# Patent

## Title
Enhanced disease resistance by introduction of NH3

## Abstract

Methods and compositions for improving plant resistance by expression of NPR homolog 3 (NH3) polypeptides are provided.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
The present patent application claims benefit of priority to U.S. Provisional Patent Application No. 61/348,618, filed May 26, 2010, the contents of which are incorporated by reference herein for all purposes.


STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
This invention was made with Government support under Contract No. 2006-35604-16640 awarded by the United States Department of Agriculture. The Government has certain rights in this invention.


BACKGROUND OF THE INVENTION
Plants survive pathogen attack by employing various defense strategies, including strengthening of cell walls, the accumulation of phytoalexins, synthesis of salicylic acid (SA), and induction of pathogenesis-related (PR) genes. A hypersensitive response (HR) is often associated with the defense response and limits pathogen growth to the infected site. After an initial local infection, Systemic Acquired Resistance (SAR) often occurs which coordinately induces expression of a set of PR genes, leading to a long-lasting enhanced resistance against a broad spectrum of pathogens (Durrant and Dong, 2004). In dicots, like Arabidopsis and tobacco, SA and its synthetic analogs, such as 2,6-dichloroisonicotinic acid (INA), benzothiadiazole (BTH), and probenazole are potent inducers of SAR (Ward et al., 1991; Friedrich et al., 1996; Yoshioka et al., 2001). In monocots, SAR can be induced by BTH in wheat (Gorlach et al., 1996) and by Pseudomonas syringae in rice (Smith and Metraux, 1991). BTH can also induce disease resistance in rice (Schweizer et al., 1999; Rohilla et al., 2002; Shimono et al., 2007) and maize (Morris et al., 1998).
The NPR1 (also known as NIM1 and SAI1) gene is a key regulator of SA-mediated SAR in Arabidopsis (Cao et al., 1994; Delaney et al., 1995; Glazebrook et al., 1996; Ryals et al. 1997; Shah et al., 1997). Upon induction by SA, INA, or BTH, NPR1 expression levels are elevated (Cao et al., 1997). NPR1 affects the SAR pathway downstream of the SA signal. Arabidopsis npr1/nim1 mutants are impaired in their ability to induce PR gene expression and mount a SAR response even after treatment with SA or INA. NPR1 encodes a protein with a bipartite nuclear localization sequence and two protein-protein interaction domains: an ankyrin repeat domain and a BTB/POZ domain (Cao et al., 1997). Nuclear localization of NPR1 protein is essential for its function (Kinkema et al., 2000). The ankyrin domain is required for interaction with TGA transcription factors (Zhang et al., 1999; Despres et al., 2000) and the BTB/POZ domain interacts with the repression domain of TGA2 to negate its function (Boyle et al., 2009). During non-induced states, NPR1 protein forms an oligomer and is excluded from the nucleus. Upon SAR induction, monomeric NPR1 emerges through redox changes, accumulates in the nucleus, and activates PR gene expression (Mou et al. 2003). NPR1 also appears to modulate the cross-talk between SA- and JA-dependent pathways; the antagonistic effect of SA on JA signaling requires NPR1, but not nuclear localization of the NPR1 protein (Spoel et al., 2003).
Overexpression of NPR1 in Arabidopsis leads to enhanced disease resistance to both bacterial and oomycete pathogens in a dose-dependent manner (Cao et al., 1998). Similarly, overexpression of Arabidopsis NPR1 or the rice NPR1 ortholog, NH1, in rice results in enhanced resistance to rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) and blast pathogen Magnaporthe grisea (Chern et al. 2001; Yuan et al., 2007), indicating the presence of a similar defense pathway in rice. Although transgenic Arabidopsis plants over-expressing NPR1 acquire enhanced sensitivity to SA and BTH (Freidrich et al., 2001), they display no obvious detrimental morphological changes and do not have elevated PR gene expression until activated by inducers or by infection of pathogens (Cao et al., 1998). However, in rice, overexpression of rice NH1 results in a development- and environment-dependent lesion-mimic phenotype, which can be further enhanced by application of BTH (Chern et al., 2005a). These results suggest that overexpression of NH1 in rice activates the defense response in the absence of inducer treatment or pathogen challenge, an undesirable consequence in terms of practical application. Thus, although rice possesses a pathway similar to the NPR1-mediated one in Arabidopsis, there may be significant differences in their regulation.
There are six NPR1-like genes in Arabidopsis (Liu et al., 2005; Zhang et al., 2006) and five NPR1-like genes in rice (Yuan et al., 2007). Despite extensive investigations done on NPR1, very little is known concerning the NPR1-like genes with regards to their possible involvement in plant defense. Arabidopsis NPR5 and NPR6 have recently been named BOP2 (Blade-On-Petiole2) and BOP1 (Blade-On-Petiole1), respectively. BOP1 and BOP2 regulate Arabidopsis leaf formation. Like NPR1, these proteins function as transcriptional coactivators targeting the AS2 (Asymmetric Leaves2) gene (Jun et al., 2010). Thus, NPR5 (BOP2) and NPR6 (BOP1) are mainly involved in regulating plant development rather than defense. Contradictory results concerning the function of Arabidopsis NPR4 have been reported. Liu et al. (2005) reported that Arabidopsis NPR4 is required for basal resistance to Pseudomonas syringae pv. tomato (Pst) DC3000 and Erysiphe cichoracearum because the npr4-1 mutant is more susceptible to these two pathogens. This group suggested that NPR4 may be also involved in the cross-talk between SA- and JA-dependent signaling pathways since expression of the JA-dependent marker gene PDF1.2 is compromised in npr4-1 leaves following application of methyl-JA. However, Zhang et al. (2006) reported that Arabidopsis NPR3 and NPR4 are negative regulators of PR gene expression and disease resistance. They showed that npr3 mutants have slightly increased basal PR-1 expression and the npr3npr4 double mutant shows even higher PR-1, PR-2, and PR-5 expression. The double mutant plants display enhanced resistance against virulent bacterial (including Pst DC3000) and oomycete pathogens (Zhang et al., 2006). Thus, the roles of NPR4 in disease resistance from these two reports contradict each other. In rice, Yuan et al. (2007) have overexpressed OsNPR1/NH1, OsNPR2, and OsNPR3 in rice and tested for enhanced resistance to Xoo and rice blast. These authors found that only OsNPR1 (but not OsNPR2 or OsNPR3) overexpression conferred enhanced resistance, leaving in doubt whether any rice NPR1 paralogs are involved in defense against pathogens.
BRIEF SUMMARY OF THE INVENTION
The present invention provides for plants (or a plant cell, seed, flower, leaf, fruit, or other plant part from such plants or processed food or food ingredient from such plants) comprising a heterologous expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide encoding a NPR1 homolog 3 (NH3) polypeptide, wherein the plant expresses the NH3 polypeptide at a higher level than a plant lacking the expression cassette and wherein the plant has enhanced disease resistance compared to the plant lacking the expression cassette.
In some embodiments, the promoter is substantially similar (e.g., has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity) to a native NH3 promoter of the plant. In some embodiments, the promoter is substantially similar (e.g., has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity) to SEQ ID NO:3. In some embodiments, the promoter is tissue-specific, organ-specific, or inducible.
In some embodiments, the polynucleotide encoding the NH3 polypeptide is substantially similar (e.g., has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity) to SEQ ID NO:1. In some embodiments, the polynucleotide encoding the NH3 polypeptide comprises SEQ ID NO:1.
In some embodiments, the polynucleotide encodes a NH3 polypeptide that is substantially similar (e.g., has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity) to any of SEQ ID NOs:2 or 4-19 (i.e., any of SEQ ID NOs:2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19). In some embodiments, the polynucleotide encodes a NH3 polypeptide comprising any of SEQ ID NOs:2 or 4-19 (i.e., any of SEQ ID NOs:2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19).
In some embodiments, the plant comprising an expression cassette as described herein expresses the NH3 polypeptide at a level from about two-fold higher to about ten-fold higher (e.g., about two-fold, about three-fold, about four-fold, about five-fold, about six-fold, about seven-fold, about eight-fold, about nine-fold, or about ten-fold higher) than the plant lacking the expression cassette.
In some embodiments, the plant (or plant cell, seed, flower, leaf, fruit, or other plant part from such plant or processed food or food ingredient from such plant) comprising an expression cassette as described herein is selected from the group consisting of rice, maize, wheat, soybeans, cotton, canola, turfgrass, and alfalfa. In some embodiments, the plant (or plant cell, seed, flower, leaf, fruit, or other plant part from such plant or processed food or food ingredient from such plant) comprising an expression cassette as described herein is rice.
The present invention also provides for expression cassettes comprising a promoter operably linked to an isolated polynucleotide encoding a NPR1 homolog 3 (NH3) polypeptide, wherein expression of the expression cassette in a plant increases the level of NH3 polypeptide expression of the plant and enhances disease resistance of the plant as compared to a plant in which the expression cassette is not expressed.
In some embodiments, the expression cassette comprises a promoter operably linked to an isolated polynucleotide encoding a NH3 polypeptide, wherein the polynucleotide encoding the NH3 polypeptide is substantially similar (e.g., has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity) to SEQ ID NO:1. In some embodiments, the polynucleotide encoding the NH3 polypeptide comprises SEQ ID NO:1. In some embodiments, the polynucleotide encodes a NH3 polypeptide that is substantially similar (e.g., has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity) to any of SEQ ID NOs:2 or 4-19 (i.e., any of SEQ ID NOs:2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19). In some embodiments, the polynucleotide encodes a NH3 polypeptide comprising any of SEQ ID NOs:2 or 4-19 (i.e., any of SEQ ID NOs:2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19).
In some embodiments, the expression cassette comprises a promoter operably linked to an isolated polynucleotide encoding a NH3 polypeptide, wherein the promoter is substantially similar (e.g., has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity) to a native NH3 promoter of a wild-type plant. In some embodiments, the promoter is substantially similar (e.g., has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity) to SEQ ID NO:3. In some embodiments, the promoter is tissue-specific, organ-specific, or inducible.
In another aspect, the present invention provides for expression vectors comprising an expression cassette as described herein.
In yet another aspect, the present invention provides for methods of enhancing plant resistance to a pathogen. In some embodiments, the method comprises:

    
    
        introducing a nucleic acid comprising a expression cassette into a plant, wherein the expression cassette comprises a promoter operably linked to a polynucleotide encoding a NPR1 homolog 3 (NH3) polypeptide; and
        from the one or more plants into which the nucleic acid comprising the expression cassette has been introduced, selecting a plant with increased resistance to a pathogen or pathogens as compared to resistance of a plant lacking the expression cassette.
    
    


In some embodiments, the expression cassette of the method comprises a polynucleotide encoding the NH3 polypeptide that is substantially similar (e.g., has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity) to SEQ ID NO:1. In some embodiments, the polynucleotide encoding the NH3 polypeptide comprises SEQ ID NO:1. In some embodiments, the polynucleotide encodes a NH3 polypeptide that is substantially similar (e.g., has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity) to any of SEQ ID NOs:2 or 4-19 (i.e., any of SEQ ID NOs:2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19). In some embodiments, the polynucleotide encodes a NH3 polypeptide comprising any of SEQ ID NOs:2 or 4-19 (i.e., any of SEQ ID NOs:2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19).
In some embodiments, the expression cassette of the method comprises a promoter that is substantially similar (e.g., has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity) to a native NH3 promoter of a wild-type plant. In some embodiments, the promoter is substantially similar (e.g., has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity) to SEQ ID NO:3. In some embodiments, the promoter is tissue-specific, organ-specific, or inducible.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. A phylogenic tree of rice NPR 1-like proteins. Sequences of the five rice NPR1-like proteins are based on TIGR annotations and confirmed by isolated cDNA clones. Sequences are aligned using BlastP. The percentage of amino acid (aa) identity was calculated by comparing individual protein to NH1.
FIG. 2. Lesion length measurements of NH3 transgenic plants. A. Resistance analysis of T0 plants. Four Ubi-NT-NH3 (grey bars) and 12 nNT-NH3 (filled bars) T0 transgenic plants were inoculated with PXO99 and lesion lengths were measured two weeks after inoculation. Wild-type Kitaake (Kit) plants were included as control (open bars). Each bar represents the average and standard deviation of two to five leaves. B. Enhanced resistance and transgene co-segregation analysis. T1 progeny from three T0 lines (#3A, #6A, and #13A) of nNT-NH3 transgenic plants were genotyped by PCR for the presence of the nNT-NH3 transgene. The progeny containing the transgene are presented as filled bars and null segregants, which no longer contain the gene, are as open bars. Kit plants were included as control (open bars). Six weeks old plants were inoculated with PXO99 and lesion lengths measured in two weeks. On average three to four leaves were inoculated. The error bars represent standard deviations.
FIG. 3. Analysis of nNT-NH3 lines #6A and #13A. A. Lesion development. Progeny of nNT-NH3 lines #6A and #13A (labeled NH3-6 and NH3-13) were first genotyped. Those containing the nNT-NH3 transgene were pooled to represent the line and inoculated with PXO99. Lesion lengths were measured at days 0, 4, 8, and 12, after inoculation. Each data point represents the average and standard deviation of three leaf samples. B. Bacterial growth curves. Inoculated leaf samples described in (a) were used to extract Xoo for growth curve analysis. C. Leaf lesions caused by Xoo. Two representative leaves, each from Kit control, NH3-6, and NH3-13, two weeks after PXO99 inoculation are shown. D. RT-PCR results. Total RNA samples were extracted from NH3-6, NH3-13, and Kit. An equal amount of RNA was reverse-transcribed and the cDNA was used for RT-PCR. The amounts of input cDNA were further equalized based on expression of actin. The expression of the NH3 genes (including endogenous and the nNT-NH3 transgene) was measured by targeting the NH3 transcripts. The expression of the nNT-NH3 transgene (labeled NTAP-NH3) was measured by using one primer targeting the NTAP tag and the other targeting NH3.
FIG. 4. Responses of nNT-NH3 plants to plant defense activators. A. Responses to 10 mM BTH. nNT-NH3 and NTAP control plants were foliar sprayed with 10 mM BTH. Three representative leaves each from the NTAP control and nNT-NH3 plants one week after the treatment. B. Responses to 1 mM BTH and INA. Plants were foliar sprayed with either 1 mM BTH, 1 mM INA, or mock solution. Two typical leaves from each combination of plant and treatment are shown. Symptoms on leaves were allowed to develop for one week following the treatment.
FIG. 5. Effects of introduction of nNT-NH3 on expression of defense-related genes. A. Effects of nNT-NH3 on induction by BTH treatment. Kit control and nNT-NH3 plants were foliar sprayed with 1 mM BTH or with mock solution. Leaf samples were collected at days 0, 1, 2, 4, and 8. Total RNA was extracted and equal amount of RNA was used to synthesize cDNA for RT-PCR. The amounts of cDNA input were further equalized using actin as the reference. The same amount of cDNA was then used in the RT-PCR reactions for NH3 and defense-related genes: NH1, PR1a, PR3, PR10 and peroxidase (POX22.3). B. Quantitative real time RT-PCR. Real time qPCR experiments were performed using the cDNA prepared in A. Open bars represent Kit treated with 1 mM BTH; filled bars represent nNT-NH3 treated with 1 mM BTH; gray bars represent nNT-NH3 with mock treatment. Days after BTH application are depicted under each panel. The targeted gene is indicated in each panel. The numbers on the X-axis in each graph represent the expression levels normalized to actin expression. Each bar represents three technical replications and its standard deviation. C. Effects of nNT-NH3 on induction by INA. Kit control and nNT-NH3 plants were treated with 1 mM INA or mock solution. Leaf samples were collected at days 0, 1, 2, 4, and 8. Total RNA samples were extracted. The amounts of cDNA input were equalized using actin as the reference. RT-PCR reactions were performed for genes NH3, PR1a, PR3, PR10, and peroxidase (POX22.3).
FIG. 6. Lesion length measurement of NH3 Myc transgenic plants. Three Kit control (open bars) and 23 independent NH3Myc (filled bars) T0 transgenic plants were inoculated with PXO99 and lesion length measured two weeks post inoculation. On average four leaves were measured for each plant. The error bars represent standard deviations.



DEFINITIONS
The term “enhanced disease resistance” refers to an increase in the ability of a plant to prevent pathogen infection or pathogen-induced symptoms. Enhanced resistance can be increased resistance relative to a particular pathogen species or genus or can be increased resistance to all pathogens (e.g., systemic acquired resistance).
The terms “NPR1 homolog 3 polypeptide,” “OsNPR3 polypeptide,” and “NH3 polypeptide” refer to a protein characterized in part by the presence of a bipartite nuclear localization sequence and two protein-protein interaction domains, an ankyrin repeat domain and a BTB/POZ domain, and which, when introduced into a plant, results in enhanced disease resistance. In some embodiments, a NH3 polypeptide comprises a rice NH3 polypeptide (e.g., SEQ ID NO:2) or a polypeptide that is substantially similar to SEQ ID NO:2. In some embodiments, a NH3 polypeptide comprises an ortholog of rice NH3, for example a polypeptide that is identical or substantially similar to any of SEQ ID NOs:4-19.
The term “nucleic acid” or “polynucleotide” refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′ end. Nucleic acids may also include modified nucleotides that permit correct read through by a polymerase and do not significantly alter expression of a polypeptide encoded by that nucleic acid.
The phrase “nucleic acid encoding” or “polynucleotide encoding” refers to a nucleic acid which directs the expression of a specific protein or peptide. The nucleic acid sequences include both the DNA strand sequence that is transcribed into RNA and the RNA sequence that is translated into protein. The nucleic acid sequences include both the full length nucleic acid sequences as well as non-full length sequences derived from the full length sequences. It should be further understood that the sequence includes the degenerate codons of the native sequence or sequences which may be introduced to provide codon preference in a specific host cell.
The phrase “host cell” refers to a cell from any organism. Exemplary host cells are derived from plants, bacteria, yeast, fungi, insects or other animals. Methods for introducing polynucleotide sequences into various types of host cells are well known in the art.
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., USA).
The phrase “substantially similar” or “substantially identical,” used in the context of two nucleic acids or polypeptides, refers to a sequence that has at least 50% sequence identity with a reference sequence. Alternatively, percent identity can be any integer from 50% to 100%. Some embodiments include at least: 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, compared to a reference sequence using the programs described herein, e.g., BLAST using standard parameters, as described below. Embodiments of the present invention provide for nucleic acids comprising a sequence that is substantially similar to SEQ ID NOs:1 or 3 across the entire length of the sequence, or polypeptides comprising a sequence that is substantially similar to SEQ ID NOs:2 or 4-19 across the entire length of the sequence.
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.
Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The 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 acts as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. 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 BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=−2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
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 0.01, more preferably less than about 10−5, and most preferably less than about 10−20.
“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)).
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, when a promoter is said to be operably linked to a heterologous coding sequence, it means that the coding sequence is derived from one species whereas the promoter sequence is derived another, different species; or, if both are derived from the same species, the coding sequence is not naturally associated with the promoter (e.g., is a genetically engineered coding sequence, e.g., from a different gene in the same species, or an allele from a different ecotype or variety).
An “expression cassette” refers to a nucleic acid construct that, when introduced into a host cell, results in transcription and/or translation of an RNA or polypeptide, respectively. Antisense or sense constructs that are not or cannot be translated are expressly included by this definition. In the case of both expression of transgenes and suppression of endogenous genes (e.g., by antisense, or sense suppression) one of skill will recognize that the inserted polynucleotide sequence need not be identical, but may be only substantially similar to a sequence of the gene from which it was derived. As explained herein, these substantially similar variants are specifically covered by reference to a specific nucleic acid sequence.
The term “promoter,” as used herein, refers to a polynucleotide sequence capable of driving transcription of a coding sequence in a cell. Thus, promoters used in the polynucleotide constructs of the invention include cis-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene. For example, a promoter can be a cis-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5′ and 3′ untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) gene transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells. A “constitutive promoter” is one that is capable of initiating transcription in nearly all tissue types, whereas a “tissue-specific promoter” initiates transcription only in one or a few particular tissue types.
The term “plant” includes whole plants, shoot vegetative organs and/or structures (e.g., leaves, stems and tubers), roots, flowers and floral organs (e.g., bracts, sepals, petals, stamens, carpels, anthers), ovules (including egg and central cells), seed (including zygote, embryo, endosperm, and seed coat), fruit (e.g., the mature ovary), seedlings, plant tissue (e.g., vascular tissue, ground tissue, and the like), cells (e.g., guard cells, egg cells, trichomes 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.
“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)).
DETAILED DESCRIPTION OF THE INVENTION
I. Introduction
As described in the Examples section, the inventors have surprisingly discovered that introduction of an extra copy of NPR homolog 3 (NH3) driven by its own promoter into plants results in plants with enhanced disease resistance. Generally, the plants of the invention express NH3 at a higher level, in some cases two-fold, three-fold, or four-fold, higher, than plants into which the extra copy of NH3 has not been introduced.
II. NH3 Proteins
In rice, there are five NPR1-like proteins, namely NH1, NH2, NH3, NH4, and NH5 (NPR1 homologs 1-5), encoded by six genes (TIGR gene ID Os01g09800, Os01g56200, Os03g46440, Os01g72020, Os11g04600, and Os12g04410) in the genome. Protein NH5 is encoded by two duplicate genes, Os11g04600 and Os12g04410, which are recently duplicated genes due to chromosomal segmental duplication (Jacquemin et al., 2009). In Arabidopsis, the six NPR1-like proteins are divided into three groups with each group containing two proteins (Liu et al., 2005). As depicted in FIG. 1, the five rice NPR1-like proteins can also be divided into three groups based on BLAST search results: NH1 in group 1, NH2 and NH3 in group 2, and NH4 and NH5 in group 3. Thus, unlike in Arabidopsis, rice NH1 is most unique in sequence among rice NPR1-like proteins.
The identity levels across different groups of rice NPR1-like proteins are higher than those in Arabidopsis. For example, in Arabidopsis NPR1 shares about 35% identity with group 2 and 20% identity with group 3, while in rice NH1 shares 43% identity with group 2 and 31% identity with group 3. These data indicate that the five rice NPR1-like proteins are closer in identity to each other than the six Arabidopsis NPR1-like proteins are to each other. Within groups, rice NH2 shares 54% identity with NH3 while NH4 shares 62% identity with NH5. Between rice and Arabidopsis, rice NH1 shares the highest homology with Arabidopsis NPR1 and NPR2, carrying 46% identity and 66% similarity with NPR1 and 46% identity and 64% similarity with NPR2. NH2 has the highest homology to Arabidopsis NPR3 and NPR4, sharing 51% identity and 69% similarity with NPR3 and 51% identity and 70% similarity with NPR4; NH3 also shares the highest homology with Arabidopsis NPR3 and NPR4, carrying 48% identity and 65% similarity with NPR3 and 46% identity and 65% similarity with NPR4. NH4 is most homologous to Arabidopsis BOP2 (NPR5), sharing 70% identity and 78% similarity; NH5 is most homologous to Arabidopsis BOP1 (NPR6), sharing 65% identity and 78% similarity.
In some embodiments, the NH3 protein comprises SEQ ID NO:2, the rice NH3 protein sequence. Those of skill in the art will appreciate that variants of the rice NH3 protein sequence can be obtained either by identifying additional NH3 ortholog sequences from other plants, or by generating directed or random mutations in the sequences. In some embodiments, the NH3 protein is substantially similar to (e.g., at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO:2. In some embodiments, the NH3 protein is identical or substantially similar to any of SEQ ID NOs:4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19.
Embodiments of the present invention provide for use of the above proteins and/or nucleic acid sequences, encoding such polypeptides, in the methods and compositions (e.g., expression cassettes, plants, etc.) of the present invention. The isolation of a polynucleotide sequence encoding a NH3 protein may be accomplished by a number of techniques. For instance, oligonucleotide probes based on the NH3 coding sequences disclosed (e.g., as listed in the SEQUENCE LISTING) here can be used to identify the desired NH3 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 tissue, such as a leaf from a particular plant species, and a cDNA library containing the gene transcript of interest is prepared from the mRNA. Alternatively, cDNA may be prepared from mRNA extracted from other tissues in which the NH3 gene is expressed.
The cDNA or genomic library can then be screened using a probe based upon the sequence of a NH3 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 polypeptide can be used to screen an mRNA expression library.
Alternatively, the nucleic acid encoding the NH3 protein can be amplified from nucleic acid samples using amplification techniques. For instance, polymerase chain reaction (PCR) technology can be used to amplify the coding sequences of NH3 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 polynucleotide sequences encoding NH3 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 sequences from plant tissues are generated from comparisons of the sequences provided here with other related genes.
In some embodiments, the partial or entire genome of a number of plants has been sequenced and open reading frames identified. By a BLAST search, one can identify the coding sequence for a NH3 protein in various plants.
III. Recombinant Expression Vectors
Once a polynucleotide sequence encoding a NH3 polypeptide is obtained, it can be used to prepare an expression cassette for expressing the NH3 polypeptide in a transgenic plant, directed by a native or heterologous promoter. Increased expression of the NH3 polypeptide is useful, for example, to produce plants that have enhanced disease resistance, such as resistance to diseases induced by pathogens or resistance to diseases induced by chemicals such as BTH, compared to plants that do not have increased expression of the NH3 polypeptide.
Any of a number of means well known in the art can be used to drive NH3 expression in plants. 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, expression of the NH3 can be conditioned to only occur under certain conditions (e.g., using an inducible promoter).
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, e.g., Weising et al. Ann. Rev. Genet. 22:421-477 (1988). A DNA sequence coding for the desired polypeptide, for example the NH3 polypeptides as described herein, will 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, a plant promoter fragment may be employed to direct expression of the NH3 polypeptide 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.
Alternatively, the plant promoter may direct expression of the NH3 polypeptide in a specific tissue (tissue-specific promoters) or may be otherwise under more precise environmental control (inducible promoters). Examples of tissue-specific promoters under developmental control include promoters that initiate transcription only in certain tissues, such as leaves or guard cells (including but not limited to those described in WO/2005/085449; U.S. Pat. No. 6,653,535; Li et al., Sci China C Life Sci. 2005 April; 48(2):181-6; Husebye, et al., Plant Physiol, April 2002, Vol. 128, pp. 1180-1188; and Plesch, et al., Gene, Volume 249, Number 1, 16 May 2000, pp. 83-89(7)). Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, elevated temperature, or the presence of light.
If proper protein expression is desired, a polyadenylation region at the 3′-end of the coding region should be included. The polyadenylation region can be derived from a NH3 gene, from a variety of other plant genes, or from T-DNA.
Optionally, the polypeptide may comprise a protein tag that facilitates detection or purification of the polypeptide. The tag may be added to the N-terminal or C-terminal region of the polypeptide or internally within the polypeptide. Examples of suitable tags include, but are not limited to, Myc, FLAG, HA, His, glutathione-S-transferase (GST), tandem affinity purification (TAP), and fluorescent protein (e.g., GFP, YFP, EGFP, RFP, DsRed) tags.
The vector comprising the sequences (e.g., promoters or NH3 coding regions) 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 chlorosluforon or Basta.
In some embodiments, the promoter that is operably linked to a polynucleotide encoding a NH3 polypeptide increases the level of expression of NH3 in a transgenic plant into which the promoter operably linked to the polynucleotide encoding the NH3 polypeptide has been introduced. In some embodiments, the level of expression increases from about two-fold to about ten-fold in a transgenic plant comprising the promoter operably linked to the polynucleotide encoding the NH3 polypeptide, compared to a plant lacking the promoter operably linked to the polynucleotide encoding the NH3 polypeptide.
Native Promoters
In some embodiments, expression of a NH3 nucleic acid may be directed by its own native promoter or a portion thereof. In some embodiments, a “portion” of a promoter comprises a continuous length of a promoter sequence that is from about 100 nucleotides in length to about 10,000 nucleotides in length. In some embodiments, a portion of a promoter comprises a continuous length of a promoter sequence that is from about 500 nucleotides in length to about 5,000 nucleotides in length. The term “native” means the naturally occurring promoter sequence that directs naturally occurring NH3 expression in a plant. A native promoter that is operably linked to a polynucleotide of interest, e.g. a NH3 polynucleotide of the present invention, will direct expression of that polynucleotide of interest in those cell and tissue types, or during those environmental conditions and states of development or cell differentiation, in which the native promoter would drive expression under physiological conditions.
A native promoter or promoter element or portion thereof may be located either upstream or downstream of the gene coding sequence which is controlled by the promoter. In some embodiments, the native promoter comprises the nucleotide sequence upstream of the NH3 gene (also called OsNPR3) in rice. In some embodiments, the native promoter comprises the nucleotide sequence of SEQ ID NO:3 or a portion thereof (e.g., about 100, 200, 300, 400, 500, 600, 700, 800, or 900 contiguous nucleotides of SEQ ID NO:3) or a nucleotide sequence substantially similar to SEQ ID NO:3 or a portion thereof (e.g., about 100, 200, 300, 400, 500, 600, 700, 800, or 900 contiguous nucleotides of SEQ ID NO:3).
A native promoter operably linked to a polynucleotide encoding a polypeptide of interest, e.g. a NH3 polypeptide of the present invention, will express the NH3 polynucleotide under those physiological conditions in which the native promoter would normally direct gene expression. The native promoter may be operably linked to an identical or substantially similar polynucleotide as is normally expressed by the native promoter. For example, the native NH3 promoter in rice may be operably linked to a polynucleotide encoding the rice NH3 polypeptide; the native NH3 promoter in maize may be operably linked to a polynucleotide encoding the maize NH3 polypeptide ortholog; the native NH3 promoter in soybean may be operably linked to a polynucleotide encoding the soybean NH3 polypeptide ortholog; the native NH3 promoter in wheat may be operably linked to a polynucleotide encoding the wheat NH3 polypeptide ortholog; etc. Alternatively, the native promoter may be operably linked to a polynucleotide that is not identical, but is substantially similar, to the polynucleotide that is normally expressed by the native promoter. For example, the native NH3 promoter in rice may be operably linked to a polynucleotide encoding the maize NH3 polypeptide ortholog, soybean NH3 polypeptide ortholog, wheat NH3 polypeptide ortholog, etc.; or alternatively, the native NH3 promoter in maize, soybean, wheat, etc., may be operably linked to a polynucleotide encoding the rice NH3 polypeptide such as the polynucleotide of SEQ ID NO:1.
Inducible Promoters
Alternatively, a plant promoter may direct expression of the NH3 polynucleotide under the influence of changing environmental conditions or developmental conditions. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions, elevated temperature, drought, or the presence of light. Such promoters are referred to herein as “inducible” promoters.
Exemplary inducible promoters include those promoters that are specifically induced upon infection by a virulent pathogen. Selected promoters useful in the invention are discussed in PCT application WO 99/43824, and include promoters from:

    
    
        a. lipoxygenases (e.g., Peng et al, J. Biol. Chem. 269:3755-3761 (1994)),
        b. peroxidases (e.g., Chittoor et al. Molec. Plant-Microbe Interact. 10:861-871 (1997)),
        c. hydroxymethylglutaryl-CoA reductase,
        d. phenylalanine ammonia lyase,
        e. glutathione-S-transferase,
        f. chitinases (e.g., Zhu et al. Mol. Gen. Genet. 226:289-296 (1991)),
        g. genes involved in the plant respiratory burst (e.g., Groom et al. Plant J. 10(3):515-522 (1996)); and
        h. pathogenesis-related (PR) protein promoters.
    
    


Alternatively, plant promoters which are inducible upon exposure to plant hormones, such as auxins, are used to express the NH3 polynucleotide. For example, the invention can use the auxin-response elements E1 promoter fragment (AuxREs) in the soybean (Glycine max L.) (Liu (1997) Plant Physiol. 115:397-407); the auxin-responsive Arabidopsis GST6 promoter (also responsive to salicylic acid and hydrogen peroxide) (Chen (1996) Plant J. 10: 955-966); the auxin-inducible parC promoter from tobacco (Sakai (1996) 37:906-913); a plant biotin response element (Streit (1997) Mol. Plant. Microbe Interact. 10:933-937); and, the promoter responsive to the stress hormone abscisic acid (Sheen (1996) Science 274:1900-1902).
Plant promoters inducible upon exposure to chemical reagents that may be applied to the plant, such as herbicides or antibiotics, are also useful for expressing the NH3 polynucleotide. For example, the maize In2-2 promoter, activated by benzenesulfonamide herbicide safeners, can be used (De Veylder (1997) Plant Cell Physiol. 38:568-577); application of different herbicide safeners induces distinct gene expression patterns, including expression in the root, hydathodes, and the shoot apical meristem. A NH3 coding sequence can also be under the control of, e.g., a tetracycline-inducible promoter, e.g., as described with transgenic tobacco plants containing the Avena sativa L. (oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 11:465-473); or, a salicylic acid-responsive element (Stange (1997) Plant J. 11:1315-1324; Uknes et al., Plant Cell 5:159-169 (1993); Bi et al., Plant J. 8:235-245 (1995)).
Examples of useful inducible regulatory elements include copper-inducible regulatory elements (Mett et al., Proc. Natl. Acad. Sci. USA 90:4567-4571 (1993); Furst et al., Cell 55:705-717 (1988)); tetracycline and chlor-tetracycline-inducible regulatory elements (Gatz et al., Plant J. 2:397-404 (1992); Roder et al., Mol. Gen. Genet. 243:32-38 (1994); Gatz, Meth. Cell Biol. 50:411-424 (1995)); ecdysone inducible regulatory elements (Christopherson et al., Proc. Natl. Acad. Sci. USA 89:6314-6318 (1992); Kreutzweiser et al., Ecotoxicol. Environ. Safety 28:14-24 (1994)); heat shock inducible regulatory elements (Takahashi et al., Plant Physiol. 99:383-390 (1992); Yabe et al., Plant Cell Physiol. 35:1207-1219 (1994); Ueda et al., Mol. Gen. Genet. 250:533-539 (1996)); and lac operon elements, which are used in combination with a constitutively expressed lac repressor to confer, for example, IPTG-inducible expression (Wilde et al., EMBO J. 11:1251-1259 (1992)). An inducible regulatory element useful in the transgenic plants of the invention also can be, for example, a nitrate-inducible promoter derived from the spinach nitrite reductase gene (Back et al., Plant Mol. Biol. 17:9 (1991)) or a light-inducible promoter, such as that associated with the small subunit of RuBP carboxylase or the LHCP gene families (Feinbaum et al., Mol. Gen. Genet. 226:449 (1991); Lam and Chua, Science 248:471 (1990)).
Tissue-Specific Promoters
Alternatively, the plant promoter may direct expression of the NH3 polypeptide in a specific tissue (tissue-specific promoters). Tissue specific promoters are transcriptional control elements that are only active in particular cells or tissues at specific times during plant development, such as in vegetative tissues or reproductive tissues.
Examples of tissue-specific promoters under developmental control include promoters that initiate transcription only (or primarily only) in certain tissues, such as vegetative tissues, e.g., roots or leaves, or reproductive tissues, such as fruit, ovules, seeds, pollen, pistols, flowers, or any embryonic tissue, or epidermis or mesophyll. Reproductive tissue-specific promoters may be, e.g., ovule-specific, embryo-specific, endosperm-specific, integument-specific, seed and seed coat-specific, pollen-specific, petal-specific, sepal-specific, or some combination thereof. In some embodiments, the promoter is cell-type specific, e.g., guard cell-specific.
Other tissue-specific promoters include seed promoters. Suitable seed-specific promoters are derived from the following genes: MAC1 from maize (Sheridan (1996) Genetics 142:1009-1020); Cat3 from maize (GenBank No. L05934, Abler (1993) Plant Mol. Biol. 22:10131-1038); vivparous-1 from Arabidopsis (Genbank No. U93215); atmyc1 from Arabidopsis (Urao (1996) Plant Mol. Biol. 32:571-57; Conceicao (1994) Plant 5:493-505); napA from Brassica napus (GenBank No. J02798, Josefsson (1987) JBL 26:12196-1301); and the napin gene family from Brassica napus (Sjodahl (1995) Planta 197:264-271).
A variety of promoters specifically active in vegetative tissues, such as leaves, stems, roots and tubers, can also be used to express polynucleotides encoding NH3 polypeptides. For example, promoters controlling patatin, the major storage protein of the potato tuber, can be used, see, e.g., Kim (1994) Plant Mol. Biol. 26:603-615; Martin (1997) Plant J. 11:53-62. The ORF13 promoter from Agrobacterium rhizogenes that exhibits high activity in roots can also be used (Hansen (1997) Mol. Gen. Genet. 254:337-343. Other useful vegetative tissue-specific promoters include: the tarin promoter of the gene encoding a globulin from a major taro (Colocasia esculenta L. Schott) corm protein family, tarin (Bezerra (1995) Plant Mol. Biol. 28:137-144); the curculin promoter active during taro corm development (de Castro (1992) Plant Cell 4:1549-1559) and the promoter for the tobacco root-specific gene TobRB7, whose expression is localized to root meristem and immature central cylinder regions (Yamamoto (1991) Plant Cell 3:371-382).
Leaf-specific promoters, such as the ribulose biphosphate carboxylase (RBCS) promoters can be used. For example, the tomato RBCS1, RBCS2 and RBCS3A genes are expressed in leaves and light-grown seedlings, only RBCS1 and RBCS2 are expressed in developing tomato fruits (Meier (1997) FEBS Lett. 415:91-95). A ribulose bisphosphate carboxylase promoters expressed almost exclusively in mesophyll cells in leaf blades and leaf sheaths at high levels, described by Matsuoka (1994) Plant J. 6:311-319, can be used. Another leaf-specific promoter is the light harvesting chlorophyll a/b binding protein gene promoter, see, e.g., Shiina (1997) Plant Physiol. 115:477-483; Casal (1998) Plant Physiol. 116:1533-1538. The Arabidopsis thaliana myb-related gene promoter (Atmyb5) described by Li (1996) FEBS Lett. 379:117-121, is leaf-specific. The Atmyb5 promoter is expressed in developing leaf trichomes, stipules, and epidermal cells on the margins of young rosette and cauline leaves, and in immature seeds. Atmyb5 mRNA appears between fertilization and the 16 cell stage of embryo development and persists beyond the heart stage. A leaf promoter identified in maize by Busk (1997) Plant J. 11:1285-1295, can also be used.
Another class of useful vegetative tissue-specific promoters are meristematic (root tip and shoot apex) promoters. For example, the “SHOOTMERISTEMLESS” and “SCARECROW” promoters, which are active in the developing shoot or root apical meristems, described by Di Laurenzio (1996) Cell 86:423-433; and, Long (1996) Nature 379:66-69; can be used. Another useful promoter is that which controls the expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase HMG2 gene, whose expression is restricted to meristematic and floral (secretory zone of the stigma, mature pollen grains, gynoecium vascular tissue, and fertilized ovules) tissues (see, e.g., Enjuto (1995) Plant Cell. 7:517-527). Also useful are knl-related genes from maize and other species which show meristem-specific expression, see, e.g., Granger (1996) Plant Mol. Biol. 31:373-378; Kerstetter (1994) Plant Cell 6:1877-1887; Hake (1995) Philos. Trans. R. Soc. Lond. B. Biol. Sci. 350:45-51. For example, the Arabidopsis thaliana KNAT1 promoter (see, e.g., Lincoln (1994) Plant Cell 6:1859-1876).
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, but may also lead to some expression in other tissues as well.
In another embodiment, a nucleic acid described in the present invention is expressed through a transposable element. This allows for constitutive, yet periodic and infrequent expression of the constitutively active polypeptide. The invention also provides for use of tissue-specific promoters derived from viruses which can include, e.g., the tobamovirus subgenomic promoter (Kumagai (1995) Proc. Natl. Acad. Sci. USA 92:1679-1683) the rice tungro bacilliform virus (RTBV), which replicates only in phloem cells in infected rice plants, with its promoter which drives strong phloem-specific reporter gene expression; the cassava vein mosaic virus (CVMV) promoter, with highest activity in vascular elements, in leaf mesophyll cells, and in root tips (Verdaguer (1996) Plant Mol. Biol. 31:1129-1139).
IV. Production of Transgenic Plants
As detailed herein, embodiments of the present invention provide for transgenic plants comprising recombinant expression cassettes for expressing a NH3 polypeptide as described herein. In some embodiments, a transgenic plant is generated that contains a complete or partial sequence of a polynucleotide that is derived from a species other than the species of the transgenic plant. It should be recognized that transgenic plants encompass the plant or plant cell in which the expression cassette is introduced as well as progeny of such plants or plant cells that contain the expression cassette, including the progeny that have the expression cassette stably integrated in a chromosome.
In some embodiments, the transgenic plants comprising recombinant expression cassettes for expressing a NH3 polypeptide express NH3 at higher levels than a plant lacking the recombinant expression cassette for expressing a NH3 polypeptide. In some embodiments, the transgenic plants comprising recombinant expression cassettes for expressing a NH3 polypeptide express NH3 at levels that are from about two-fold to about four-fold higher than a plant lacking the recombinant expression cassette for expressing a NH3 polypeptide.
In some embodiments, the transgenic plants comprising recombinant expression cassettes for expressing a NH3 polypeptide express NH3 at a level that results in increased disease resistance compared to a plant lacking the recombinant expression cassette for expressing a NH3 polypeptide, wherein the transgenic plants comprising recombinant expression cassettes for expressing a NH3 polypeptide have about the same growth as a plant lacking the recombinant expression cassette for expressing a NH3 polypeptide. In some embodiments, the transgenic plant comprising a recombinant expression cassette for expressing a NH3 polypeptide has no more than about a 10% reduction in growth compared to a plant lacking the recombinant expression cassette for expressing a NH3 polypeptide. In some embodiments, the transgenic plant comprising a recombinant expression cassette for expressing a NH3 polypeptide does not produce significantly more lesion mimics compared to a plant lacking the recombinant expression cassette for expressing a NH3 polypeptide.
A recombinant expression vector comprising a NH3 coding sequence driven by promoter 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 construct can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment. Alternatively, the DNA construct 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. While transient expression of NH3 is encompassed by the invention, generally expression of construction of the invention will be from insertion of expression cassettes into the plant genome, e.g., such that at least some plant offspring also contain the integrated expression cassette.
Microinjection techniques are also useful for this purpose. These techniques are well known in the art and thoroughly described in the 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).
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).
Transformed plant cells derived by any of the above transformation techniques can be cultured to regenerate a whole plant that possesses the transformed genotype and thus the desired phenotype such as enhanced disease resistance. Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker which 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).
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.
The expression cassettes of the invention can be used to confer disease resistance on essentially any plant. Thus, the invention has use over a broad range of plants, including species from the genera Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucumis, Cucurbita, Daucus, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Oryza, Panieum, Pannesetum, Persea, Pisum, Pyrus, Prunus, Raphanus, Secale, Senecio, Sinapis, Solanum, Sorghum, Trigonella, Triticum, Vitis, Vigna, and, Zea. In some embodiments, the plant is selected from the group consisting of rice, maize, wheat, soybeans, cotton, canola, turfgrass, and alfalfa. In some embodiments, the plant is an ornamental plant. In some embodiments, the plant is a vegetable- or fruit-producing plant. In some embodiments, the plant is a monocot. In some embodiments, the plant is a dicot.
V. Selecting for Plants with Enhanced Disease 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)). Alternatively, another method of selecting plants with enhanced resistance is to determine resistance of a plant to a specific compound that induces Systemic Acquired Resistance. Such compounds include, but are not limited to, salicylic acid, 2,6-dichloroisonicotinic acid (INA), benzothiadiazole (BTH), and probenazole (see, e.g., Ward et al., 1991; Gorlach et al., 1996; Schweizer et al., 1999; and Morris et al., 1998)). One of skill in the art will recognize that resistance responses of plants vary depending on many factors, including what pathogen, compound, 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. (1995) Science 268(5211): 661-7). 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 defense related promoter. Measurement of such expression can be measured by quantifying 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: β-gal, GUS (see, e.g., Jefferson, R. A., et al., (1987) EMBO J. 6:3901-3907), green fluorescent protein, luciferase, and others.
EXAMPLES
The following examples are offered to illustrate, but not limit the claimed invention.
NPR1 (nonexpresser of pathogenesis related genes 1) is the master regulator of salicylic acid-mediated systemic acquired resistance. Overexpression of Arabidopsis NPR1 and rice NH1 (NPR1 homolog1)/OsNPR1 in rice results in enhanced resistance. While there are four rice NPR1 paralogs in the rice genome, none have been demonstrated to function in disease resistance. To study rice NPR1 paralog 3, we introduced constructs into rice and tested for effects on resistance to infection by Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight. While overexpression of NH3 using the maize ubiquitin-1 promoter failed to enhance resistance, introduction of an extra copy of NH3 driven by its own promoter (nNT-NH3) resulted in clear, enhanced resistance. Progeny analysis confirms that the enhanced resistance phenotype, measured by Xoo-induced lesion length, is associated with the NH3 transgene. Bacterial growth curve analysis indicates that bacterial population levels are reduced 10-fold in nNT-NH3 lines compared to control rice lines. The transgenic plants exhibit higher sensitivity to BTH (benzothiadiazole) and INA (2,6-dichloroisonicotinic acid) treatment as measured by increased cell death. Expression analysis of pathogenesis-related (PR) genes showed that nNT-NH3 plants display greatly enhanced induction of PR genes only after treatment with BTH. Our study demonstrates an alternative method to employ a regulatory protein to enhance plant defense. This approach avoids using undesirable constitutive, high-level expression and may prove to be more practical for engineering resistance.
Construct Using the Ubi-1 Promoter to Overexpress NH3 Yielded No Transgenic Plants with Enhanced Resistance
Overexpression is a useful tool to study of the possible function of a gene of interest. The Ubi-1 promoter is commonly used for such purpose because of its ability to express a gene to very high levels, especially in monocots, including rice. In an attempt to study possible involvement of rice NH1 paralogs in disease resistance, we tried to overexpress the rice NH3 (also called OsNPR3) gene using the maize Ubi-1 promoter in the Kitaake (Kit) rice variety. A 1.8 kb rice NH3 cDNA was introduced into a Ubi-C1300-based binary vector. We were able to produce many (approximately 20) transgenic green calli and generated more than 10 transgenic rice seedlings. Upon transfer to greenhouse, most of these transgenic rice plants developed lesion mimics and dwarfism and eventually died. Only four plants eventually survived. These plants were challenged with Xoo pathogen PXO99 to test for possible effects on disease resistance. No obvious enhanced disease resistance was observed in these transgenic plants, as shown in FIG. 2A.
Enhanced Disease Resistance is Accomplished by Introduction of a Modified Copy of NH3
In the same attempt to study the function of NH3, we also created a binary construct based on the C1300 vector in which expression of the NH3 cDNA is directed by its own native promoter, contained in a one-kb DNA fragment. An N-terminal tag (NTAP:N-terminus tandem affinity purification) was added to the NH3 protein to potentially facilitate detection of the protein. This construct is designated nNT-NH3. When this construct was introduced into the Kit recipient, 12 transgenic plants were obtained and eventually survived. These plants were subject to challenge by Xoo. Our results (FIG. 2a) showed that about half (7 out of 12) of the TO transgenic plants exhibit obvious enhanced resistance to Xoo.
Progeny segregation analysis was performed subsequently to test if the observed enhanced resistance phenotype is associated with the nNT-NH3 transgene. The presence of the nNT-NH3 transgene was detected by conducting polymerase-chain-reactions (PCR) targeting the NH3 mini-gene, which differs from the endogenous NH3 gene in lacking introns. Shown in FIG. 2b are progeny segregation analysis results of such three (lines #3A, 6A, and 13A) transgenic lines. The results clearly show that all progeny plants containing the nNT-NH3 gene (closed bars in FIG. 2b) are resistant to PXO99. On the contrary, null segregants (open bars), which no longer contain the transgene, are as susceptible to PXO99 as the wild type control Kit plants.
A more detailed analysis of the progeny of lines #6A and 13A was conducted to study the resistance phenotype. Progeny plants were genotyped for the presence of the nNT-NH3 gene. Those that contained the gene (both homozygous and heterozygous) were pooled together to represent lines #6A (labeled NH3-6) and #13A (labeled NH3-13), respectively. FIG. 3a shows the lesion development and 3b the growth curves of Xoo. Consistent with previous results, FIG. 3a shows that NH3-6 and NH3-13 plants clearly developed much shorter lesions compared with the Kit control at day 8 and day 12. Bacterial growth curves in FIG. 3b also show clear differences between the NH3-6 and NH3-13 transgenic plants and the Kit control at day 8 and day 12. The bacterial populations in Kit are more than 10 fold higher than those in the transgenic plants. T-tests on NH3-6 vs Kit and NH3-13 vs Kit at day 12 yield p values=0.0036 and 0.0042, respectively, indicating that the differences are highly statistically significant. Two typical leaves (12 days after Xoo inoculation) from each line and the Kit control are shown in FIG. 3c. An RT-PCR was conducted to confirm that transgenic lines NH3-6 and NH3-13 express the nNT-NH3 gene. RT-PCR results shown in FIG. 3d reveal that the NH3 transcript level is higher in both NH3-6 and NH3-13 (including both native NH3 and NTAP-NH3 transcripts) than in Kit (by approximately 3-fold) and that the NTAP-tagged NH3 is expressed only in the transgenic plants. These results clearly demonstrate that introduction of the nNT-NH3 transgene into wild type rice enhances resistance to the Xoo pathogen.
NH3 Transgenic Plants Show Strong Responses to Plant Defense Activators
We have previously reported that application of BTH to the Ubi-NH1 transgenic plants induces/enhances lesion mimic development (Chern et al., 2005). We tested if the nNT-NH3 transgenic plants would have a similar response to BTH treatment. To our surprise, when sprayed with 10 mM BTH, the nNT-NH3 plants responded to BTH very strongly, leading to extremely severe cell death. As shown in FIG. 4a, the severity of the cell death induced by BTH treatment is extremely high, as evidenced by the dry-out of whole leaves within 3-4 days. On the contrary, plants that carry an NTAP control driven by the Ubi-1 promoter (Park et al., 2008) are completely clear of such cell death phenotype. These results suggest that the development of the severe cell death phenotype after BTH application is due to the NH3 transgene.
We then treated the nNT-NH3 plants as well as the Kit control with 1 mM BTH or 1 mM INA to test for effects. When treated with 1 mM BTH, the nNT-NH3 plants developed lesion mimic spots in general together with some leaves showing bleached areas, representing severe cell death, as shown by the two representative leaves in FIG. 4b (labeled NH3, BTH). In contrast, when treated with 1 mM BTH, Kit developed no lesion mimic spots at all and was free of bleached areas (see Kit, BTH). Mock nNT-NH3 plants treated with no BTH only occasionally carry some small lesion mimic spots (NH3, mock). When treated with 1 mM INA, the nNT-NH3 plants developed typical lesion-mimic spots, but no bleached areas on leaves (NH3, INA). Treatment with 1 mM INA had little effects on Kit plants (Kit, INA). These results suggest that the nNT-NH3 plants respond to plant defense activators, such as BTH and INA, with clearly elevated sensitivity.
Defense-Related Genes are Induced More Strongly in the NH3 Transgenic Plants
To investigate the BTH- and INA-induced responses at the molecular level, we looked at the transcript expression levels of several rice defense-related genes, including PR1a, PR3, PR10, POX (peroxidase), and NH1. RT-PCR results of these genes for response to BTH treatment are shown in FIG. 5a and real-time quantitative PCR results in FIG. 5b. The input cDNA amounts were equalized first by using actin as the reference in FIG. 5a. Quantitative expression data in FIG. 5b are expressed as fold of actin gene expression. As expected, the NH3 levels in the nNT-NH3 plants (filled bar) are elevated by about 3- to 4-fold compared to Kit (open bar) control before treatment. Interestingly, in Kit control plants (open bars), the NH3 transcript levels appear slightly reduced after BTH application, especially at day 4 and day 8. In nNT-NH3 plants, the NH3 levels appear further modestly induced at day 1, but otherwise remain constantly elevated compared to Kit after BTH treatment. Mock treatment appears also has effects on the NH3 levels. In Kit controls, NH1 levels remain relatively constant, except at day 4 and day 8 where the NH1 levels appear slightly reduced. Interestingly in untreated nNT-NH3 plants, the NH1 level is reduced by half compared to Kit and is induced by about 3.5-fold (2-fold compared to untreated Kit) one day after BTH treatment; the NH1 levels then remain at levels compatible to that of untreated Kit control, except at day 8, where it is significantly higher.
Results in FIGS. 5a and 5b show that expression of defense gene PR1a is at comparable levels in Kit and in nNT-NH3 plants before treatment, both at very low levels. Upon 1 mM BTH treatment, PR1a expression is induced up to 11-fold in Kit. PR1a is induced dramatically more by BTH (up to 244-fold) in the nNT-NH3 plants than in Kit. The PR3 expression level in nNT-NH3 plants is about two fold that in Kit. PR3 expression is slightly (up to 2-fold) induced by BTH in Kit, but strongly induced (up to 10-fold) by BTH in nNT-NH3 plants. The PR10 level in nNT-NH3 plants is lower than in Kit. Similarly, expression of PR10 is slightly induced (up to 2-fold) in Kit but strongly induced (up to 51-fold over untreated nNT-NH3 or 12-fold over untreated Kit) in nNT-NH3 plants. Expression levels of peroxidase genes POX8.1 and POX22.3 in nNT-NH3 are comparable to those in Kit. BTH treatment significantly induces POX8.1 and POX22.3 expression in both Kit and nNT-NH3 plants, but more strongly for POX22.3 in nNT-NH3 plants. However, BTH treatment results in a faster drop in the POX8.1 expression level, leading to a lower level of POX8.1 in nNT-NH3 than in Kit at day 4.
Results in FIG. 5c show that 1 mM INA treatment also slightly reduces the levels of NH3 expression. However, the NH3 levels in the nNT-NH3 plants remain constantly elevated. Treatment with INA slightly induces expression of PR1a, PR3, and PR10 in Kit plants. The elevated levels of NH3 have no obvious effects on induction of these genes by INA. INA also induces expression of POX in Kit and nNT-NH3 plants. The elevated levels of NH3 appear to have similar but mild effects on peroxidase gene expression.
These results suggest that elevated NH3 levels, in general, enhance induction of defense-related genes, but may have the opposite effect on some other genes, such as peroxidases, at certain stages. Nevertheless, these results support the observation that higher levels of NH3 transcript enhance responses to BTH treatment, which induces PR gene expression and disease resistance. More importantly, even at these higher levels of NH3 transcript, the defense-related genes are not highly induced without inducer treatment.
The Enhanced Disease Resistance Phenotype is Due to the NH3 protein itself
There are no reports so far describing possible involvement of rice NH3 in plant defense or any function of NH3. The enhanced disease resistance effects and elevated responses to BTH and INA, conferred by the nNT-NH3 plants, are most likely due to the function of the NH3 protein itself because control transgenic plants expressing the NTAP tag alone show no such effects (see FIG. 4a above; Park et al., 2008). However, the possibility remains that introducing the NTAP tag to the N-terminus of the NH3 protein may cause the protein to function differently, leading to the observed phenotypes. To rule out this possibility, we created another modified NH3 construct, NH3Myc, which would produce an NH3 protein fused to the c-Myc tag at the C-terminus and its expression driven by the same 1 kb NH3 promoter. We transformed Kit with this construct and generated more than 20 TO transgenic plants. These plants were challenged with PXO99 along with Kit as control. Approximately half of the NH3Myc transgenic plants show high levels of enhanced resistance to Xoo challenge, as shown in FIG. 6, similar to the case of nNT-NH3 transgenic plants. These results show that the NH3Myc construct has the same function as the nNT-NH3 construct. Because the NH3Myc protein carries a tag different from the TAP tag at a totally different location, these results suggest that the observed effects are from the function of the NH3 protein itself.
Discussion
Despite numerous studies using NPR1 or its orthologs to achieve enhanced disease resistance in many plant species (Cao et al., 1998; Friedrich et al., 2001; Chern et al., 2001; Lin et al., 2004; Chern et al., 2005a; Le et al., 2009), for application purposes, no NPR1 paralogous genes have been shown to be able to enhance disease resistance until now. In Arabidopsis, BOP1 (NPR6) and BOP2 (NPR5) were shown to regulate leaf development. Both Arabidopsis and the rice NPR1-like proteins form three groups, with BOP1 and BOP2 being the most distant members from NPR1. Thus, this group of NPR1-like proteins may function to regulate leaf development rather than defense. In support of this hypothesis, we have shown that increased expression of rice NH4 or NH5, using either their native promoters or the maize Ubi-1 promoter, do not confer enhanced resistance in transgenic plants (M. S. Chern and P. Ronald, unpublished). These observations are consistent with Arabidopsis results supporting the notion that this group of NPR1-like proteins may not be involved in regulating the plant defense response.
Liu et al. (2005) reported that Arabidopsis NPR4 is required for basal resistance to Pseudomonas syringae pv. tomato (Pst) DC3000 and Erysiphe cichoracearum because they observed that the npr4-1 mutant is more susceptible to these two pathogens. However, Zhang et al. (2006) reported that Arabidopsis NPR3 and NPR4 are negative regulators of PR gene expression and disease resistance. Zhang et al. showed that npr3 mutants have slightly increased basal PR-1 expression and the npr3npr4 double mutant shows higher PR-1, PR-2, and PR-5 expression. The double mutant plants display enhanced resistance against virulent bacteria, such as Pst DC3000, and oomycete pathogens (Zhang et al., 2006). The conclusions of these two reports contradict each other. Moreover, other than NPR1 itself, no Arabidopsis NPR1-like genes have been shown to enhance disease resistance when expressed at higher levels.
Yuan et al. (2007) showed that overexpression of OsNPR2 and OsNPR3 in rice had no significant effects on resistance to Xoo. Thus, whether any of the rice NPR1 paralogs is similarly involved in regulating defense responses was in serious doubt. We tried to overexpress NH3 in the Kit rice variety by using the maize Ubi-1 promoter, which has been successfully used in many cases to overexpress a gene in rice (Chern et al., 2001; Chern et al., 2005a; Chern et al., 2005b). We failed to obtain plants carrying enhanced resistance. Instead, most of the transgenic plants died before or after transfer to greenhouse. Presumably ultra high levels of NH3 expression, as driven by the Ubi-1 promoter (whose expression often reaches 10-fold or higher than the endogenous gene), cause toxicity leading to lethality. A similar situation may have occurred in Yuan's overexpression experiment with the OsNPR3 gene when they used the Ubi-1 promoter.
When we used the NH3 native promoter to drive the expression, many of the transgenic plants exhibited enhanced resistance. These native promoter NH3 transgenic plants survived, most likely because they do not express extremely high levels of NH3, but only moderate levels (about 3- to 4-fold higher than the endogenous level) sufficient to enhance resistance. In addition, the native promoter NH3 constructs may express NH3 only in relevant tissues at the right developmental stages and the right timing, minimizing possible detrimental effects. This is supported by the observation that expression of PR genes in nNT-NH3 plants is not highly elevated until induction by BTH (FIGS. 5a and 5b). However, once it is triggered by BTH, PR1a induction reaches as high as 244-fold in the nNT-NH3 plants whereas induction by BTH reaches 11-fold in Kit plants. Thus, the NH3 transgene has little effects before BTH induction but magnifies the BTH effect by 20-fold. Practically, this likely means that less BTH is needed to induce same levels of defense response. In contrast, rice transgenic plants carrying the Ubi-NH1 transgene, which exhibit normal developmental phenotypes, show elevated levels of PR gene expression without induction (Chern et al., 2005). Thus, the use of native promoter to express an extra copy of NH3 clearly carries advantages. In Arabidopsis, the NPR1 transcript level is elevated only two to three-fold after induction (Cao et al., 1997). These levels of NPR1 transcript are high enough to enhance disease resistance significantly. In the results presented here, introduction of an extra copy of NH3 is able to achieve similar levels (3- to 4-fold) of NH3 transcript, leading to enhanced disease resistance. Similarly, we have also used the same strategy to introduce an extra copy of NH1 into Kit rice and observed enhanced resistance (Chern and Ronald, unpublished). Therefore, this strategy may be widely applicable when using regulatory genes, such as NH3 and NH1, to engineer plants with enhanced disease resistance and other desirable traits.
The dramatic cell death responses of the nNT-NH3 plants to BTH are striking. At 1 mM of BTH, these plants start to show bleached areas on leaves, a symptom of severe cell death and strongly activated defense response. At 10 mM of BTH, treated leaves are bleached and dried out completely. These symptoms are more severe than those observed on NH1 over-expression plants treated with the same concentrations of BTH. Thus, NH3 may respond to BTH more dramatically than NH1 in certain ways. Additional molecular characterization is needed to reveal the mechanism leading to the difference in response to BTH.
It is interesting to notice that while the NH1 transcript level is lower in nNT-NH3 than in Kit plants without induction, it is induced to a level twice as high as that in Kit plants one day after BTH treatment (FIG. 5b). In contrast, in Kit plants after application of 1 mM BTH, NH1 expression levels are relatively unchanged. These data suggest that the levels of NH1 and NH3 expression may be highly coordinated in order to maintain a cellular homeostatic state. In support of this notion, our microarray results show that, in the NH1 over-expression plants, NH3 expression is up-regulated by 1.6 fold (unpublished). This data is consistent with the idea that NH3 is involved in plant defense responses. Also in support of a positive role for NH3 in plant defense response is that our microarray data on NRR over-expressing (NRRox) plants show a 2.4 fold down-regulation of NH3 expression (unpublished). NRRox plants exhibit super-susceptibility, evidenced by long lesions and few HR-like spots following Xoo challenge (Chern et al., 2005b). Down-regulation of NH3 expression in NRRox plants may contribute to the super-susceptible phenotype.
Experimental Procedures
Plant Materials, Growth Conditions, and Pathogen Challenge
The Kitaake (Kit) japonica rice (Oryza sativa L) cultivar was used for this study. Kit rice is susceptible to the Philippine Xoo strain PXO99AZ. Rice plants were grown in green houses at UC Davis at 27-32° C. under sunlight. For Xoo inoculation, 5-6 weeks old plants were transferred to a growth chamber and inoculated with PXO99AZ by the scissor-dip method (Kauffman et al., 1973). Xoo growth curve measurements were conducted as described before (Chern et al., 2005b). Growth chambers were set at the same temperatures with a day/night time cycle of 14 h/10 h.
Gene Isolation and Plasmid Construction
NH3 cDNA was amplified from a Nipponbare rice cDNA pool with primers NH3TAP1 (5′CACCGAGACGTCCACCATAAGCTTCTC3; SEQ ID NO:20) and NH3TAP3 (5′ACTGCAGATTAGACTTAACTGCTG3; SEQ ID NO:21). The NH3 cDNA PCR product was cloned into the pENTR-D vector and confirmed by sequencing. The one-kb NH3 promoter was amplified with primers NH3P-1 (5′TTTTAAGCTTCGTTGGATGAACTACATTGCTGAT3′; SEQ ID NO:22) and NH3P-2 (5′TTGGATCCAGATCTTATCCGGAAATTTCGCGCGTGT3; SEQ ID NO:23) and cloned into pBluescript II SK—using HindIII+BamHI. The insert was sequence-confirmed. The NH3 cDNA was cloned into the Gateway-compatible Ubi-NTAP-1300 vector (Rohila et al., 2006) by LR recombination to generate the Ubi-NT-NH3 over-expression construct. To create a native promoter NH3 construct, the NH3 promoter was first cloned into the Ubi-NTAP-1300 vector using HindIII+BglII to replace the Ubi-1 promoter. The resultant plasmid was used to accommodate the NH3 cDNA by recombination, yielding the nNT-NH3 construct.
For the NH3-Myc construction, the same one-kb NH3 promoter fragment (NH3P1.0) was amplified with primers NH3P-3 (5′CACCTCGTTGGATGAACTACATTGCTGAT3; SEQ ID NO:24) and NH3P-NcoI (5′TCCATGGCTCTTATCCGGAAATTTCGCGCGTGT3; SEQ ID NO:25) and cloned into the pENTR-D vector. The NH3 cDNA was re-amplified sequentially, first with primers NH3ATG (5′CACCATGGAGACGTCCACCATAAG3; SEQ ID NO:26) and NH3-cMyc (5′GGAGATGAGCTTCTGCTCCCGTGATAGCTTCCCTTTCTTG3′; SEQ ID NO:27), then with primers NH3ATG and cMyc-SpeI (5′ACTAGTTATTTCTCCAACAGGTCTTCCTCGGAGATGAGCTTCTGCTC3′; SEQ ID NO:28). The NH3Myc PCR product was cloned into the pENTR-D vector and confirmed by sequencing. The NH3P1.0 fragment was excised with NotI+NcoI and cloned into the NH3Myc/pENTR plasmid, pre-digested with NcoI+NotI, generating the P1-NH3Myc/pENTR construct. This construct was used to transfer the P1-NH3Myc fragment into a Gateway-C1300 vector by recombination, creating P1-NH3Myc/C1300. The P1-NH3Myc construct was used to transform Kit rice, generating NH3Myc transgenic plants.
Treatment with Plant Defense Activators
For 10 mM BTH treatment, the commercial product Actigard (Syngenta) was first used for foliar spray on the rice plants. Pure BTH chemical Acibenzolar-5-methyl (Wako) was subsequently used in place of Actigard. One mM of BTH and INA solutions were prepared in 0.05% Tween 20 for foliar spray.
RNA Extraction and RT-PCR
Leaf samples were collected and frozen immediately in liquid nitrogen. Leaf samples were stored at −80° C. until use. Total RNA was extracted using the Trizol reagent (Sigma) according to the manufacturer's instruction. RNA was reverse-transcribed using reverse transcriptase Superscript (Invitrogen). Approximately 10 μg of RNA was used for each reverse transcription. RT-PCR was first performed with primers targeting an actin gene as the reference. An equal amount of cDNA input was then used for RT-PCR for NH3 or defense-related genes. RT-PCR for NH3 used primers NH3-RT1 (5′GTGCATTGGCGTCTTACAGCA3′; SEQ ID NO:29) and NH3-RT2 (5′GGGAAGTATCGTCGTCCGAGT3; SEQ ID NO:30). RT-PCR for NTAP-NH3 used primers NH3-2 (5′GTGGCTGCAGCCGTCGTCCA3; SEQ ID NO:31) and NTAP-5 (5′ATGCCCAAGCCCCAAAGGACTACG3; SEQ ID NO:32). RT-PCR for NH1 used primers NH1-RT1 (5′ACTTAGCTCGGATGACGGCAC3; SEQ ID NO:33) and NH1-TAP2 (5′AGCAATGGTGTTCATCTCCTTGGT3; SEQ ID NO:34). Genes PR1a (Os07g03710) and PR10 (Os12g36830) as markers for plants defense have been described before (Park et al., 2008).
Quantitative real time PCR were performed on a Bio-Rad CFX96 Real-Time System coupled to a C1000 Thermal Cycler (Bio-Rad). For qPCR reactions, the Bio-Rad SsoFast EvaGreen Supermix was used. QPCR primers used are as follows: Actin-Q1 (TCGGCTCTGAATGTACCTCCTA; SEQ ID NO:35) and Actin-Q2 (CACTTGAGTAAAGACTGTCACTTG; SEQ ID NO:36) for actin gene; NH1-RT3 (CTGATCCGTTTCCCTCGGA; SEQ ID NO:37) and NH1-RT4 (GACCTGTCATTCTCCTCCTTG; SEQ ID NO:38) for the NH1 gene; NH3-RT3 (TGCTACACCTCTGCTGGTTGA; SEQ ID NO:39) and NH3-RT4 (GACCAGCAAACTCTTGAGTTGAG; SEQ ID NO:40) for the NH3 gene; PR3-1 (CTTGGACTGCTACAACCAGA; SEQ ID NO:41) and PR3-2 (CATTGTGGGCATTACTGATG; SEQ ID NO:42) for the PR3 gene; POX8.1-1 (CAAACTGGATACAAAAGCAAACAC; SEQ ID NO:43) and POX8.1-2 (CATGGGCTTCCTGATCTG; SEQ ID NO:44) for the POX8.1 gene; and POX22.3-1 (ATCGTGTCGACGACGACAT; SEQ ID NO:45) and POX22.3-2 (CTCTGCTCCATACACTTGATG; SEQ ID NO:46) for the POX22.3 gene. QPCR reactions were run at 56° C. annealing for 12 sec and 95° C. for 8 sec for 40 cycles.
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It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

## Claims

1. A transgenic rice plant comprising a heterologous expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide encoding a polypeptide comprising a NPR1 homolog 3 (NH3) polypeptide, wherein the NH3 polypeptide is a polypeptide encoded by SEQ ID NO:1 and wherein the promoter comprises SEQ ID NO:3, wherein the plant expresses the NH3 polypeptide at a higher level than a plant lacking the expression cassette and wherein the plant has enhanced disease resistance compared to the plant lacking the expression cassette.

2. The plant of claim 1, wherein the polynucleotide is at least 70% identical SEQ ID NO:1.

3. The plant of claim 1, wherein the polynucleotide is at least 90% identical to SEQ ID NO:1.

4. The plant of claim 1, wherein the plant expresses the NH3 polypeptide at a level from about two-fold higher to about four-fold higher than the plant lacking the expression cassette.

5. A plant cell from the plant of claim 1, wherein the plant cell comprises the heterologous expression cassette.

6. A seed, flower, leaf, fruit, processed food, or food ingredient from the plant of claim 1, wherein the seed, flower, leaf, fruit, processed food, or food ingredient comprises the heterologous expression cassette.

7. A method of enhancing plant resistance to a pathogen, the method comprising:
introducing a nucleic acid comprising an expression cassette into one or more rice plants, wherein the expression cassette comprises a promoter operably linked to a polynucleotide encoding a NPR1 homolog 3 (NH3) polypeptide; wherein the NH3 polypeptide is a polypeptide encoded by SEQ ID NO:1 and wherein the promoter comprises SEQ ID NO:3, and
from the one or more plants into which the nucleic acid comprising the expression cassette has been introduced, selecting a plant having increased resistance to a pathogen or pathogens as compared to the resistance of a plant lacking the expression cassette.

8. The method of claim 7, wherein the polynucleotide encoding the NH3 polypeptide has at least 70% sequence identity to SEQ ID NO:1.

9. The method of claim 8, wherein the polynucleotide encoding the NH3 polypeptide comprises SEQ ID NO:1.

10. The plant of claim 1, wherein the polynucleotide is at least 95% identical to SEQ ID NO:1.

11. The plant of claim 1, wherein the polynucleotide comprises SEQ ID NO:1.