Progress 10/01/02 to 09/30/07
Outputs
The overall goal of this project was to identify genes involved in the plant defense signaling pathways. We have identified more than 30 novel genes that are differentially expressed in tomato plants overexpressing the wound signal systemin. Key objectives of this research were to (1)identify plant defense-related genes that are differentially regulated by the systemic wound signal systemin, and (2) To characterize biological roles for selected candidate sequences having putative regulatory roles in plant defense gene signaling. The first objective has been accomplished and characterization of several newly-identified genes is on-going. Significant results from this work included the identification of over two dozen novel genes, including a novel calmodulin isoform that is alternatively spliced. This gene will be used as a model system to trace the evolution of a new gene function (i.e., nuclear targeting of normally-cytosolic calmodulin) and test the "Gene Shuffling"
theory. Further work is also expected to lead to a better understanding of the role of introns and alternative splicing in gene evolution and differential gene expression. I have recently moved to Black Hills State University (Spearfish, S.D.) and will be submitting (one re-submission) at least two manuscripts describing this work sometime this summer (2004).
Impacts
Benefits of this work have already provided insight into the evolution of new gene funtion on eukaryotes, and generated a model experimental system for testing hypotheses involving evolution within the plant kingdom. This work has resulted in the identification of many novel, differentially regulated genes. Several of these gene are key regulatory genes that serve as reporters indicating specific plant processes and biochemical pathways selectively modified during plant defense responses.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
OVERVIEW: Virtually all plant and animal genes that encode proteins consist of a series of different alternating intron and exon sequences. Therefore, for "normal" gene expression to occur in higher organisms introns must be removed, and exons must be spliced back together precisely, and in correct sequence, before an intact functional protein can be produced. Science has not yet reached a consensus, and perhaps never will, on whether introns evolved in eukaryotes, or were lost from prokaryotes, over the past many thousands of years. RECENT PROGRESS: As a result of our SAGE analyses, we recently discovered and cloned a novel tomato calmodulin transcript, which is a member of a 6-gene family in tomato. This novel isoform contains a hybrid DNA sequence that consists of both intron and exon sequences, relative to the genomic DNA (i.e., the "gene") the transcript is derived from. Discovery of this unusual transcript provides a valuable experimental system that can be
exploited for investigating the molecular mechanisms that alter gene expression via alternative splicing, and the evolutionary processes that lead to new gene function (i.e., the "Gene Shuffling" theory). We have characterized the exression patterns of all tomato calmodulin gene family members (including the novel transcript described above), and are currently finishing up a phylogenetic study of the calmodulin gene family to get insight into the evolutionary history of this major gene shuffling event. A manuscript describing this work is being written and is anticipated to be submitted in Spring 2003.
Impacts
This work will shed light light on the regulation of genes controlling a wide range of defense-, stress-, and development-related functions. An additional benefit of this work will be novel insight into how genes evolve in eukaryotes. Potential practical applications of this knowledge include enhancing crop defense in response to biotic and abiotic stresses, and manipulation of gene expression strategies.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
Our SAGE results resolved dozens of apparently novel defense gene sequences, and we have confirmed differential expression of candidate genes via Northern analysis. Two manuscripts describing the results of this work have been submitted for publication in 2002. Continuing work will include further characterization of novel resolved sequences. However, the primary emphasis in our continuing research efforts will be the functional characterization of 3-4 recently resolved candidate genes that apparently play key regulatory roles in both wound- and pathogen-mediated defense signaling. All these putative regulatory genes are up-regulated very early upon activation of the wound-response pathway, and all have concensus regulatory domains in their deduced amino acid sequences. We have cloned full-length cDNA's for 2 of the 4 genes. Detailed functional characterization these putative regulatory genes will lead to significant progress toward elucidating molecular and
biochemical mechanisms that govern the regulatory interplay between wound- and pathogen-mediated defense signaling pathways. Additional findings from the SAGE analysis revealed several genes that are alternatively spliced, some example include prosystemin, threonine deaminase, a dehydrin protein, and a chlorophyll A/B binding protein involved in photosynthesis. Surprisingly, some of the alternative splice products identified were regulated reciprocally, with one transcript induced and the other transcript suppressed. Why plant cells would regulated alternative splice products in a reciprocal manner is unknown, and as far as we know unprecedented, and we will explore this phenomenon further.
Impacts
This work has identified numerous novel genes involved in plant defense-and stress-related responses. Continuing work will provide significant insight into the regulatory mechanisms that govern the interplay between wound- and pathogen-mediated signaling pathways.
Publications
- (1) Hernandez M-J, Moffet M, Bergey DR (2002) A novel alternative splicing strategy in Solanaceous plants results in a unique calmodulin isoform encoding a C-terminal nuclear localization signal and prenylation motif. The Plant Journal (submitted 4/02).
- (2) Bergey DR Cook BE, Madden SS, Hernandez M-J (2002). Transcript profiling of tomato plant defense-related genes using serial analysis of gene expression (SAGE). The Plant Cell (submitted 3/02).
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Progress 01/01/00 to 12/31/00
Outputs
SUMMARY: Systemin, jasmonic acid (JA), and salicylic acid (SA) mediate plant defense responses to pests and pathogens, and overlap between the signaling pathways directed by these potent gene regulators exist. We are using serial analysis of gene expression (SAGE) to identify and characterize genes involved in the regulatory cross-talk among these gene activators. Physical damage by pests or wounding initiates the processing and rapid mobilization of a polypeptide signal, systemin, throughout plant tissues. After interacting with target cells, systemin activates the octadecanoid pathway, which results in biosynthesis of JA and regulation of defense-related genes encoding systemic wound responsive proteins (SWRPss). Transgenic tomato plants overexpressing prosystemin appear normal, but constitutively express SWRP genes in the absence of wounding. These transgenic plants thus provide a unique experimental system for investigating and characterizing a constitutively
activated defense signaling circuit. Salicylic acid (SA) plays a key role in many pathogen-mediated defense responses by activating a distinct group of genes encoding pathogenesis-related (PR) proteins. Application of SA to plants before wounding blocks systemin-mediated SWRP gene expression and, in general, SA- and systemin-mediated plant defense pathways are mutually antagonistic. Other lines of evidence also confirm that cross-talk between these major defense pathways occurs, yet genes governing the interaction between these vital signaling circuits have not been identified. OBJECTIVES: (1) An additional 800 clones from each of the SAGE libraries (representing wild-type and transgenic tomato plants) will be sequenced. Sequence data generated during this second phase will establish detailed gene expression profiles for the libraries, and allow comprehensive comparative differential expression studies to be performed. (2) A new SAGE library will be prepared from leaves of tomato
plants treated with a mixed elicitor preparation, including salicylic acid. This library will mimic and represent a pathogen-induced plant system. 1500 clones will be sequenced and a detailed gene expression profile will be established. (3) Pair-wise comparative analyses among all three SAGE libraries (wild-type, transgenic, and "pathogen-induced") will be performed and differentially expressed sequences identified and characterized. Northern analysis will be performed to confirm the expression patterns of all candidate sequences. PROGRESS TO DATE: (1) Identification of over 2 dozen novel, differentially induced gene sequences (2) Identification of differentially regulated alternative splice variants derived from potential regulatory genes (i.e., kinases, homeotic genes, etc.) (3) Confirmation of all resolved differentially expressed sequence tags by Northern analysis
Impacts
Identification and characterization of genes that coordinate the interplay between the SA- and systemin-mediated defense pathways will provide valuable insight into plant defense responses to a broad range of biological, physical, and environmental stresses.
Publications
- Systemin is a Master Regulator of Plant Defense Gene Expression: Resolution of Systemic Wound Response Genes in Tomato Leaves Using Serial Analysis of Gene Expression (2001). --- Daniel R. Bergey, Brian Cook, Maria Jesus-Hernandez, Steve Madden --- This manuscript is in final review and will be submitted this month (Apr 2001)
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Progress 01/01/99 to 12/31/99
Outputs
The overall goal of this project is to establish a comprehensive expression profile and initiate analysis of genes tregulate, and are regulated by, the plant octadecanoid signaling pathway. Gene expression profiles from tomato leaf tissue from wild-type plants, and from transgenic plants overexpressing the wound hormone systemin, will be analyzed and compared using the method of Serial Analysis of Gene Expression (SAGE). Comparative analysis of wild-type and transgenic plants will yield comprehensive differential expression profiles for genes regulated through the octadecanoid pathway. Identification and cloning of these genes will provide a framework for long-term efforts in my laboratory to determine functional roles for these genes in plant defense, development, and environmental stress tolerance. RESULTS--Within the last year we have sequenced over nine thousand SAGE clones each from both wild-type and transgenic SAGE libraries. Preliminary comparative analysis
between the sequence tags expressed by these plants has identified of dozens of differentially regulated genes. Many of these genes have been previously identified, but dozens of others are potentially novel. As expected, the vast majority of gene sequences resolved in the preliminary analysis are constitutively expressed house-keeping genes. However, nearly two-dozen previously described systemin- and wound-inducible genes have been identified. Many of the resolved systemin-regulated genes were described in a previous PNAS report (Bergey et al., 1995). The sequences encoding highly expressed systemic wound responsive proteins (SWRPs) provide a useful internal control for the integrity of this SAGE study. The rapid identification of previosuly described differentially regulated genes validates SAGE as an effective method for plant gene expression analysis, and supports the experimental design used for this project. As our research progresses and more sequence tags are accumulated,
increasing numbers of differentially regulated tomato sequences expressed at lower absolute levels will be resolved. Recent NSF funding of a major tomato EST effort will allow rapid assignment of resolved SAGE tags to longer tomato gene sequences, and thus provide ready access to larger gene fragments. This will facilitate expression studies and gene identification. We have recently submitted major grants to NSF and USDA to support a comprehensive extension of this research project. In addition to the identification of known systemin- and wound-regulated genes, we have resolved nearly three dozen sysstemin-regulated sequences that have no current sequence database matches. Furthermore, several of the sequences resolved are tomato homologues of various stress- or signal transduction-related genes from other plant species. These newly identified genes include both up- and down-regulated sequences. More detailed SAGE studies related to this project are in progress. We are currently
preparing a manuscript to be submitted this summer describing results from the preliminary SAGE analysis describe above.
Impacts
Detailed characterization of biochemical events mediating lipid-based plant defense responses will contribute to overall understanding of analogous lipid-based signaling pathways in animals, and provide novel insight into the biochemical strategies used by plants to develop effective tolerance to a wide range of environmental stresses including ozone, UV light, high salt, etc.
Publications
- No publications reported this period
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