Source: MICHIGAN STATE UNIV submitted to
THE IMPACT OF MULES ON GENOME EVOLUTION AND THE MECHANISM UNDERLYING PACK-MULE FORMATION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
REVISED
Funding Source
Reporting Frequency
Annual
Accession No.
0205538
Grant No.
(N/A)
Project No.
MICL02120
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2010
Project End Date
Sep 30, 2015
Grant Year
(N/A)
Project Director
Jiang, N.
Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824
Performing Department
Horticulture
Non Technical Summary
For crops and other organisms, genetic materials are passed by genes or pieces of DNA in the genomes. The presence of genes in the production of specific proteins which determined the growth and product of plants. As could be imagined, the improvement of crops relies on the modification of the genome, either naturally or artificially. This project studies transposable elements - the "jumping genes" of plants. The genome sequence is in a certain order, and change of the order will lead to change of the function, or the emergence of new traits, which is important in agriculture. Transposable elements are one of the factors that change the order of the genome sequence. The transposable elements involved in this project are called "Pack-MULEs", which are abundant inthe genome of plants. A unique feature of Pack-MULEs is that they are capable of duplicating, reorganizing, and shuffling the DNA pieces in the genome. As a result, Pack-MULEs are potential "gene machines" for generating new genes with novel functions. Despite the abundance of Pack-MULEs and their potential in generating new traits, not much is known about how frequently Pack-MULE serve as new genes and the mechanism how they duplicate DNA pieces. With this proposal, we will determine how many of the 3,000 Pack-MULEs in rice are making protein products, which imply they are true genes. In addition, we will utilize bread yeast and genetic markers to observe how Pack-MULEs duplicate genomic sequences. Finally, we will further characterize a Pack-MULE which is shown to improve the stress tolerance of model plants and test its application in crop plants. This study may enable more powerful breeding methods and the emergence of new crop varieties.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011530108070%
2011460108030%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1460 - Tomato; 1530 - Rice;

Field Of Science
1080 - Genetics;
Goals / Objectives
1. Continuation on functional analysis of the PM-CBF gene in rice as well as its possible application in agriculture practice. 2. Determination of the translational status of Pack-MULEs in rice by measuring mRNAs associated with polysomes. 3. Establishment of a yeast assay system for transposition and sequence acquisition of Mutator elements and Pack-MULEs.
Project Methods
Methods for each objective Objective 1: Continuation on functional analysis of the PM-CBF gene in rice as well as its possible application in agriculture practice. In our previous study we over-expressed the coding region of a Pack-MULE element that contains a novel CBF gene in rice and Arabidopsis. CBF genes encode transcription factors that are involved in stress tolerance. In this study, we will use yeast two hybrid assay to determine the biochemical function of the domains harbored by this Pack-MULE elements. Moreover, we will over-express this Pack-MULE gene in two dicot crop plants, tomato and petunia. The transgenic progeny will be examined to test whether this gene will enhance stress tolerance but without reducing growth potential. Objective 2: Determination of the translational status of Pack-MULEs in rice by measuring mRNAs associated with polysomes. Since actively translated mRNAs are associated with multiple ribosomes in large polyribosome (polysome) complexes, an mRNA is very likely to be translated if it is associated with polysomes. The isolation of polysomes will be facilitated by immunoprecipitation of intact ribosomes using a transgenic line where the large subunit if ribosomes is tagged with a FLAG epitope at a position at the base of the ribosome. The frozen tissue will be homogenized and centrifuged, and the supernatant will be incubated with anti-FLAG agarose beads (Invitrogen) at 4oC. Thereafter, the agarose beads will be eluted with buffer containing RNase inhibitor. RNA will be extracted from the eluted material using the Qiagen RNA Easy kit. The mRNAs will be processed and paired end sequenced using the Illumina GA2 sequencer. The sequencing data derived from RNA-seq will be mapped to the rice genome using a variety of software written for use with next generation sequencing data. After the reads are mapped, Pack-MULE derived mRNAs can be identified by comparing the position of Pack-MULEs and that of the sequencing reads. Objective 3: Establishment of a yeast assay system for transposition and sequence acquisition of Mutator elements and Pack-MULEs. We will express the transposase of autonomous Mutator like elements in yeast and engineer a Pack-MULE inside a reporter gene to test its excision. Transposon display will be used to detect new insertions of the element. PCR will be used to amplify the newly formed element to test whether there is any new sequence acquired. Thereafter, swapping and deletion experiments will be conducted to test which factors that are important for transposition and acquisition.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Our target audience includes both researchers and the broader community. In addition to the scientific value, understanding how TEs constantly shape their host genomes promotes the acceptance of genetically engineered organisms. Our research can educate the broader public as our experiments will reveal that "genetic engineering" in nature is associated with alterations in both molecular and phenotypic level. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? The results have been published as Journal articles and book Chapters. Jiang gave two seminars in March 2013 in Northest Institute of Geography and Agroecology, Chinese Academy of Science and Northest normal University, China. Jiang also presented a poster at 56th maize genetic conference, Beijing, China. What do you plan to do during the next reporting period to accomplish the goals? 1. Completion of analysis of data collected from RNA-seq, ChIP-seq, methylation to reveal the epigenetic impact of Pack-MULEs on genome evolution. 2. Completion RNA-seq from His-RPL18 rice lines, analyzing data to estimate how many Pack-MULEs are translated. 3. Backcross of Pack-MULE insertion lines to determine whether the mutation phenotype is caused exclusively by interruption of Pack-MULEs. 4. Publication of 2 to 3 research articles.

Impacts
What was accomplished under these goals? Mutator-like transposable elements (MULEs) are widespread in plants and the acvitity of MULEs results in the formation of Pack-MULEs, which carry gene or gene fragments. However, it is unclear what determines the abudance or amplification of MULEs in different plant genomes. MULEs were first discovered in maize where there are a total of 12,900 MULEs. In comparison, rice, with a much smaller genome, harbors over 30,000 MULEs. Since maize and rice are close relatives, the differential amplification of MULEs raised an inquiry into the underlying mechanism. We hypothesize this is partly attributed to the differential copy number of autonomous MULEs which have the potential to generate the transposase that is required for transposition. To test our hypothesis, we mined the two genomes and detected 530 and 476 MULEs containing transposase sequences (candidate coding-MULEs) in maize and rice, respectively. Over 1/3 of the candidate coding-MULEs harbor nested insertions and the ratios are similar in the two genomes. Among the maize elements with nested insertions, 24% have insertions in coding regions and over half of them harbor two or more insertions. In contrast, only 12% of the rice elements have insertions in coding regions and 19% have multiple insertions, suggesting that nested insertions in maize are more disruptive. This is because most nested insertions in maize are from LTR retrotransposons, which are large in size and are prevalent in the maize genome. Our results suggest that the amplification of retrotransposons may limit the amplification of DNA transposons but not vice versa. In addition, more indels are detected among maize elements than rice elements whereas defects caused by point mutations are comparable between the two species. Taken together, more disruptive nested insertions combined with higher frequency of indels resulted in few (6%) coding-MULEs that may encode functional transposases in maize. In contrast, 35% of the coding-MULEs in rice retain a putative intact transposase. This is in addition to the higher expression frequency of rice coding-MULEs, which may explain the higher occurrence of MULEs in rice than that in maize. When studying the transposition behavior of a MULE from rice (called Os3378), we found its target sequence is highly AT rich, which is in constrast to the Mutator elements in maize that specifically target GC-rich sequences. In addition, we discovered that fusion of a fluorescent protein (EYFP) to the Os3378 transposase significantly improve its transposition activity. Finally, it is not rare for Os3378 to leave a single terminus after excision (a long “footprint”) in yeast. Given the fact that the retention of single termini of Mutator elements was previously described; it suggests this is a common feature for MULEs and a Pack-MULE may arise when two adjacent elements flanking a gene both leave a single terminus in an appropriate orientation. To determine the impact of MULEs on gene expression, we conducted RNA-seq, ChIP-seq, methylation analysis from rice cultivars containing polymorphic Pack-MULE insertion sites. We are analyzing the data to construct a model for Pack-MULE action and deduce the impact of Pack-MULE on their adjacent genes and parental genes. To determine translation status of Pack-MULEs, we bulked seeds for His-RPL18 rice lines, prepared the RNA-seq libraries from the lines and the RNA-library is under sequencing. To further determine the coding capapcity of Pack-MULEs, we phenotyped tagging lines of Pack-MULEs and over half of them demonstrated phonotypes.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Ferguson, A. A., Zhao, D and Jiang, N. 2013 Selective acquisition of genomic sequences by Pack-MULEs based on GC content and breadth of expression. Plant Physiology. 163:14191432.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Zhao, D., Jiang, N. 2014. Nested Insertions and Accumulation of Indels are Negatively Correlated with Abundance of Mutator-Like Transposable Elements in Maize and Rice. PLoS ONE 9 (1): e87069.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Campbell, M. S., Law, M., Holt, C., Stein, J. C., Moghe, G. D., Hunagel, D. E., Lei, J., Achawanantakun, R., Jiao, D., Lawrence, C. J., Ware, D., Shiu, S-H., Childs, K. L., Sun, Y., Jiang, N, Yandell, M. 2014. MAKER-P: a tool-kit for the rapid creation, management, and quality control of plant genome annotations. Plant Physiology 164 513-524.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Xu, Y., Jiang, N., Zou, Z., Tu, Z., Chen, A., Zhao, Q., Xiang, Z., and He, N. 2014. Retrotransposon "Qian" mediated segmental duplication in silkworm, Bombyx mori, Insect Biochem Mol Biol 46, 9-16.
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2014 Citation: Jiang, N. 2014. Plant Transposable Elements: Beyond Insertions and Interruptions. In Molecular Life Sciences. Springer Science+Business Media DOI 10.1007/978-1-4614-6436-5_104-2 (In Press).


Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Our target audience includes both researchers and the broader community. In addition to the scientific value, understanding how TEs constantly shape their host genomes promotes the acceptance of genetically engineered organisms. Our research can educate the broader public as our experiments will reveal that “genetic engineering” in nature is associated with alterations in both molecular and phenotypic level. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? No, this project does not directly support any of my lab membmers. How have the results been disseminated to communities of interest? The results have been published as Journal articles and book chapters. Jiang gave an invited talk at the XXIst International Plant and Animal Genome Meeting in San Diego, CA in January 2013. What do you plan to do during the next reporting period to accomplish the goals? 1. Integrate data collected from RNA-seq, ChIP-seq, methylation to construct a model for Pack-MULE action and deduce the impact of Pack-MULE on their adjacent genes and parental genes. 2. Bulking seeds for His-RPL18 rice lines, preparing RNA-seq libraries from the lines and conduct RNA-seq to determine the translation profile of Pack-MULEs. 3. Completion of genotyping and screening for homozygous lines of transgenic rice with putative transposon insertions inside Pack-MULEs. The homozygous lines will be phenotyped for developmental variations from the parental line, Nipponbare. 4. Publication of 2 to 3 research articles.

Impacts
What was accomplished under these goals? Despite the abundance of Pack-MULEs in plants, the mechanism by which parental genes or parental sequences are captured by Pack-MULEs remains largely an enigma except that Pack-MULEs prefer to duplicate GC-rich sequences. To identify factors important for sequence acquisition, we characterized all MULEs in rice carrying recognizable non-transposon genomic sequences and discovered that MULEs are capable of capturing or duplicating both genic and intergenic sequences. Among all MULEs carrying genomic sequences, only 3% carry intergenic squences, which is much less compared to the number of MULEs (97%) carrying genic sequences. Comparsion between parental genes of Pack-MULEs and other genes indicate that Pack-MULE do not have a preference for specific gene function; however, they prefer genes with a wide breadth of expression. This feature, combined with their preference for GC-rich sequences, enables them to nearly exclusively duplicate bona fide genes, which provides new resources for the evolution of novel genes as well as the regulation of expression of existing genes. MULEs are widespred in plants and well known for their high transposition activity. Nevertheless, few MULEs have been shown to be currently active and there is no report regarding MULE activity in a non-native host. It has been suggested that host factors are involved in the transposition of Mutator element in maize. In our study, we tested the transposition activity of a MULE from rice in yeast. Both excisions and new insertions were detected, suggesting yeast harbors all the host factors required for the transposition of this specific MULE. Although the transposition efficiency caused by wild-type transposase is very low, we discovered a critical region within the transposase for manipulation of transposition activity. The excision frequency can be increased 20 fold or higher through deletion, substitution, and fusion of amino acid sequences in this region. The establishment of a MULE transposition system in yeast provides a foundation for further studying the transposition mechanism of MULEs as well as how they duplicate/acquire genomic sequences.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Smith, J. J., Kuraku, S., Holt, C., Sauka-Spengler, T., Jiang, N., Campbell, M. S., Yandell, M. D., Manousaki, T., Meyer, A., Bloom, O. E., Morgan, J. R., Buxbaum, J. D., Sachidanandam, R., Sims, C., Garruss, A. S., Cook, M., Krumlauf, R., Wiedemann, L. M., Sower, S. A., Decatur, W. A., Hall, J. A., Amemiya, C. T., Saha, N. R., Buckley, K. M., Rast, J. P., Das, S., Hirano, M., McCurley, N., Guo, P., Rohner, N., Tabin, C. J., Piccinelli, P., Elgar, G., Ruffier, M., Aken, B. L., Searle, S. M., Muffato, M., Pignatelli, M., Herrero, J., Jones, M., Brown, C. T., Chung-Davidson, Y. W., Nanlohy, K. G., Libants, S. V., Yeh, C. Y., McCauley, D. W., Langeland, J. A., Pancer, Z., Fritzsch, B., de Jong, P. J., Zhu, B., Fulton, L. L., Theising, B., Flicek, P., Bronner, M. E., Warren, W. C., Clifton, S. W., Wilson, R. K., and Li, W. (2013) Sequencing of the sea lamprey (Petromyzon marinus) genome provides insights into vertebrate evolution, Nat Genet 45, 415-421. Ming, R., Vanburen, R., Liu, Y., Yang, M., Han, Y., Li, L. T., Zhang, Q., Kim, M. J., Schatz, M. C., Campbell, M., Li, J., Bowers, J. E., Tang, H., Lyons, E., Ferguson, A. A., Narzisi, G., Nelson, D. R., Blaby-Haas, C. E., Gschwend, A. R., Jiao, Y., Der, J. P., Zeng, F., Han, J., Min, X. J., Hudson, K. A., Singh, R., Grennan, A. K., Karpowicz, S. J., Watling, J. R., Ito, K., Robinson, S. A., Hudson, M. E., Yu, Q., Mockler, T. C., Carroll, A., Zheng, Y., Sunkar, R., Jia, R., Chen, N., Arro, J., Wai, C. M., Wafula, E., Spence, A., Xu, L., Zhang, J., Peery, R., Haus, M. J., Xiong, W., Walsh, J. A., Wu, J., Wang, M. L., Zhu, Y. J., Paull, R. E., Britt, A. B., Du, C., Downie, S. R., Schuler, M. A., Michael, T. P., Long, S. P., Ort, D. R., Schopf, J. W., Gang, D. R., Jiang, N., Yandell, M., Depamphilis, C. W., Merchant, S. S., Paterson, A. H., Buchanan, B. B., Li, S., and Shen-Miller, J. (2013) Genome of the long-living sacred lotus (Nelumbo nucifera Gaertn.), Genome Biol 14, R41. Ferguson, A. A., Zhao, D., and Jiang, N. (2013) Selective acquisition and retention of genomic sequences by pack-mutator-like elements based on Guanine-Cytosine content and the breadth of expression, Plant Physiol 163, 1419-1432.
  • Type: Book Chapters Status: Published Year Published: 2013 Citation: Jiang, N., and Panaud, O. (2013) Transposable element dynamics in rice and its wild relatives. , In Genetics and Genomics of Rice (Wing, Q. Z. a. R., Ed.), pp 55-70, Springer, New York. Jiang, N. (2013) Overview of Repeat Annotation and De Novo Repeat Identification., In Plant Transposable Elements: Methods and Protocols (Peterson, T., Ed.), pp 275-287, Springer Science+Business Media, New York. Jiang, N. (2013) Computational Methods for Identification of DNA transposons, In Plant Transposable Elements: Methods and Protocols (Peterson, T., Ed.), pp 289-304, Springer Science+Business Media, New York.
  • Type: Journal Articles Status: Under Review Year Published: 2013 Citation: Campbell, M. S., Law, M. Y., Holt, C., Stein, J. C., Moghe, G., Hunagel, D. E., Lei, J., Achawanantakun, R., Jiao, D., Lawrence, C. J., Ware, D., Shiu, S. H., Childs, K., Sun, Y., Jiang, N., and Yandell, M. (2013) MAKER-P: a tool-kit for the rapid creation, management, and quality control of plant genome annotations, Plant Physiology, In revision. Zhao, D., and Jiang, N. (2013) Nested insertions and accumulation of indels are negatively correlated with abundance of Mutator-like transposable elements in maize and rice, PLOS One In revision.


Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: The project is associated with two goals: 1) to test the functional potential of MULEs including Pack-MULEs; 2) explore the mechanism underlying Pack-MULE formation. Coding capacity of Pack-MULEs: to test the coding potential of Pack-MULE genome-widely, we ordered ~80 rice FST (Flanking Sequence Tag) lines that contain transposon or T-DNA insertion inside the Pack-MULEs. Theoretically, if the Pack-MULEs are functional, the presence of insertion will cause certain phenotype. At this stage we are verifying the presence of insertion as well as screening for homozygous mutants. Since most of the Pack-MULEs are not associated with a FST line, we are seeking an alternative approach to evaluate the function of Pack-MULEs. If a Pack-MULE has coding capacity, it must be first translated into protein. Since actively translated mRNAs are associated with multiple ribosomes in large polyribosome (polysome) complexes, an mRNA associated with polysomes is very likely to be translated. The isolation of polysomes can be facilitated by immunoprecipitation of intact ribosomes if a ribosomal protein of the large subunit is tagged with a FLAG epitope at a position at the base of the ribosome. The advantage of this approach is that mRNAs in polysomes are separated from those in mRNPs (messenger ribonucleoproteins). When this technique is combined with high-throughput transcriptome profiling technologies, it is possible to monitor the translation status of genes in a genome-wide fashion. To facilitate the isolation of polysomes, we have constructed a rice 35S:His6-FLAG-RPL18 (epitope tagged rice Ribosome Protein L18 cDNA) line and is in the process to verify whether the tagged protein is associated with polysomes. The mechanism(s) involved in the formation of Pack-MULEs: to study the mechanism of sequence acquisition of Pack-MULEs, We are establishing a yeast system to observe the transposition of MULEs and possible acquisition events. We synthesized transposase coding region from this MULE and constructed a non-autonomous element and successfully introduced to yeast. Transposition events, including both excision and reinsertion events, are observed. However, the efficiency is rather low. Through a series of deletion test, we observed that if the 5 end of the transposase is deleted, the transposition frequency is significantly increased. In addition, we demonstrated that the concentration of galactose, the substance that is used to induce the expression of transposase, influences the transposition frequency. The fact that there is an optimal concentration indicates the abundance of transposase protein has an impact on transposition. When the transposase protein level is too high, transposition reaction was suppressed. PARTICIPANTS: Ning Jiang: project director, designing experiments and conducting experiments TARGET AUDIENCES: Through my efforts, post-doctorial associates, graduate students and undergraduate students are trained as next generation leaders for science. Our research results are inspiring for people how work on transposon biology and evolutionary biology. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
MULEs play important roles in plant genome evolution due to their high activity and potential to acquire and amplify gene fragments. Despite the abundance of Pack-MULEs and their potential functional roles, Pack-MULE research to date has been limited to descriptive and static characterization studies. Little is known about how Pack-MULEs acquire genomic sequences including genes. The establishment of a transposition system in yeast provided a foundation for establishing an acquisition system for Pack-MULEs and testing various models for sequence acquisition. Accordingly, this study is important for both theoretical research and practical applications. Theoretically, gene duplication is an important means for evolution of new functions. Unlike segmental duplication or genome-wide duplication, gene duplication mediated by Pack-MULEs (and other transposons) is often accompanied by modification or rearrangement of the duplicated sequences. As a result, understanding how Pack-MULEs acquire genomic sequence will provide new insights into how new genes evolve and how transposons contribute to this process. Practically, my long term goal is to build a "gene-shuffling machine" for the generation of novel phenotypes and for studying gene functions. All existing mutation systems, such as T-DNA insertion, radiation, EMS, and transponson-tagging, are largely disruptive, i.e., the mutation is created through the abortion of gene function. In contrast, the activity of Pack-MULEs resembles a natural "gene recombination lab". In addition to its possible disruptive effect (like other transposons), it may bring "constructive" features, such as the formation of new genes. Thus the action of Pack-MULEs may lead to phenotypes that cannot be produced in other mutation systems. Understanding the acquisition mechanism is the foundation for control of the acquisition process and for establishing a novel mutation system that complements current mutation systems. This may result in novel strategies for future breeding or engineering in crops, microbes, and maybe animals.

Publications

  • Vieler, A., Wu, G., Tsai, C.H., Bullard, B., Cornish, A.J., Harvey, C., Reca, I., Thornburg, C.1, Achawanantakun, R.5, Buehl, C.J., Campbell, M.S.6, Cavalier, D., Childs, K.L., Clark, T.J., Deshpande, R., Erickson, E., Ferguson, A.A., Handee, W.2, Kong, Q., Li, X., Liu, B., Lundback, S., Peng, C., Roston, R., Simpson, S.J.P.3, TerBush, A., Warakanont, J., Zauner, S., Farre, E.M., Hegg, E.L., Jiang, N., Kuo, M.H.1, Lu, Y., Niyogi, K.K, Ohlrogge, J., Osteryoung, K.W., Shachar-Hill, Y., Sears, B.B, Sun, Y., Takahashi, H.,Yandell, M., Shiu, S.H., Benning, C. 2012. Genome, Functional Gene Annotation, and Nuclear Transformation of the Heterokont Oleaginous Alga Nannochloropsis oceanica CCMP1779. PLoS. Genetics. 8(11):e1003064.
  • Davidson, R.M., Gowda, M., Moghe, G., Lin, H., Vailancourt B., Shiu, S.H., Jiang, N. and Buell, C.R. 2012. Comparative transcriptomics of three Poaceae species reveals patterns of gene expression evolution. Plant J. 71:492-502
  • Ferguson, A., Jiang, N. 2012. Mutator-like elements with multiple long terminal inverted repeats (TIR) in plants. Comparative and Functional Genomics. 2012.


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: The project is associated with two goals: 1) to test the functional potential of MULEs including Pack-MULEs; 2) explore the mechanism underlying Pack-MULE formation. Coding capacity of Pack-MULEs: for the first task, we are continuing to characterize the function of a Pack-MULE-CBF gene. Most known CBF genes are capable of enhancing stress tolerance of plants. This particular Pack-MULE harbors a DNA domain from a known CBF gene. Nevertheless, it does not contain the coding region for the activation domain from the same gene; instead, the putative activation domain was replaced by a DNA fragment from the downstream region of the relevant CBF gene. In this case, the PM-CBF either has a novel or non-functional activation domain. The over-expression of this PM-CBF enhances the drought and salt tolerance in transgenic rice, which mimic the phenotype generated with a normal CBF gene. As a result, either the activation domain of the PM-CBF is capable of activating the transcription of target genes, or it may form a heterodimer with other CBF proteins. To test the hypothesis, we established a yeast system to determine the activation activity of the Pack-MULE. Nevertheless, our analysis analysis indicates that the Pack-MULE has no detectable activation activity, nor it can form heterodimers with other CBFs. To resolve these apparently controversial results, we are transforming the Pack-MULE under native promoter to a cultivar that lacks this Pack-MULE, and we already obtained seedlings. We will conduct stress tolerance and other tests using the progeny of the seedlings to elucidate the function of this Pack-MULE. Duplication of Pack-MULE terminal inverted repeats: Mutator-like transposable elements (MULEs) are widespread in plants and majority of them have long terminal inverted repeats (TIRs), which distinguish them from other DNA transposons. It is known that the long TIRs of Mutator elements harbor transposase binding sites and promoters for transcription, indicating that TIR sequence is critical for transposition and for expression of sequences between the TIRs. During our course of study, we detected MULEs with multiple TIRs mostly located in tandem. These elements are detected in the genomes of maize, tomato, rice, and Arabidopsis. Some of these elements are present in multiple copies, suggesting their mobility. For those elements that have amplified, sequence conservation was observed for both of the tandem TIRs. For one MULE family carrying a gene fragment (a Pack-MULE), the elements with tandem TIRs are more prevalent than their counterparts with a single TIR. The successful amplification of this particular MULE demonstrates that MULEs with tandem TIRs are functional in both transposition and duplication of gene sequences. The mechanism(s) involved in the formation of Pack-MULEs: to study the mechanism of sequence acquisition of Pack-MULEs, We are establishing a yeast system to observe the transposition of MULEs and possible acquisition events. We synthesized transposase coding region and constructed a non-autonomous element and successfully introduced to yeast. We are screening for the putative transposition events. PARTICIPANTS: Ning Jiang: project director, designing experiments and conducting experiments TARGET AUDIENCES: Through my efforts, post-doctorial associates, graduate students and undergraduate students are trained as next generation leaders for science. Our research results are inspiring for people how work on transposon biology and evolutionary biology. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
MULEs play important roles in plant genome evolution due to their high activity and potential to acquire and amplify gene fragments. We uncovered that formation of duplicated TIRs may have contributed to the success of some MULE elements including Pack-MULEs. The formation of elements with additional TIR is not a rare event but only elements with duplicated TIR on both termini have significant mobility. The genome of dicots harbors more elements with duplicated TIRs than that of monocots, and such difference might be attributed to the presence of GC-rich sequences in the genomes of monocots. The distribution of size vs. copy number of MULEs (or Pack-MULEs) is periodic, suggesting the distance between the TIRs or the relative spatial position of TIRs may have a role in transposition. In the elements with tandem TIRs, both TIRs appear to be subject to certain constraints, and the presence of duplicated TIRs may confer certain mechanistic advantages for transposition. Such features may be utilized to create elements with elevated transposition activity.

Publications

  • Davidson, R.M., Candice N. Hansey, C.N., Gowda, M., Childs, K.L., Lin, H., Vaillancourt, B., Sekhon, R.S., de Leon, N., Kaeppler, S.M., Jiang, N., Buell, C.R. 2011. Utility of RNA seq for Analysis of Maize Reproductive Transcriptomes. The Plant Genome. 4:191-203.
  • Ferguson, A. A., Jiang, N. 2011. Pack-MULEs: recycling and reshaping genes through GC-biased acquisition. Mobile Genetic Elements 1:135-138.
  • Jiang, N., Furgonson, A. A., Slotkin, R. K., Lisch, D. 2011 Pack-Mutator like transposable elements (Pack-MULEs) induce directional modification of genes through biased insertion and DNA acquisition. Proc Natl Acad Sci USA 108:1537-1542.


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: The project is associated with two goals: 1) to test the functional potential of MULEs including Pack-MULEs; 2) explore the mechanism underlying Pack-MULE formation. Coding capacity of Pack-MULEs: for the first task, we are continuing to characterize the function of a Pack-MULE-CBF gene. Most known CBF genes are capable of enhancing stress tolerance of plants. This particular Pack-MULE harbors a DNA domain from a known CBF gene. Nevertheless, it does not contain the coding region for the activation domain from the same gene; instead, the putative activation domain was replaced by a DNA fragment from the downstream region of the relevant CBF gene. In this case, the PM-CBF either has a novel or non-functional activation domain. The over-expression of this PM-CBF enhances the drought and salt tolerance in transgenic rice, which mimic the phenotype generated with a normal CBF gene. As a result, either the activation domain of the PM-CBF is capable of activating the transcription of target genes, or it may form a heterodimer with other CBF proteins. In either case, it demonstrates that the Pack-MULE can create novel functional genes with a novel evolutionary mechanism. To test the hypothesis, we established a yeast system to determine the activation activity of the Pack-MULE. Our preliminary analysis indicates that the Pack-MULE has no detectable activation activity. As a consequence, we will test whether it forms dimers with other CBF proteins. Pack-MULE modifying the structure of existing genes: in monocots, many genes demonstrate a significant negative GC-gradient, meaning that the GC content declines along the orientation of transcription. Such a gradient is not observed in the genes of the dicot plant Arabidopsis. In addition, a lack of homology is often observed at the 5 end of the coding region of orthologous genes between rice and Arabidopsis. The reasons for these differences have been enigmatic. Our analysis with rice and maize genomic sequences indicates that Pack-MULEs specifically acquire GC rich fragments and preferentially insert into the 5 end of genes. The resulting Pack-MULEs may form independent, GC rich transcripts with a negative GC gradient. Alternatively, the Pack-MULEs can evolve into additional or novel exons at the 5 end of existing genes, thus altering the GC content in those regions. In this ways, Pack-MULEs have been modifying the 5 end of genes and are at least partially responsible for the negative GC gradient of genes in grasses. Such a trend is not obvious in Arabidopsis due to the lack of GC-rich islands. The capability for Pack-MULEs to acquire GC rich sequence and modify 5 end of genes provides novel insights how TEs may shape their host genomes. The mechanism(s) involved in the formation of Pack-MULEs: to study the mechanism of sequence acquisition of Pack-MULEs, I plan to establish a yeast system to observe the transposition of MULEs and possible acquisition events. As the first step, we are cloning the transposase domain and build artificial non-autononmous elements for the test. PARTICIPANTS: Ning Jiang: project director, designing experiments and conducting experiments TARGET AUDIENCES: Through my efforts, post-doctorial associates, graduate students and undergraduate students are trained as next generation leaders for science. Our research results are inspiring for people how work on transposon biology and evolutionary biology. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our finding indicates that Pack-MULE can influence their genomes in multiple ways and has profound impact on genome evolution. In addition to their coding capacity to serve as independent genes, the amplification of Pack-MULEs will result in the increase of GC content of the genome as well as the GC gradient of genes. The presence of GC rich Pack-MULEs at the 5 end of genes, either through fusion to their adjacent gene, or as independent elements with close proximity to the end of genes, may have further evolutionary impact beyond the alteration of gene sequences. For instance, it is known that in many organisms the recombination rate is correlated with local GC content. Thus, the insertion of Pack-MULEs may alter the local recombination rate. In mammalian cells, GC rich genes are associated with elevated expression levels compared to their GC poor counterparts, even if they encode the same protein and are driven by the same promoters. GC rich sequences also provide more targets for DNA methylation, which can contribute to the regulation of gene expression. GC rich regions are associated with more CG and CHG sites (where H=A, T or C), and therefore have the potential to maintain epigenetic patterns of DNA methylation more efficiently than GC poor regions. In plants, only cytosines in CG or CHG contexts can propagate the DNA methylation state from one cell to the next or from parent to daughter generation. GC sparse regions have larger amounts of CHH, which are incapable of maintaining the methylation pattern upon S-phase DNA replication. Therefore, GC rich sequences retain heritable DNA methylation levels more efficiently, and the presence of Pack-MULEs may stabilize epigenetic control of gene expression of the neighboring genes. Finally, GC rich sequences tend to augment bendability and ability to undergo a B-Z transition, which is often associated with open chromatin and active transcription.

Publications

  • Ammiraju J.S., Fan C., Yu Y., Song X., Cranston K.A., Pontaroli A.C., Lu F., Sanyal A., Jiang N., Rambo T., Currie J., Collura K., Talag J., Bennetzen J.L., Chen M., Jackson S., Wing R.A. 2010 (Aug). Spatio-temporal patterns of genome evolution in allotetraploid species of the genus Oryza. Plant J. 63:430-442.
  • Zhang, H., Liang, W., Yang, X., Luo, X., Jiang, N., Ma, H., Zhang, D. 2010. Carbon Starved Anther (CSA) Encoding a MYB Domain Protein Regulates Sugar Partitioning Required for Rice Pollen Development. Plant Cell 22(3):672-89.
  • Vogel, J.P., Bevan, M.W., Garvin, D.F., Mockler, T.C., Lindquist, E., Grigoriey, I., Tice, H., Wang, M., Barry, K., Schmutz, J., Grimwood, J., McKenzie, N., Huo, N., Gu, Y.Q., Luo, M., Dvorak, J., Anderson, O.D., Haberer, G., Wright, J., Febrer, M., Spannagl, M., Idziak, D., Hasterok, R., Mayer, K., Wicker, T., Fox, S.E., Priest, H.D., Filichkin, S.A., Givan, S.A., Bryant, D.W., Chang, J.H., Mockler, T.C., Wu, H., Wu, W., Hsia, A., Schnable, P.S., Kalyanaraman, A., Barbazuk, B., Michael, T.P., Hazen, S., Rattei, T., Bragg, J., Laudencia, D., Mitros, T., Weng, Y., Rokhsar, D., Buchmann, J.P., Tanskanen, J., Schulman, A.H., Gundlach, H., Costa de Oliveira, A., Carlos de Maia, L., Jiang, N., Lai, J., Ma, J., Salse, J., Murat, F., Abrouk, M., Bruggmann, R., Dvorak, J., Fahlgren, N., Fox, S.E., Sullivan, C.M., Carrington, J.C., Chapman, E., Zhai, J., Ganssmann, M., Ranjan, G.S., German, M., May, G.D., Meyers, B.C., Green, P.J., Debodt, S., Verelst, W., Inze, D., Heese, M., Schnittger, A., Hazen, S., Pelloux, J., Sedbrook, J., Cass, .M., Ronald, P., Carrington, J., Fahlgren, N., Fox, S., Filchin, S., Priest, H.D., Chang, J.H., Kimbel, J.A., Cui, Y., Ouyang, S., Sun, Q., Liu, Z. 2010. Genome Sequence Analysis of the Model Grass Brachypodium Distachyon: Insights Into Grass Genome Evolution. Nature 463:763-768.
  • Tsukamoto, T., Hauck, N.R., Tao, R., Jiang, N. and Iezzoni A.F. 2010. Molecular and Genetic Analyses of Four Non-Functional S Haplotype Variants Derived from a Common Ancestral S Haplotype Identified in Sour Cherry (Prunus cerasus L.) Genetics 184 (2):411-27.


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: The project is associated with two goals: 1) to test the function of MULEs including Pack-MULEs; 2) explore the mechanism(s) underlying Pack-MULE formation. For the first task, we are continuing to characterize the function of a Pack-MULE-CBF gene. Most known CBF genes are capable of enhancing stress tolerance of plants, but such enhancement is often associated with growth inhibition, which hindered the application of this gene family in crop breeding. This particular Pack-MULE harbors a DNA domain from a known CBF gene. Nevertheless, it does not contain the coding region for the activation domain from the same gene; instead, the putative activation domain was replaced by a DNA fragment from the downstream region of the relevant CBF gene. In this case, the PM-CBF either has a novel or non-functional activation domain. The over-expression of the PM-CBF enhances the cold hardiness and salt tolerance in transgenic Arabidopsis plants, but without the usual associated growth retardation. In fact, the transgenic plants flower earlier than wild-type plants. This phenotype is distinct from any of the CBF genes characterized to date and could be very useful in crop improvement. To understand the mechanism(s) involved in the formation of Pack-MULEs, the best approach is to study a Pack-MULE with current activity, and characterize the process of how a novel genomic sequence is captured. To this end, we screened maize populations with active Mutator elements. Mutator elements are known to carry gene fragments but it is not clear whether such activity (to acquire new gene fragments) is still an on-going process. For this purpose, we developed a genome-wide approach to amplify elements with novel internal sequences (putatively through new acquisition events) which are called variants. Through such variants, we now know what we cannot learn from existing sequences. For example, the internal regions of Pack-MULEs are frequently associated with deletions, insertions, and reversions compared to their parental genes. However, such rearrangements were not observed with the new variants. This implies that the initial acquisition by the Pack-MULE is a process with high fidelity, and the rearrangements observed are likely generated in the subsequent transposition process. PARTICIPANTS: Ning Jiang: project director, designing experiments and conducting experiments. TARGET AUDIENCES: Through my efforts, post-doctorial associates, graduate students and undergraduate students are trained as next generation leaders for science. Especially, we worked together with McNair program to encourage minority, low-income, and first-generation college juniors and seniors to enter graduate study. This year we accepted a student, Tanisha Vazquez, from McNair program for summer research internship. Now she is preparing for application for graduate school. Students of transposable element structure and function will find my results of interest and value in their own programs; as will evolutionary biologists interested in plant genes/genomes. Also, the identified gene(s) can be useful for improvements in crop production through biotechnology PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
These findings are significant for many reasons, both theoretically and practically. First of all, our results prove that Pack-MULEs do encode functional proteins, so this closes the long-term dispute whether any Pack-MULE represents a functional protein coding gene. Second, this further indicates that Pack-MULEs represent an important mechanism for gene, and thus genome, evolution. Third, the novel phenotype caused by the PM-CBF indicates that what Pack-MULEs mediate is not a simple duplication process. Rather, it is a creative pathway that may lead to the generation of novel functions. Fourth, it is very likely the gene duplication by Pack-MULE is an on-going process so Pack-MULEs are shaping the plant genome at current time. Finally, as mentioned above, the fact that the Pack-MULE-CBF can enhance the stress tolerance but without inhibiting growth should it is a very good candidate gene for engineering crop plants with better stress tolerance. Several relevant manuscripts have been published this year.

Publications

  • Gao, D., Gill, N., Kim, H.R., Walling, J.G., Zhang, W., Fan, C., Yu, Y., Ma, J., Sanmiguel, P., Jiang, N., Cheng, Z., Wing, R.A., Jiang, J.and Jackson, S.A. 2009. A lineage-specific centromere retrotransposon in Oryza brachyantha. Plant J. 60 (5): 820-831.
  • Jiang, N., Gao, D., Xiao, H. and van der Knaap, E. 2009. Genome organization of the tomato sun locus and characterization of the unusual retrotransposon Rider. Plant J. 60: 181-193.
  • Wei, .F., Stein, J., Liang, C., Zhang, J., Fulton, R.S., Baucom, R.S., Paoli, E.D., Zhou, S., Yang, L., Han, Y., Pasternak, S., Narechania, A., Zhang, L., Yeh, C., Ying, K., Nagel, D.H., Collura, K., Kudrna, D., Currie, J., Lin, J., Kim, H., Angelove, A., Scara, G., Wissotski, M., Golser, W., Courtney, L., Kruchowski, S., Graves, T., Rock, S., Adams, S., Fulton, L., Fronick, C., Courtney, W., Kramer, M., Spiegel, L., Nascimento, L., Kalyanaraman, A., Chaparro1, C., Deragon, J., SanMiguel1, P., Jiang, N., Wessler, S.R., Green, J., Soderlund1, C., Yu, Y., Schwartz, D.C., Meyers, B.C., Bennetzen, J., Martienssen, R., McCombie, W.R., Aluru1, S., Clifton, S.W., Schnable, P.S., Ware, D., Wilson, R.K., Wing, R.A. 2009. Detailed Analysis of a Contiguous 22-Mb Region of the Maize Genome. PLoS Genetics 5 (11): e1000728.
  • Schnable, P.S., Ware, D., Pasternak, R.S., Liang, C., Zhang, J., Fulton, L., Graves, T.A., Minx, P., Reily, A.D., Courtney, L., Kruchowski, S.S., Tomlinson, C., Strong, C., Delehaunty, K., Fronick, C., Courtney, B., Rock, S., Belter, E., Du, F., Kim, K., Abbott, R.M., Cotton, M., Levy, A., Marchetto, P., Ochoa, K., Jackson, S.M., Gillam, B., Chen, W., Yan, L., Higginbotham, J., Cardenas, M., Waligorski, J., Applebaum, E., Phelps, L., Falcone, J., Kanchi, K., Thane, T., Scimone, A., Thane, N., Henke, J., Wang, T., Ruppert, J., Shah, N., Rotter, K., Hodges, J., Ingenthron, E., Cordes, M., Kohlberg, S., Sgro, J., Delgado, B., Mead, K., Chinwalla, A., Leonard, S., Crouse, K., Collura, K., Kudrna, D., Currie, J., He, R., Angelova, A., Rajasekar, S., Mueller, T., Lomeli, R., Scara, G., Ko, A., Delaney, K., Wissotski, M., Lopez, G., Campos, D., Braidotti, M., Ashley, E., Golser, W., Kim, H., Lee, S., Lin, J., Dujmic, Z., Kim, W., Talag, J., Zuccolo, A., Fan, C., Sebastian, A., Kramer, M., Spiegel, L., Nascimento, L., Zutavern, T., Miller, B., Ambroise, C., Muller, S., Spooner, W., Narechania, A., Ren, L., Wei, S., Kumari, S., Faga, B., Levy, M., McMahan, L., Buren, P.V., Vaughn, M.W., Ying, K., Yeh, C., Emrich, S.J., Jia, Y., Kalyanaraman, A., Hsia, A., Barbazuk1, W.B., Baucom1, R.S., Brutnell, T.P., Carpita, N.C., Chaparro, C., Chia, J., Deragon, J., Estill, J.C., Fu, Y., Jeddeloh, J.A., Han, Y., Lee, H., Li, P., Lisch, D., Liu, S., Liu, Z., Nagel, D.H., McCann, M.C., San Migue, P.S., Myers, A.M., Nettleton, D., Nguyen, J., Penning, B.W., Ponnala, L., Schneider, K.L., Schwartz, D.C., Sharma, A., Soderlund, C., Springer, N.M., Sun, Q., Wang, H., Waterman, M., Westerman, R., Wolfgruber, T.K., Yang, L., Yu, Y., Zhang, L., Zhou, S., Zhu, Q., Bennetzen, J.L., Dawe, R.K., Jiang, J., Jiang, N., Presting, G.G., Wessler, S., Aluru, S., Martienssen, R.A., Clifton, S.W., McCombie, W.R., Wing, R.A. and Wilson, R.K. 2009. The B73 maize genome: complexity, diversity and dynamics. Science 326 (5956): 1112-1115 (Cover story).
  • Lisch, D., and Jiang, N. 2009. Mutator and MULE transposons. In Handbook of Maize: Genetics and Genomics, J.L. Benntzen and S. Hake, eds (New York: Springer), pp. 277-306.


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: MULEs refer to Mutator-like transposable elements, and they often carry fragment of host genes (called Pack-MULEs). We performed a genome-wide analysis using existing transcription dataset. Our analysis indicates that Pack-MULEs are frequently associated with small RNAs. The presence of these small RNAs are associated with a reduction in expression of both the Pack-MULEs and their parental genes. Moreover, such suppression seems to be correlated with the age of Pack-MULEs. Younger Pack-MULEs have lower expression level and share more sRNAs with their parental genes. To test the function of Pack-MULE-CBF gene and its relationship with endogenous CBF genes, we built over-expression lines in Arabidopsis and rice as well RNAi knock-out line in rice. In Arabidopsis, the over-expression of Pack-MULE-CBF not only suppresses the expression of downstream gene, but also affects the development of the plants. In rice, the over-expression of Pack-MULE-CBF completely shut down the expression of one of the target genes of normal CBFs. The above evidence clearly indicates that the Pack-MULE-CBF gene is a functional gene. In addition, our genome wide analysis indicates that the percent of expressed Pack-MULEs is much higher than that of other transposable elements as well as pseudogenes, and Pack-MULEs are subject to purifying selection, which suggests that a subset of Pack-MULEs could be functional genes. To date, maize is the only plant with active Pack-MULEs. To study the mechanism of gene acquisition by Pack-MULEs, we designed a genome-wide approach to screen for the Mutators with altered internal regions. Using this strategy, we did recover elements with dramatic rearrangements, suggesting constant DNA breakage/recombination occurring within the Mutator elements. We are optimizing the approach to catch the acquisition in action. PARTICIPANTS: Individuals who worked on this project: Veronica Vallejo: postdoctoral associate, the function of a Pack-MULE CBF gene in rice and Arabidopsis. Ann Armenia: acquisition mechanism of Pack-MULEs Collaborators: Kousuke Hanada and Shin-Han Shiu (Michigan State University), purifying selection on Pack-MULEs Kan Nobuta and Blake C. Meyers (University of Delaware), expression of Pack-MULEs Keith Slotkin and Damon Lisch (University of California at Berkeley), the relationship between inverted repeat and small RNAs derived from Pack-MULEs. Patrick Schnable (Iowa State University), Provision of Mutator active lines. TARGET AUDIENCES: Plant researchers and scientists who are interested in evolution PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We released the inventory of 2809 Pack-MULEs in the genome of Nipponbare. This will facilitate the gene annotation in rice as well as provide basis for future functional analysis. Our survey clarified an important issue in evolution, i.e., the formation of Pack-MULEs generates various impacts on the genomes, including the creation of novel functional genes.

Publications

  • Hanada K., Vallejo V., Nobuta K.,R. Slotkin R.K., Lisch D., Meyers, B.C., Shiu, S.H., and Jiang N. 2009. The functional role of Pack-MULEs in rice inferred from purifying selection and expression profile. The Plant Cell (In press.)
  • Lisch D and Jiang N. Mutator and MULE transposons 2008. In Bennetzen J. and Hake S (ed.), Domestication, Genetics and Genomics of Maize. (In press).
  • Ammiraju, J.S.S, Lu F., Sanyal A., Yu Y., Song X., Jiang N, Pontaroli A.C., Rambo T., Currie J., Collura K., Talag J, Fan C., Goicoechea J.L., Zuccolo A., Chen J, Bennetzen J.L., Chen M., Jackson, S., and Wing R.A. 2008. Dynamic Evolution of Oryza Genomes Is Revealed by Comparative Genomic Analysis of a Genus-Wide Vertical Data Set. The Plant Cell. 20:
  • Xiao H., Jiang N., Schaffner E., Stockinger E.J., van der Knaap E. 2008. A retrotransposon-mediated gene duplication underlies morphological variation of tomato fruit. Science 319:1527-1530.


Progress 01/01/07 to 12/31/07

Outputs
1. We performed a genome-wide characterization of Pack-MULEs using the inventory of 2,800 Pack-MULEs that we generated in the previous year. At least 20% of Pack-MULEs are expressed, yet the expression of Pack-MULEs is less ubiquitous than that of their parent genes, and the level of expression is lower than that of the parent genes in general. Interestingly, most Pack-MULEs are transcribed in the tissues (or conditions) where transcripts from their parent genes are not detectable, suggesting the duplication of genes by Pack-MULEs facilitate novel tissue specific expression patterns. Pack-MULEs are frequently associated with small RNAs, and a large part of the small RNAs could be attributed to the presence of inverted repeat of Pack-MULEs. Those small RNAs seems to suppress the expression of the element themselves and the expression of the parent genes. The transcription of Pack-MULEs could be in both sense direction and anti-sense direction; however, there are much more elements with sense transcripts than that with ant-sense transcripts, suggesting that there is a selective pressure against anti-sense transcription. Moreover, the elements with sense transcripts have smaller ka/ks (non-synonymous to synonymous substitution rate), suggesting that they have coding potentials. Taken together, our analysis indicates that Pack-MULEs are capable of regulating the expression of parent genes as well as encoding functional proteins. 2. To experimentally test the function of genes carried by Pack-MULEs, we built both the over-expression and knock-down lines (using inverted repeat for part of the coding region) of a CBF gene carried by a Pack-MULE. As a first step to characterize the transgenic plants, the copy number of the transgene was tested by DNA blot experiments and the expression level of transgene was verified by northern blot and RT-PCR. The next experiment will be testing the expression of putative downstream genes as well as phenotypic changes, using lines with few copies of transgenes and high expression levels.

Impacts
Our genome-wide analysis of Pack-MULEs benefits the relevant research in the community. For example, we have collaborated with Robin Buell and her group to map all the Pack-MULEs in the rice genome so to facilitate gene annotation. Interestingly, we found that Pack-MULEs are over-represented in Conserved Poaceae Specific Genes, provided a possible mechanism how those genes arose. The whole Pack-MULE dataset will be available to public after the relevant manuscript is published.

Publications

  • Zhang S., Gu Y.Q., Singh J., Coleman-Derr D., Brar, D.S., Jiang, N. and Lemaux, P.G. 2007. New insights into Oryza genome evolution: high gene colinearity and differential retrotransposon amplification. Plant Mol Biol. 64:589-600.
  • Ammiraju, J.S.S., Zuccolo, A., Yu, Y., Song, X., Piegu, B., Chevalier, F., Walling, J.G., Ma, J., Talag, J., Brar, D.S., SanMiguel, P.J., Jiang, N., Jackson, S.A., Panaud, O. and Wing, R.A.. 2007. Evolutionary dynamics of an ancient retrotransposon family provides insights into evolution of genome size in the genus Oryza. Plant J. 52:342-51.
  • Campbell, M.A. Zhu, W., Jiang, N., Haas, B.J., Lin, H., Ouyang, S., Childs, K.L., Hamilton, J.P. and Buell, C.R.. 2007. Identification and characterization of lineage-specific genes within the Poaceae. Plant Physiol. 145:1311-22.


Progress 01/01/06 to 12/31/06

Outputs
1. We have generated an inventory of Pack-MULEs in the rice genome. The 28,000 Pack-MULEs are located in all 12 chromosomes but the density of Pack-MULEs varies from chromosome to chromosome. An examination of genes located in Pack-MULEs indicated that genes related to plant defense processes (such as heat shock, cold shock and pathogenesis-related) are over represented. Our analysis also indicated that rice Pack-MULEs, like the original Mutator elements in maize, preferentially inserted into the promoter regions of genes. Nevertheless, the target site sequence of Pack-MULEs in rice is different from that in maize. Whereas the new insertions of maize Mutator elements are inserted into GC-rich regions, the target site sequences of rice Pack-MULEs are AT-rich. This suggests either the rice MULEs recognize different sequence motifs or there is selective pressure against the insertions in GC-rich regions in rice. A relevant manuscript is in preparation and all the data will be publicly available. 2. To test the function of genes carried by Pack-MULEs, we generated over-expression lines of a CBF gene carried by a Pack-MULE in rice, using Arabidopsis and rice plants. Due to the length of life cycle, the transgenic rice lines will be ready in 2007. Our experiments with transgenic Arabidopsis suggest that when there are no endogenous CBF transcripts, the PM-CBF can induce the expression of downstream genes, to a certain level. However, the level of expression is not sufficient to result in a dwarf phenotype, as that would be caused by the over-expression of an endogenous CBF gene. On the other hand, when the endogenous CBF transcripts are at a moderate level, the presence of PM-CBF antagonizes the effect of endogenous CBF genes, reflected by the relative low expression of downstream genes. When endogenous CBF transcripts are expressed at high level, no detectable difference was observed. Based on those results and the sequence comparison between PM-CBF and its genomic copy, we hypothesize that the PM-CBF has a functional DNA binding domain, but the activity of its activation domain is limited. To test the hypothesis, we generated constructs contain individual domains of the PM-CBF and will further test their function in vitro using yeast one-hybrid system.

Impacts
The comprehensive list of Pack-MULEs in rice will facilitate gene annotation in rice and benefit the research of the whole rice community. Our analysis on the Pack-MULE-CBF gene will provide first evidence whether Pack-MULEs generate novel genes. It will add to our knowledge about how new genes evolved and what is the impact of transposable elements on host organisms.

Publications

  • No publications reported this period