can be used to screen an mRNA expression 
library. 
Alternatively, the nucleic acids of interest can be amplified from nucleic 
acid samples using amplification techniques. For instance, polymerase 
chain reaction (PCR) technology can be used to amplify the sequences of 
bZIP genes directly from genomic DNA, from cDNA, from genomic libraries or 
cDNA libraries. PCR and other in vitro amplification methods may also be 
useful, for example, to clone nucleic acid sequences that code for 
proteins to be expressed, to make nucleic acids to use as probes for 
detecting the presence of the desired mRNA in samples, for nucleic acid 
sequencing, or for other purposes. For a general overview of PCR see PCR 
Protocols: A Guide to Methods and Applications. (Innis, M, Gelfand, D., 
Sninsky, J. and White, T., eds.), Academic Press, San Diego (1990). 
Appropriate primers and probes for identifying bZIP sequences from plant 
tissues are generated from comparisons of the sequences provided here 
(e.g. SEQ ID NO: 1, SEQ ID NO:3, etc.). 
Polynucleotides may also be synthesized by well-known techniques as 
described in the technical literature. See, e.g., Carruthers et al., Cold 
Spring Harbor Symp. Quant. Biol. 47:411-418 (1982), and Adams et al., J. 
Am. Chem. Soc. 105:661 (1983). Double stranded DNA fragments may then be 
obtained either by synthesizing the complementary strand and annealing the 
strands together under appropriate conditions, or by adding the 
complementary strand using DNA polymerase with an appropriate primer 
sequence. 
Preparation of Recombinant Vectors 
To use isolated sequences in the above techniques, recombinant DNA vectors 
suitable for transformation of plant cells are prepared. Techniques for 
transforming a wide variety of higher plant species are well known and 
described in the technical and scientific literature. See, for example, 
Weising et al. Ann. Rev. Genet. 22:421-477 (1988). A DNA sequence coding 
for the desired polypeptide, for example a cDNA sequence encoding a full 
length protein, will preferably be combined with transcriptional and 
translational initiation regulatory sequences which will direct the 
transcription of the sequence from the gene in the intended tissues of the 
transformed plant. 
For example, for overexpression, a plant promoter fragment may be employed 
which will direct expression of the gene in all tissues of a regenerated 
plant. Such promoters are referred to herein as "constitutive" promoters 
and are active under most environmental conditions and states of 
development or cell differentiation. Examples of constitutive promoters 
include the cauliflower mosaic virus (CaMV) 35S transcription initiation 
region, the 1'- or 2'-promoter derived from T-DNA of Agrobacterium 
tumafaciens, and other transcription initiation regions from various plant 
genes known to those of skill. Such genes include for example, ACT11 from 
Arabidopsis (Huang et al. Plant Mol. Biol. 33:125-139 (1996)), Cat3 from 
Arabidopsis (GenBank No. U43147, Zhong et al., Mol. Gen. Genet. 
251:196-203 (1996)), the gene encoding stearoyl-acyl carrier protein 
desaturase from Brassica napus (Genbank No. X74782, Solocombe et al. Plant 
Physiol. 104:1167-1176 (1994)), GPc1 from maize (GenBank No. X15596, 
Martinez et al. J. Mol. Biol 208:551-565 (1989)), and Gpc2 from maize 
(GenBank No. U45855, Manjunath et al., Plant Mol. Biol. 33:97-112 (1997)). 
Alternatively, the plant promoter may direct expression of bZIP nucleic 
acid in a specific tissue, organ or cell type (i.e. tissue-specific 
promoters) or may be otherwise under more precise environmental or 
developmental control (i.e. inducible promoters). Examples of 
environmental conditions that may effect transcription by inducible 
promoters include anaerobic conditions, elevated temperature, the presence 
of light, or sprayed with chemicals/hormones. Tissue-specific promoters 
can be inducible. Similarly, tissue-specific promoters may only promote 
transcription within a certain time frame of developmental stage within 
that tissue. Other tissue specific promoters may be active throughout the 
life cycle of a particular tissue. One of skill will recognize that a 
tissue-specific promoter may drive expression of operably linked sequences 
in tissues other than the target tissue. Thus, as used herein a 
tissue-specific promoter is one that drives expression preferentially in 
the target tissue or cell type, but may also lead to some expression in 
other tissues as well. 
A number of tissue-specific promoters can also be used in the invention. 
For instance, promoters that direct expression of nucleic acids in leaves, 
roots or flowers are useful for enhancing resistance to pathogens that 
infect those organs. For expression of a bZIP polynucleotide in the aerial 
vegetative organs of a plant, photosynthetic organ-specific promoters, 
such as the RBCS promoter (Khoudi, et al., Gene 197:343, 1997), can be 
used. Root-specific expression of bZIP polynucleotides can be achieved 
under the control of the root-specific ANR1 promoter (Zhang & Forde, 
Science, 279:407, 1998). Any strong, constitutive promoters, such as the 
CaMV 35S promoter, can be used for the expression of bZIP polynucleotides 
throughout the plant. 
If proper polypeptide expression is desired, a polyadenylation region at 
the 3'-end of the coding region should be included. The polyadenylation 
region can be derived from the natural gene, from a variety of other plant 
genes, or from T-DNA. 
The vector comprising the sequences (e.g., promoters or coding regions) 
from genes of the invention will typically comprise a marker gene that 
confers a selectable phenotype on plant cells. For example, the marker may 
encode biocide resistance, particularly antibiotic resistance, such as 
resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide 
resistance, such as resistance to chlorosulfuron or Basta. 
Production of Transgenic Plants 
DNA constructs of the invention may be introduced into the genome of the 
desired plant host by a variety of conventional techniques. For example, 
the DNA construct may be introduced directly into the genomic DNA of the 
plant cell using techniques such as electroporation and microinjection of 
plant cell protoplasts, or the DNA constructs can be introduced directly 
to plant tissue using ballistic methods, such as DNA particle bombardment. 
Microinjection techniques are known in the art and well described in the 
scientific and patent literature. The introduction of DNA constructs using 
polyethylene glycol precipitation is described in Paszkowski et al. Embo. 
J 3:2717-2722 (1984). Electroporation techniques are described in Fromm et 
al. Proc. Natl. Acad. Sci. USA 82:5824 (1985). Ballistic transformation 
techniques are described in Klein et al. Nature 327:70-73 (1987). 
Alternatively, the DNA constructs may be combined with suitable T-DNA 
flanking regions and introduced into a conventional Agrobacterium 
tumefaciens host vector. The virulence functions of the Agrobacterium 
tumefaciens host will direct the insertion of the construct and adjacent 
marker into the plant cell DNA when the cell is infected by the bacteria. 
Agrobacterium tumefaciens-mediated transformation techniques, including 
disarming and use of binary vectors, are well described in the scientific 
literature. See, for example Horsch et al. Science 233:496-498 (1984), and 
Fraley et al. Proc. Natl. Acad. Sci. USA 80:4803 (1983) and Gene Transfer 
to Plants, Potrykus, ed. (Springer-Verlag, Berlin 1995). 
Transformed plant cells which are derived by any of the above 
transformation techniques can be cultured to regenerate a whole plant 
which possesses the transformed genotype and thus the desired phenotype 
such as increased seed mass. Such regeneration techniques rely on 
manipulation of certain phytohormones in a tissue culture growth medium, 
typically relying on a biocide and/or herbicide marker that has been 
introduced together with the desired nucleotide sequences. Plant 
regeneration from cultured protoplasts is described in Evans et al., 
Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 
124-176, MacMillilan Publishing Company, New York, 1983; and Binding, 
Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca 
Raton, 1985. Regeneration can also be obtained from plant callus, 
explants, organs, or parts thereof. Such regeneration techniques are 
described generally in Klee et al. Ann. Rev. of Plant Phys. 38:467-486 
(1987). 
The nucleic acids of the invention can be used to confer desired traits on 
essentially any plant. Thus, the invention has use over a broad range of 
plants, including species from the genera Anacardium, Arachis, Asparagus, 
Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, 
Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium, 
Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, 
Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, 
Olea, Oryza, Panieum, Pannesetum, Persea, Phaseolus, Pistachia, Pisum, 
Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum, 
Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, and Zea. 
One of skill will recognize that after the expression cassette is stably 
incorporated in transgenic plants and confirmed to be operable, it can be 
introduced into other plants by sexual crossing. Any of a number of 
standard breeding techniques can be used, depending upon the species to be 
crossed. 
Using known procedures one of skill can screen for plants of the invention 
by detecting the increase or decrease of bZIP mRNA or protein in 
transgenic plants. Means for detecting and quantitating mRNAs or proteins 
are well known in the art. 
Methods of Enhancing Plant Resistance to Pathogens 
The present invention provides for method of enhancing plant resistance to 
pathogens by modulating the expression and/or activity of bZIP 
polynucleotides and/or polypeptides. Without limiting the invention to a 
particular mechanism of operation, increased expression of bZIP 
polynucleotides or bZIP polypeptides can be used to enhance resistance of 
plants to pathogens. Resistance can be enhanced, for instance, relative to 
a pathogen species or genus or systemic acquired resistance can be induced 
by increased expression of bZIP polynucleotides or bZIP polypeptides. 
Alternatively, or in combination, bZIP polynucleotides or bZIP 
polypeptides can be modified to enhance resistance, e.g., by increasing or 
decreasing bZIP polypeptides' interactions with other components important 
in plant pathogen resistance. 
Without limiting the invention to a particular mechanism of operation, one 
possible mechanism by which bZIP polypeptides modulate resistance, for 
example, is by interacting with the promoters of defense-related genes. 
Interaction of bZIP polypeptides with these promoters may lead directly to 
increased transcription of defense-related transcripts, thereby enhancing 
resistance to pathogens. Alternatively, bZIP polypeptides may interact 
with promoters of other genes as well as with other regulatory factors, 
thereby modulating expression of defense related genes or other genes 
involved in resistance. For instance, bZIP polypeptides may interact with 
a transcriptional repressor, thereby allowing for the expression of 
defense-related genes. 
Selecting for Plants With Enhanced Resistance 
Plants with enhanced resistance can be selected in many ways. One of 
ordinary skill in the art will recognize that the following methods are 
but a few of the possibilities. One method of selecting plants with 
enhanced resistance is to determine resistance of a plant to a specific 
plant pathogen. Possible pathogens include, but are not limited to, 
viruses, bacteria, nematodes, fungi or insects (see, e.g., Agrios, Plant 
Pathology (Academic Press, San Diego, Calif.) (1988)). One of skill in the 
art will recognize that resistance responses of plants vary depending on 
many factors, including what pathogen or plant is used. Generally, 
enhanced resistance is measured by the reduction or elimination of disease 
symptoms when compared to a control plant. In some cases, however, 
enhanced resistance can also be measured by the production of the 
hypersensitive response (HR) of the plant (see, e.g., Staskawicz et al. 
Science 268(5211): 661-7 (1995)). Plants with enhanced resistance can 
produce an enhanced hypersensitive response relative to control plants. 
Enhanced resistance can also be determined by measuring the increased 
expression of a gene operably linked a deferise related promoter. 
Measurement of such expression can be measured by quantitating the 
accumulation of RNA or subsequent protein product (e.g., using northern or 
western blot techniques, respectively (see, e.g. Sambrook et al. and 
Ausubel et al.). A possible alternate strategy for measuring defense gene 
promoter expression involves operably linking a reporter gene to the 
promoter. Reporter gene constructs allow for ease of measurement of 
expression from the promoter of interest. Examples of reporter genes 
include: .beta.-gal, GUS (sec, e.g., Jefferson, R. A., et al., EMBO J 6: 
3901-3907 (1987), green fluorescent protein, luciferase, and others.