Progress 07/01/07 to 06/30/10
Outputs
OUTPUTS: Gene pairs including divergent gene pairs which harbor putative bidirectional promoters in their intergenic regions were analyzed from rice, sorghum, maize, Brachypodium, Arabidopsis and Populus genomes. This comparative analysis of gene pairs was performed to identify conservation and rearrangement during the evolution of these plant genomes and to identify cis-regulatory elements in putative bidirectional promoters. Coexpression of gene pairs was also studied in order to identify gene pairs with similar expression patterns regulated by bidirectional promoters. We developed a vector with a novel dual reporter gene system (rfp on one side and egfp/gus on the other side) for the analysis of bidirectional promoters by modifying a binary Gateway vector, pBGWFS7 (Institute of Plant Systems Biology, Ghent University, Belgium) carrying egfp/gus. Six full-length promoters were cloned in this vector to drive the expression of enhanced green fluorescent protein and red fluorescent protein/gus genes in forward and reverse orientations, respectively. We developed an Agrobacterium-mediated transient expression assay to study the activity of these promoters. This transient assay also works with other cereals such as maize, barley, oats and wheat. In addition to full-length promoters, 14 deletion constructs were generated in order to identify regions and cis-regulatory elements responsible for bidirectional activity. Further, a few cis-regulatory elements were deleted to identify their effect on reporter gene expression. Real-time RT-PCR was used to evaluate the expression of reporter genes and correlate these results with histochemical assays for GUS and fluorescent microscopic analysis of eGFP and RFP. The results from this project were shared with the scientific community through three peer-reviewed publications in Plant Physiology, Gene and DNA Research, and presentations in Maize Genetics meeting, Plant and Animal Genome conference and USDA annual project directors meeting. We expect to publish two more peer-reviewed research papers based on this project. We shared the modified binary vector with Dr. Erich Grotewold's group in Ohio State University. We will deposit this vector with Institute of Plant Systems Biology, Ghent University, Belgium for public distribution. Agrobacterium-mediated transient expression assay for rice developed in this project was introduced in Molecular Biology techniques laboratory course (BL4840) for undergraduate and graduate students. Real-time RT-PCR was also introduced in this course as part of the above experiment. PARTICIPANTS: Ramakrishna Wusirika is the project director (PD). Surendar Reddy Dhadi is a graduate student (Ph.D.) carrying out most of the experimental work. This student has developed expertise in molecular biology and has developed Agrobacterium-mediated transient expression assay. Nicholas Krom (Ph.D.) who graduated recently carried out most of the bioinformatics work. Two undergraduate students were also trained. This project has contributed towards the professional development of two graduate students and imparting training to undergraduate students. TARGET AUDIENCES: Target audiences include those working on rice and gene regulation, comparative and functional genomics in plants. We have introduced experiments (transient expression assays and real-time RT-PCR) used in this project in an undergraduate molecular biology teaching laboratory. Graduate students who worked on this project served as instructors in Summer Youth Programs for high school students. PROJECT MODIFICATIONS: Nothing significant to report.
Impacts
Coexpression patterns of divergent gene pairs harboring putative bidirectional promoter in rice were identified. Several coexpressed gene pairs with shared GO terms were identified which suggest functional correlation. Rice gene pairs including divergent genes with strongly correlated expression levels were found to be more likely to be conserved in three other grass genomes, sorghum, maize, and Brachypodium. This pattern was also observed in comparison of mammalian gene clusters suggesting it to be a universal phenomenon in eukaryotes. A few over- and under-represented cis-regulatory elements in the intergenic regions of genes (putative bidirectional promoters) arranged in a divergent manner were identified. In addition, we identified similarities and differences with human bidirectional promoters based on sequence patterns. Bidirectional promoters provide a new toolkit for manipulating rice and other plant genomes. Five out of six promoters studied showed bidirectional activity. Promoter activities were experimentally verified under normal and various stress (drought, salt, cold and abscissic acid) conditions. We have further developed and optimized the Agrobacterium mediated transient expression assay for cereal plants, without the necessity for pre-processing of Agrobacterium cultures and wounding of plants. Three regions were deleted for each promoter to evaluate its effect on activity of reporter genes. Deletion of 75 bp out of 388 bp promoter region close to rfp gene abolished the bidirectional activity of promoter 4-431 for putative pentatricopeptide (LOC_Os04g41140) and expressed protein (LOC_Os04g41150). Similarly, deletion of 115 bp out of 355 bp promoter close to rfp gene resulted in significant reduction in bidirectional activity of promoter 6-5 for two expressed proteins (LOC_Os06g01460 and LOC_Os06g01470). Deletion of middle fragment of 180 bp out of 455 bp promoter 6-8 for myb family transcription factor (LOC_Os06g01670) and expressed protein (LOC_Os06g01680) showed a considerable increase (~3 fold) in bidirectional expression. Deletion of the cis-regulatory elements SITEIIATCYTC, SORLIP2AT and CGACGOSAMY3 showed a two fold increase in the bidirectional expression under cold and salt stress conditions driven by the promoter, 3-343 for putative seed maturation protein PM36 (LOC_Os03g19390) and expressed protein (LOC_Os03g19400). Deletion of the 213 bp close to egfp gene of the promoter 2-273 for bZIP transcription factor family protein (LOC_Os02g16680) and expressed protein (LOC_Os02g16690) resulted in loss of the bidirectional activity of the promoter. Promoter 2-914 for expressed protein (LOC_Os01g59080) and thylakoid lumenal 20 kDa protein (LOC_Os01g59090) appears to be unidirectional with the deletion of 100 bp close to egfp gene reducing the expression of egfp and gus genes. Real-time RT-PCR was used to confirm the experimental observations from histochemical GUS analysis and fluorescent microscopic analysis of eGFP and RFP. Results from this project will help in expanding the study of bidirectional promoters to a genome wide scale in rice and other plants.
Publications
- Dhadi SR, Deshpande A and Ramakrishna W. (2010) A novel Agrobacterium mediated transient expression assay (AmTEA) for cereals (pending).
- Krom N and Ramakrishna W. (2010) Conservation, rearrangement, and deletion of gene pairs during the evolution of four grass genomes. DNA Research (accepted).
- Dhadi SR and Ramakrishna W. (2011) Characterization of rice bidirectional promoters (pending).
- Krom N and W. Ramakrishna. (2008) Comparative analysis of divergent and convergent gene pairs and their expression patterns in rice, Arabidopsis, and Populus. Plant Physiology 147:1763-1773.
- Krom N, Dhadi SR and W. Ramakrishna. (2008) Comparative analysis of divergent and convergent gene pairs, their expression patterns, and bidirectional promoters in rice, Arabidopsis, and Populus. 50th Annual Maize Genetics Conference, Washington D.C., February 27-March 1.
- Dhadi, SR, Krom N and W. Ramakrishna. (2008) Identification and characterization of bidirectional promoters in the rice genome: A pilot scale study. Genes to products: Agricultural plant, microbe and biobased product research, USDA project directors' meeting, Washington D.C., April 16-18.
- Dhadi SR, Krom N, Ramakrishna W. (2009) Genome-wide comparative analysis of putative bidirectional promoters from rice, Arabidopsis, and Populus. Gene 429:65-73.
- Dhadi SR and Ramakrishna W. (2009) Identification and characterization of bidirectional promoters in the rice genome: a pilot scale study. Genes to Products: Agricultural Plant, Microbe and Biobased Product Research, USDA Project Directors' Meeting, Washington D.C., May 4-6.
- Ramakrishna W and Krom N. (2009) Expression and rearrangement of gene pairs in plant genomes. Invited talk in International Grass Genome Initiative Workshop in Plant and Animal Genome XVII Conference, San Diego, CA, January 10-14.
- Krom N and Ramakrishna W. (2009) Conservation and expression of gene pairs and retrotransposon-associated genes in plant genomes. Plant and Animal Genome XVII Conference, San Diego, CA, January 10-14.
- Kumar D, Dhadi SR, Krom N and Ramakrishna W. (2009) Dissecting cis-regulatory code of putative bidirectional promoters in cereal genomes. Annual Maize Genetics Conference, St. Charles, IL, March 12-15.
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Progress 07/01/08 to 06/30/09
Outputs
OUTPUTS: We performed comparative analysis of gene pairs among four grass genomes including rice. Conservation of gene order and orientation and its correlation with expression patterns was analyzed. This includes divergent gene pairs which harbor putative bidirectional promoters in their intergenic regions. The first objective in the proposal has been accomplished. Significant progress has been made for second objective in the project. In addition to full-length promoters cloned in a binary Gateway vector driving the expression of enhanced green fluorescent protein / GUS and red fluorescent protein genes in forward and reverse orientations, respectively, 13 deletion constructs were generated. This analysis will determine the regional dependencies of the bidirectional promoter expression and the group of cis-regulatory elements responsible for their activity. Based on the overrepresented cis-regulatory elements identified in these promoters and the effect of deleting regions harboring these elements, site directed mutagenesis is being used to delete these motifs (SORLIP2AT and SITEIIATCYTC). These constructs are being tested using Agrobacterium-mediated transient expression assay developed in our laboratory. Stable transgenic lines will be generated for mutated constructs that show changes in expression patterns both in egfp / gus and rfp compared to full-length constructs. Transient expression assay developed for rice is being expanded to other grasses (maize, barley, rye, oats, sorghum) and dicots (Arabidopsis). We are also collaborating with other research groups to test this assay in other dicots such as soybean and Populus. The results from this project are being shared with the scientific community through peer-reviewed publications and presentations in Maize Genetics meeting, Plant and Animal Genome conference and USDA annual project directors meeting. We modified a Gateway binary vector with egfp / gus genes from Flanders Interuniversity Institute for Biotechnology (VIB), Belgium, by inserting rfp gene so that a promoter of our choice will drive the expression of egfp / gus genes in the forward orientation and rfp gene in the reverse orientation. We shared this vector with Dr. Erich Grotewold's group in Ohio State University. PARTICIPANTS: Ramakrishna Wusirika is the project director (PD) who is responsible for the management of the project which includes advising the personnel and planning of experimental strategy. Surendar Reddy Dhadi is a graduate student (Ph.D.) carrying out most of the experimental work. This student has developed expertise in molecular biology and has developed Agrobacterium-mediated transient expression assay. Another graduate student and an undergraduate student were also trained in this project. We are sharing a binary vector developed in this project with Dr. Erich Grotewold's group in Ohio State University in a collaborative project to study short promoters that drive the expression of two flanking genes in Arabidopsis. TARGET AUDIENCES: Target audiences include those working on gene regulation in plants. We are introducing experiments which are being used in this project in an undergraduate molecular biology teaching laboratory in fall semester. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Lower rates of conservation among rice divergent gene pairs than convergent or tandem pairs in sorghum, maize, and Brachypodium were identified. The frequency of the different types of genomic rearrangements varied significantly among the three types of rice gene pairs as also their organization in other grass genomes. In rice, gene pairs with strongly correlated expression levels were found to be conserved significantly more often than pairs with little or no correlation. This study provides information on conservation and rearrangement of gene pairs during the evolution of four grass genomes serving as basis for further investigations on functional interactions between adjacent genes. Preliminary results suggest that four out of five promoters studied show bidirectional activity. Deletion of regions close to egfp gene appears to affect bidirectional activity of most of these promoters. Further experimental analysis which is in progress will precisely identify regulatory elements responsible for bidirectional activity in a few rice promoters. Results from this project will help in expanding the study of bidirectional promoters to a genome wide scale in rice and other plants. Expression patterns using transient expression analysis developed in our laboratory will be compared to stable transgenic lines. If the transient expression assays are found to be similar to that of stable lines, rapid analysis of transgene activity can be accomplished.
Publications
- Krom N and Ramakrishna W. Conservation and expression of gene pairs and retrotransposon-associated genes in plant genomes. Plant and Animal Genome XVII Conference, San Diego, CA, January 10-14 (2009).
- Kumar D, Dhadi SR, Krom N and Ramakrishna W. Dissecting cis-regulatory code of putative bidirectional promoters in cereal genomes. Annual Maize Genetics Conference, St. Charles, IL, March 12-15 (2009).
- Dhadi SR, Krom N, Ramakrishna W. Genome-wide comparative analysis of putative bidirectional promoters from rice, Arabidopsis, and Populus. Gene (2009) 429:65-73.
- Dhadi SR and Ramakrishna W. Identification and characterization of bidirectional promoters in the rice genome: a pilot scale study. Genes to Products: Agricultural Plant, Microbe and Biobased Product Research, USDA Project Directors' Meeting, Washington D.C., May 4-6 (2009).
- Ramakrishna W and Krom N. Expression and rearrangement of gene pairs in plant genomes. Invited talk in International Grass Genome Initiative Workshop in Plant and Animal Genome XVII Conference, San Diego, CA, January 10-14 (2009).
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Progress 07/01/07 to 06/30/08
Outputs
OUTPUTS: We performed computational analysis to identify putative bidirectional promoters based on the coexpression of adjacent genes with divergent arrangement in the rice genome. We identified cis-regulatory motifs overrepresented in putative bidirectional promoters of rice which is part of the first objective in the proposal. Experimental work for the second objective has been initiated. Five full-length promoters were cloned in a binary Gateway vector to drive the expression of enhanced green fluorescent protein and red fluorescent protein genes in forward and reverse orientations, respectively. Agrobacterium-mediated transformation was used to transform rice calli and transgenic plants are being generated. These results were shared with the scientific community through presentations in Maize Genetics and USDA annual project directors meetings held in Washington DC. We also shared our research findings through publications in peer-reviewed scientific journals. Furthermore, we are in the process of developing a web site to display our results. PARTICIPANTS: Surendar Reddy Dhadi - Graduate student (Ph.D.) carrying out most of the experimental work. Nicholas Krom - Graduate student (Ph.D.) carrying out most of the bioinformatics work. Megan McQuillan - Undergraduate student helping Surendar Reddy Dhadi. Prashant Swamy - Student working on this project helping Surendar Reddy Dhadi during summer of 2008. These students were initially trained in the laboratory. The only graduate student (Surendar) who is working full-time on this project and is funded entirely by this grant has developed the ability to plan and execute experiments independently. This project is contributing towards the professional development of this student and other graduate students so that they can become independent investigators after their graduation. TARGET AUDIENCES: The output from this proposal will be of interest to those working on gene regulation and genetic manipulation of crop plants. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We studied coexpression and inter-species conservation of divergent gene pairs in rice. Strongly correlated expression levels between divergent genes were common in rice and other two plant genomes (Arabidopsis and Populus). The frequency of strong correlation was found to be independent of intergenic distance. Conservation of divergent arrangement of genes is not common. A correlation between intergenic distance of divergent genes in a pairs and shared GO classes was observed in rice. Cis-regulatory elements over-represented in putative bidirectional promoters of rice and shared with Arabidopsis, were identified. Our studies suggest that shared and unique mechanisms exist for functioning of divergent gene pairs regulated by bidirectional promoters in different plant genomes including rice. Five putative bidirectional promoters from rice were cloned in a binary Gateway vector to test their ability to drive the expression of enhanced green fluorescent protein (GFP) and red fluorescent protein (RFP) genes. These constructs are first being tested in Arabidopsis for their bidirectional activity. GFP and RFP activities were observed for two constructs tested so far. We are in the process of testing three other constructs. Agrobacterium-mediated transformation was used to transform rice calli with these five constructs and transgenic plants are being generated. We are in the process of generating deletion constructs. This project has helped us develop Gateway cloning and rice transformation technology in the lab. We also developed expertise in analyzing reporter proteins (GFP and RFP) using a fluorescent microscope.
Publications
- Krom, N., and W. Ramakrishna (2008) Comparative analysis of divergent and convergent gene pairs and their expression patterns in rice, Arabidopsis, and Populus. Plant Physiol 10.1104/pp.108.122416.
- Krom, N., S.R. Dhadi., and W. Ramakrishna (2008) Comparative analysis of divergent and convergent gene pairs, their expression patterns, and bidirectional promoters in rice, Arabidopsis, and Populus. 50th annual maize genetics conference, Washington D.C., Feb. 27 through March 1, 2008.
- Dhadi, S.R., N. Krom., and W. Ramakrishna (2008) Identification and characterization of bidirectional promoters in the rice genome: A pilot scale study. Genes to products: Agricultural plant, microbe and biobased product research, USDA project directors' meeting, Washington D.C., April 16-18, 2008.
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