Source: OKLAHOMA STATE UNIVERSITY submitted to
CLONING AND CHARACTERIZATION OF SMALL RNAS FROM MEDICAGO TRUNCATULA AND GLYCINE MAX
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0209525
Grant No.
(N/A)
Project No.
OKL02611
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jan 1, 2007
Project End Date
Sep 30, 2012
Grant Year
(N/A)
Project Director
Sunkar, R.
Recipient Organization
OKLAHOMA STATE UNIVERSITY
(N/A)
STILLWATER,OK 74078
Performing Department
BIOCHEMISTRY & MOLECULAR BIOLOGY
Non Technical Summary
Recent discovery of small RNAs with regulatory roles has uncovered an active role for RNA in regulating gene expression. Thus far, efforts in identification of small RNAs have been limited to a few model species such as Arabidopsis and rice and legume-specific small RNAs are unknown. Leguminous plants are an important source of human and dietary needs second only to cereals. Here, we propose to identify the complete set of miRNAs and other endogenous small RNAs including their targets in two legumes. In addition, we propose to identify small RNAs that might be regulated during abiotic stress and symbiosis in M. truncatula and soybean. The proposed research is directed to identify and understand the roles of microRNAs and small-interfering RNAs in M. truncatula and soybean. A long-term objective is to investigate other legumes and crop plants such as peanuts that are a major component of Oklahoma agriculture. Therefore, output from the proposed study will not only contribute to our basic knowledge of plant biology but also have potential for biotechnological applications in agriculture.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011649108040%
2011820108060%
Goals / Objectives
1) Deep profiling of small RNAs in different tissues, in response to abiotic stress and during symbiosis (nodulation and mycorrhizal associations) in Medicago truncatula and Glycine max (soybean), 2) Determine the tissue specificity, abiotic stress regulation and regulation during symbiotic associations of the miRNAs and other endogenous siRNAs, 3) Target prediction for the newly identified small RNAs and their experimental validation, 4) Determine the effect of small RNAs on spatio- and temporal-expression of the target genes, and 5).Production of small RNA microarrays for M. truncatula and soybean and high throughput expression analyses of miRNAs and other siRNAs.
Project Methods
We will use the small RNA cloning approach to identify the complete or near complete set of microRNAs in Medicago truncatula (jemalong) and soybean (Glycine max (L.). Merr, Williams 82 cultivar) from different tissues and organs and seedlings exposed to abiotic stress. We will also generate small RNA libraries from the Medicago truncatula roots treated with Sinirhizobium meliloti and soybean roots treated with Bradyrhizobium japonicus. Additionally, we will construct libraries from roots colonized with Glomus sps which will allow us to identify common as well as distinct small RNAs in two different symbiotic processes. We will perform Northern analyses for the miRNAs and selected siRNAs. We will also predict and validate the mRNA targets for these small RNAs in M. truncatula and soybean.

Progress 01/01/07 to 09/30/12

Outputs
OUTPUTS: Recently discovered small RNAs [microRNAs (miRNAs) and short interfering RNAs (siRNAs)] emerged as key regulators of gene expression. Leguminous plants are an important source of human and animal dietary needs second only to cereal plants. We have generated small RNA libraries from Medicago truncatula and Glycine max. For Glycine max, the project was focussed on identification of microRNAs, which are altered during nodulation. For Medicago truncatula, the sequences were analysed for identification of conserved as well as novel miRNAs. This analysis resulted in identification 22 conserved miRNA families in. Sequence analysis also revealed eight novel small RNAs, of which four (miR2118, miR2199, miR1507 and miR2119) are legume-specific miRNAs, because these are conserved in related legumes. Furthermore we also characterized Tas3-siRNA biogenesis in M. truncatula and validated three ARF transcripts that are targeted by tasiRNAs. We have performed Northern blot analysis to determine the expression patterns of miRNAs in Medicago truncatula. We have predicted several TIR-NBS-LRR genes as targets for miR2118, one of the legume-specific miRNA. Using 5'-RACE assay we confirmed one of the TIR-NBS-LRR genes as genuine target for miR2118. The identification of miR2118 that targets TIR-NBS-LRR genes suggested an important role for this miRNA in plant-microbe interactions or plant symbiosis. PARTICIPANTS: Ramanjulu Sunkar (PI), Guru jagadeeswaran (Post-doc), Yong-Fang Li (Post-doc), Kanchana Gowdu (Research Specialist), Lata Shukla (Visiting Scientist) and Jessica Matts (Graduate Student) Collaborataors, Dr. Rao Uppalapati, SR Noble Foundation, Ardmore, Senthil Subramanian and Oliver Yu, Donald Danforth Plant Science Center, St Louis; Bruce A Roe, University of Oklahoma, Norman; Weixiong Zhang, Washington University at St Louis. TARGET AUDIENCES: Scientists working on post-transcriptional gene regulation in Medicago, soybeand and other legumes. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Identification of four families of legume-specific miRNAs besides 22 families of conserved miRNAs in M. truncatula and soybean suggested that legumes have complex mechanisms of gene regulation involving specific and common small RNAs operating post-transcriptionally. Identification of a legume-specific miR2118, which is targeting TIR-NBS-LRR genes is a major important finding. The analyses of small RNA library generated from Glycine max roots subjected to nodulation has led to the identification of the involvement of miRNAs in nodulation process. A post-doctoral fellow/graduate students has been trained in generating microRNA libraries, sequence analysis and performing Northern blot analysis and modified 5' RACE assays to detect the miRNA-guided cleavage site on target mRNAs as well as writing manuscripts for publication in peer-reviewed journals.

Publications

  • Zheng, Y., Li, Y., Sunkar, R. and Zhang, W. (2012). Seq-Tar: An effective method for identifying microRNA guided cleavage sites from degradome of polyadenylated transcripts in plants. Nucleic Acids Research, 40(4), e28.
  • Li, Z., Zheng, Y., Jagadeeswaran, G. Li, Y., Gowdu, K and Sunkar, R. (2011). Identification and temporal expression analysis of conserved and novel miRNAs in Sorghum. Genomics, 98: 460-468.
  • Sunkar, R. (2010). MicroRNAs with macro effects on plant stress responses. Seminars in Cell and Developmental Biology, 21(8), 805-11.
  • Jagadeeswaran G, Zheng Y, Li YF, Shukla LI, Matts J, Hoyt P, Macmil SL, Wiley GB, Roe BA, Zhang W, Sunkar R. (2009). Cloning and characterization of small RNAs from Medicago truncatula reveals four novel legume-specific microRNA families. New Phytologist, 184: 85-98.
  • Sunkar, R and Jagadeeswaran, G. (2008). In silico identification of conserved miRNAs in large number of diverse plant species. BMC Plant Biology, 8:37. Subramanian, S., Fu, Y., Sunkar, R., Barbazuk, B. W., Zhu, J-K. and Yu, O. (2008). Novel and nodulation-regulated microRNAs in soybean roots. BMC Genomics, 9:160.
  • Shukla, L., Chinnusamy, V. and Sunkar, R (2008). The role of microRNAs and other endogenous small RNAs in plant stress responses. Biochem. Biophys. Acta; Gene Regulatory Mechanisms, 1779, 743-748
  • Sunkar, R., Chinnusamy, V., Zhu, J. and Zhu, J.K. (2007). Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. Trends in Plant Sciences, 12: 301-309.
  • Sunkar, R. and Zhu, J.K. (2007). Micro RNAs and short-interfering RNAs in plants. Journal of Integrative Plant Biology, 49: 817-826.
  • Yao, Y., Guo, G., Ni. Z., Sunkar, R., Du, J., Zhu, J.K. Sun, Q. (2007). Cloning and characterization of microRNAs from Wheat (Triticum aestivum L.). Genome Biology, 8: R96.
  • Sunkar, R., Li, Y. and Jagadeeswaran, G. (2012). Functions of microRNAs in plant stress responses. Trends in Plant Science, 17, 196-203.
  • Iyer, N., Jia, X., Sunkar, R., Tang, G. and Mahalingam, R. (2012). Analysis of microRNAs responsive to ozone-induced oxidative stress in Arabidopsis thaliana. Plant Signaling and Behavior 7, 484-491.
  • Fu, C., Sunkar, R., Zhou, C., Shen, H., Zhang, J., Matts, J., Wolf, J., Mann, D.G.J., Neal Stewart, C., Tang, Y. and Wang, Z-Y (2012). Overexpression of miR156 in switchgrass results in various morphological alterations and leads to improved biomass production. Plant Biotechnology Journal, 10, 443-452.


Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: The identification of miR2118 that targets TIR-NBS-LRR genes suggested an important role for this miRNA in plant-microbe inetractions or plant symbiosis. In order to investigate its precise role, we are generating transgneics overexpressing miR2118 and miR2118-resistant TIR-NBS-LRR gene. Currently these experiments are underway. PARTICIPANTS: Ramanjulu Sunkar (PI), Guru jagadeeswaran (Post-doc), Yong-Fang Li (Post-doc), Kanchana Gowdu(Research Specialist) Collaborataors, Drs. Rao Uppalapati and Kinran Mysore, SR Noble Foundation, Ardmore TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Identification of a microRNA which is legumes-epcific and targeting TIR-NBS-LRR genes is a major important finding.

Publications

  • Li, Z., Zheng, Y., Jagadeeswaran, G. Li, Y., Gowdu, K and Sunkar, R.(2011) Identification and temporal expression analysis of conserved and novel miRNAs in Sorghum. Genomics, in press.


Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: OUTPUTS: We sequenced a small RNA library generated from M. truncatula and identified eight novel small RNAs, of which four (miR2118, miR2199, miR1507 and miR2119) appears to be legume-specific. We have predicted several TIR-NBS-LRR genes as targets for miR2118, one of the legume-specific miRNA. Using 5'-RACE assay we confirmed one of the TIR-NBS-LRR genes as genuine target for miR2118. We have isolated the full length sequence of this gene as well as precursor of MIR2118 and generated bnary constructs. Along with the wild-type TIR-NBS-LRR gene, we also generated a mutant version of this gene (introduced mutations in the miRNA recognition site) and cloned into binary vector. All these constructs are used for transforming M. truncatula. Once we have the transgenics we will analyze the response of these transgenics for their resistance or sensitivity to diverse pathogens (bacterial and fungal pathogens). PARTICIPANTS: PARTICIPANTS: Ramanjulu Sunkar (PI), Guru Jagadeeswaran (Post-doc), Yong-Fang Li (Post-doc). Collaborators Dr. Rao Uppalapati, SR Noble Foundation, Ardmore. TARGET AUDIENCES: TARGET AUDIENCES: Scientists working on post-transcriptional gene regulation in Medicago and legumes. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Identification of several TIR-NBS-LRR genes as targets for a legume-specific miRNA2118 suggested that miR2118 may have an important role in legume-pathogen interactions.

Publications

  • Sunkar, R. (2010). MicroRNAs with macro effects on plant stress responses. Seminars in Cell and Developmental Biology, 21(8), 805-11.


Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: MicroRNAs (miRNAs) and small-interfering RNAs (siRNAs) have emerged as important regulators of gene expression in higher eukaryotes. Leguminous plants are grown throughout the world for food and forage production. To date the lack of genomic sequence data has prevented systematic examination of small RNAs in leguminous plants. Medicago truncatula, a diploid plant with near-completely sequenced genome has recently emerged as an important model legume. We sequenced a small RNA library generated from M. truncatula to identify not only conserved miRNAs but also novel small RNAs, if any. Sequence analysis indicated that we have identified eight novel small RNAs, of which four (miR2118, miR2199, miR1507 and miR2119) are legume-specific miRNAs, because these are conserved in related legumes. Furthermore we also characterized Tas3-siRNA biogenesis in M. truncatula and validated three ARF transcripts that are targeted by tasiRNAs. PARTICIPANTS: Ramanjulu Sunkar (PI), Guru Jagadeeswaran (Post-doc), Yong-Fang Li (Post-doc), Jessica Matts (graduate student). Collaborators Dr. Bruce Roe, University of Oklahoma, Graham B. Wiley, University of Oklahoma, Simone Macmil, University of Oklahoma, Dr. Peter Hoyt, Oklahoma State University, Yun Zheng, Washington University in St Louis and Weixiong Zhang, Washington University in St Louis TARGET AUDIENCES: Scientists working on post-transcriptional gene regulation in Medicago and legumes. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Identification of four families of legume-specific miRNAs besides 20 families of conserved miRNAs in M. truncatula suggested that legumes have complex mechanisms of gene regulation involving specific and common small RNAs operating post-transcriptionally.

Publications

  • Jagadeeswaran G, Zheng Y, Li YF, Shukla LI, Matts J, Hoyt P, Macmil SL, Wiley GB, Roe BA, Zhang W, Sunkar R. (2009). Cloning and characterization of small RNAs from Medicago truncatula reveals four novel legume-specific microRNA families. New Phytologist, 184: 85-98.


Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: We have generated small RNA libraries from Medicago truncatula and Glycine max. The libraries were sequenced and analysed for the identification of microRNAs or other classes of endogenous siRNAs. We also have predicted targets for many of the conserved and novel miRNAS we have idnetified in Medicago truncatula. We have performed Northern blot analysis to determine the expression patterns of miRNAs in Medicago truncatula. The results will be dessiminated through a publication and currently the manuscript is under revision for a peer-reviewed Plant Science Journal. For Glycine max, the project was focussed on identification of microRNAs which are altered during nodulation. Results were dessiminated through a poster presentation at the Annual meeting of American Society of Plant Biologists. Results were also published in the journal BMC Genomics, an open access journal. PARTICIPANTS: Ramanjulu Sunkar (PI); Guru Jagadeeswaran (Post-doc), Jessica Matts (Graduate Student) Collaborators, Senthil Subramanian and Oliver Yu, Donald Danforth Plant Science Center, St Louis; Bruce A Roe, University of Oklahoma, Norman; Weixiong Zhang, Washington University at St Louis. Trained Guru Jagadeeswaran (Post-doc), Lata Shukla (Visiting Scientist) and Jessica Matts (Graduate Student) TARGET AUDIENCES: Plant Molecular Biologists working on post-transcriptional regulation of gene expression medicago truncatula, Glycine max and other reltaed legumes PROJECT MODIFICATIONS: None

Impacts
The sequence analysis of Medicago small RNA library revealed novel legume-specific miRNAs as well as Medicago truncatula-specific miRNAs and this new information will be published soon. The anlsysis of small RNA library generated from Glycine max roots subjected to nodulation has led to the identification of the involvement of miRNAs in nodulation process. This has been published in BMC Genomics. A post-doctoral fellow has been trained in generating microRNA libraries, sequence analysis and perfroming Northern blot analysis and performing modified 5' RACE assays todetect the miRNA-guided cleavage site on target mRNAs as well as writing manuscripts for publication in peer-reveiwed journals.

Publications

  • Sunkar, R and Jagadeeswaran, G. (2008). In silico identification of conserved miRNAs in large number of diverse plant species. BMC Plant Biology, 8:37.
  • Subramanian, S., Fu, Y., Sunkar, R., Barbazuk, B. W., Zhu, J-K. and Yu, O. (2008). Novel and nodulation-regulated microRNAs in soybean roots. BMC Genomics, 9:160.
  • Shukla, L., Chinnusamy, V. and Sunkar, R (2008). The role of microRNAs and other endogenous small RNAs in plant stress responses. Biochem. Biophys. Acta; Gene Regulatory Mechanisms, 1779, 743-748


Progress 10/01/06 to 09/30/07

Outputs
OUTPUTS: Recently discovered small RNAs [microRNAs (miRNAs) and short interfering RNAs (siRNAs)] emerged as key regulators of gene expression. Leguminous plants are an important source of human and animal dietary needs second only to cereal plants. M. truncatula is rapidly developed as an experimental model legume to pursue a number of important biological questions unique to these plants. So far, identification of small RNAs has been limited to a few model species such as Arabidopsis and rice and very little is known about the small RNA component of a legume. Currently, our investigation has identified 20 families of conserved miRNAs in Medicago. PARTICIPANTS: Ramanjulu Sunkar (PI) Dr. Lata Shukla (Post-doc) Collaborators Dr. Senthil Subramanian, Donald Danfoth Plant Sceince Center, St.Louis Dr. Oliver Yu, Donald Danfoth Plant Sceince Center, St.Louis TARGET AUDIENCES: Scientists working on post-transcriptional gene regulation in Medicago and in legumes PROJECT MODIFICATIONS: None

Impacts
we have identified ∼20 conserved miRNA families in Medicago truncatula. Identfication of conserved miRNAs using in silico approach, confirmed that the all of the 20 well characterized miRNA families are conserved in Medicago. To probe spatio- and temporal- expression patterns of these miRNAs, we are performing Northern analysis and these experiments are in progress. Besides, we have predicted target genes for majority of these miRNAs in Medicago. Future experimental validation will reveal how many of these targets are under miRNA-directed post-transcriptional regulation in Medicago.

Publications

  • Sunkar, R., Chinnusamy, V., Zhu, J. and Zhu, J.K. (2007). Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. Trends in Plant Sciences, 12: 301-309.
  • Sunkar, R. and Zhu, J.K. (2007). Micro RNAs and short-interfering RNAs in plants. Journal of Integrative Plant Biology, 49: 817-826.
  • Yao, Y., Guo, G., Ni. Z., Sunkar, R., Du, J., Zhu, J.K. Sun, Q. (2007). Cloning and characterization of microRNAs from Wheat (Triticum aestivum L.). Genome Biology, 8: R96.