Progress 05/01/11 to 04/30/15
Outputs
Target Audience: Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Two postdoctoral fellows gained experience working with soybean. Postdoctoral fellows: Kestrel Rogers and Brandon Le Kestrel performed the wet-lab experiments and Brandon Le performed the bioinformatics analysis. The project also provided the opportunity to collaborate with the following two groups. Dr. Zhanyuan Zhang Plant Transformation Core Facility University of Missouri Columbia, MO 65211 Dr. Huixia Shou, College of Life Sciences, Zhejiang University, Hangzhou, China How have the results been disseminated to communities of interest? Results from this project were discussed at the following meetings and seminars. Seminar, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, Sept 19, 2014. Talk, Symposium of Korean Society of Plant Biology, Daejeon, South Korea, Nov 6-7, 2014. What do you plan to do during the next reporting period to accomplish the goals? This is the last year of the project. We are still working on the project in order to write up manuscripts to report our findings.
Impacts
What was accomplished under these goals?
We collaborated with Dr. Zhanyuan Zhang from the plant transformation facility at University of Missouri to transform RNAi plasmids against NRPD1, NRPE1, and NRPD2 (the common second subunit of Pol IV and Pol V). Their first round of transformation using standard conditions was not successful. As a year had passed by then, we decided to initiate another collaboration with Dr. Huixia Shou at Zhejiang University in China. Now, both these labs have been able to generate transgenic soybean with these constructs. We have obtained transgenic lines of NRPD1 RNAi and NRPE1 RNAi from the Shou lab, and transgenic lines of NRPD1 RNAi, NRPE1 RNAi, and NRPD2 RNAi from the Zhang lab. We have screened the transgenic lines to ensure that they contain the expected transgenes through genotyping in the T1 generation, and then obtained isogenic lines without the transgene and with the transgene from the segregating population in the T2 generation. We have conducted real-time RT-PCR assays to determine whether the lines show the expected reduction in the expression of the genes. We found that some lines do show reduced expression of the genes to be silenced. We have begun to analyze these lines in terms of small RNAs and DNA methylation. We performed small RNA sequencing and the results are being analyzed now. We also plan to perform methylome studies to interrogate the DNA methylation status of the RNAi lines. We expect to find genome-wide reduction in DNA methylation in the three RNAi lines.
Publications
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Progress 05/01/13 to 04/30/14
Outputs
Target Audience: The target audiences include the scientific community, biotechnology industry, and undergraduate and graduate students. Changes/Problems: As we ran into difficulty in obtaining the soybean transgenic lines, our progress has been severely delayed. We may need additional time to finish the project. What opportunities for training and professional development has the project provided? Two postdoctoral fellows and one graduate student gained experience working with soybean. Postdoctoral fellows: Lei Gao and Shengben Li Graduate student: Yu Yu Yu Yu constructed small RNA libraries; Shengben Li performed northern blots to validate the flux of siRNAs; Lei analyzed genomic datasets. The project also provided the opportunity to collaborate with the following three groups. Dr. Zhanyuan Zhang Plant Transformation Core Facility University of Missouri Columbia, MO 65211 Dr. Huixia Shou, College of Life Sciences, Zhejiang University, Hangzhou, China Dr. Wenbo Ma Department of Plant Pathology and Microbiology University of California Riverside, CA 92521 How have the results been disseminated to communities of interest? Results from this project were discussed at the following meetings and seminars. Plant and Animal Genome Conference, January 11-15, 2014. Seminar, School of Biological Sciences, Chinese University of HongKong, March 13, 2014. Seminar, College of Biological Sciences, Shenzhen University, China, March 10, 2014. 1st FAFU (Fujian Agriculture and Forestry University) Symposium of Biological Science, Fuzhou, Fujian, China, March 4, 2014. What do you plan to do during the next reporting period to accomplish the goals? Now that we have obtained the RNAi strains with reduced NRPD1 or NRPE1 expression, we will analyze these lines for endogenous small RNAs and their effects on seed maturation and seed germination. Both molecular, genomic, and phenotypic studies will be performed.
Impacts
What was accomplished under these goals?
For objectives 1-3, we profiled small RNAs and several epigenetic marks using wild type tissues (see maturation and seed germination time series). We still need to do the same for strains in which NRPD1 (the largest subunit of Pol IV) and NRPE1 (the largest subunit of Pol V) have been knocked down. Objective 3 also relies on these strains. We ran into difficulty generating these strains in the first two years of the project. Finally, we have been able to generate these strains. We first collaborated with Dr. Zhanyuan Zhang from the plant transformation facility at University of Missouri to transform RNAi plasmids against NRPD1, NRPE1, and NRPD2 (the common second subunit of Pol IV and Pol V). Their first round of transformation using standard conditions was not successful. As a year had passed by then, we decided to initiate another collaboration with Dr. Huixia Shou at Zhejiang University in China. Now, both these labs have been able to generate transgenic soybean with these constructs. The Shou lab was more advanced in generating these lines and showed that the lines had reduced expression of NRPD1 and NRPE1. They have already propagated the lines to T2 generation, such that we have both the transgenic and non-transgenic strains derived from the same T1 line; the non-transgenic strains serve as the best control for the transgenic lines as they have gone through the same tissue culture procedure. The Shou lab will be providing us with the RNA and DNA samples from these lines so that we can use them to proceed with the planned experiments in Objectives 1-3. This NIFA funding has also made it possible for us to collaborate with Dr. Wenbo Ma's lab in the department of plant pathology and microbiology at UC Riverside to work on soybean's defense against Phytophthora sojae. Root and stem rot caused by the oomycete pathogen P. sojae is one of the most destructive diseases of soybean. In this collaborative project, we showed that small RNAs are a battlefield for soybean and Phytophthora. Phytophthora infection results in the induction of microRNAs and phased secondary siRNAs (phasiRNAs) from plant resistance genes (R genes). We showed that miR393, whose expression is induced by P. sojae infection, promotes defense against P. sojae.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
James Wong, Lei Gao, Yang Yang, Jixian Zhai, Siwaret Arikit, Yu Yu, Shuyi Duan, Qin Xiong, Jun Yan, Shengben Li, Renyi Liu, Yuanchao Wang, Guiliang Tang, Blake C. Meyers, Xuemei Chen, Wenbo Ma* (2014). Roles of small RNAs in soybean defense against Phytophthora sojae infection. The Plant J., doi: 10.1111/tpj.12590.
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Progress 05/01/12 to 04/30/13
Outputs
Target Audience: The target audiences include the scientific community, biotechnology industry, and undergraduate and graduate students. Changes/Problems: The Plant Transformation Center at the University of Missouri has ran into difficulty generating transgenic soybean lines withour RNAi constructs. If this problem persists for a few more months, we will pursue other collaborations to produce theselines. What opportunities for training and professional development has the project provided? Two postdoctoral fellows and one graduate student gained experience working with soybean. Postdoctoral fellows: Shengben Li and Lin Liu Graduate student: Yu Yu Shengben Li constructed small RNA libraries; Yu Yu performed northern blots to validate the flux of siRNAs, constructed RNAi plasmids, and is currently conducting ChIP-seq and Bisulfite-seq experiments; Lin Liu worked on the joint project between the Chen and Ma labs on Phytophthora sojae. The project also provided the opportunity to collaborate with the following two groups. Dr. Zhanyuan Zhang Plant Transformation Core Facility University of Missouri Columbia, MO 65211 Dr. Wenbo Ma Department of Plant Pathology and Microbiology University of California Riverside, CA 92521 How have the results been disseminated to communities of interest? Results from this project were discussed at the following seminars. Seminar at Hunan Agricultural University, Huanan, China, June 18, 2012. Seminar at Shenzhen University, Shenzhen, China, June 17, 2012. Seminar at Center for Advanced Agricultural Genomics, Barcelona, Spain. July 13, 2012. What do you plan to do during the next reporting period to accomplish the goals? The most important goal to achieve in the next reporting period is to generate transgenic soybean lines with reducedexpression of Pol IV or Pol V. The constructs have been generated and soybean transformation is being conducted at thePlant Transformation Center at University of Missouri. But they have run into difficulties generating transgenic lines. Ifnecesary, we will initiate new collaborations to produce these lines.
Impacts
What was accomplished under these goals?
Objective 1 is nearly accomplished. We profiled small RNAs (both microRNAs and endogenous siRNAs) using highthroughput sequencing during seed germination at different time points. The dynamic changes in microRNA levels have beendetermined. Endogenous siRNAs were identified and mapped to the genome in 500bp windows. The flux of siRNAs in eachwindow during germination has been determined. The changes in microRNA and siRNA levels during germination have beenvalidated by northern blotting. RNA-seq is currently being carried out to examine changes in gene expression. Objective 2 is in progress. We have profiled two histone marks, H3K9me2 and H3K27me3, and DNA methylation using ChIPseqand Bisulfite-seq, respectively. The libraries have been made and are awaiting sequencing. Analysis of the datasets willprovide information on the dynamics of epigenetic changes in soybean germination. For objective 3, the functional dissection of the role of small RNAs in gene expression relies on genetic material in which thesmall RNAs are abrogated. We constructed RNAi plasmids that separately target the largest subunit of RNA polymerase IV(Pol IV), the largest subunit of Pol V, and the common second largest subunit in Pol IV and Pol V. From work in Arabidopsisand maize, Pol IV and Pol V are known to be major players in siRNA biogenesis and siRNA-mediated transcriptional genesilencing, respectively. Each subunit is encoded by two genes in soybean. The RNAi constructs were designed such that bothgenes are targeted by the same construct. Soybean transformation using these constructs is currently being conducted at theUniversity of Missouri. Unfortunately, the facility has run into difficulties in obtaining transgenic soybean material and iscurrently switching to another soybean cultivar. This has become a major obstacle to the progress of the project. Finally, this NIFA funding has made it possible for us to form collaboration with Dr. Wenbo Ma's lab in the department of plantpathology and microbiology at UC Riverside to work on soybean's defense against Phytophthora sojae. Root and stem rotcaused by the oomycete pathogen P. sojae is one of the most destructive diseases of soybean. In this collaborative project,we showed that two effector proteins from P. sojae, PSR1 and PSR2, are suppressors of RNA silencing. They inhibit smallRNA biogenesis via distinct mechanisms. Knocking down PSR2 in P. sojae greatly compromised the ability of the pathogen tocause symptoms in soybean. This study is the first to establish RNAi as a host defense mechanism against a eukaryoticpathogen.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Yongli Qiao, Lin Liu, Cristina Flores, James Wong, Xianbing Wang, Qijun Xiang, Xigang Liu, Shushu Jiang, Jinxia Shi, Fuchun Zhang, Howard S. Judelson, Xuemei Chen, Wenbo Ma*. (2013). Oomycete Pathogens Encode RNA Silencing Suppressors. Nature Genetics 45, 330333.
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Xurong Tang, Shaomin Bian, Mingjuan Tang, Qing Lu, Shengben Li, Xigang Liu, Gang Tian, Vi Nguyen, Edward W.T. Tsang, Aiming Wang, Steven J. Rothstein, Xuemei Chen*, and Yuhai Cui*. (2012). MicroRNA-mediated repression of the seed maturation program during vegetative development in Arabidopsis. Plos Genetics 8(11): e1003091.
doi:10.1371/journal.pgen.1003091
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Progress 05/01/11 to 04/30/12
Outputs
Target Audience: The targeted audience is the plant biology scientific community and biotechnology companies. Changes/Problems: The Plant Transformation Center at the University of Missouri has ran into difficulty generating transgenic soybean lines with our RNAi constructs. If this problem persists for a few more months, we will pursue other collaborations to produce these lines. What opportunities for training and professional development has the project provided? Two postdoctoral fellows and one graduate student gained experience working with soybean. Postdoctoral fellows: Shengben Li and Lin Liu Graduate student: Yu Yu Shengben Li constructed small RNA libraries; Yu Yu performed northern blots to validate the flux of siRNAs, constructed RNAi plasmids, and is currently conducting ChIP-seq and Bisulfite-seq experiments; Lin Liu worked on the joint project between the Chen and Ma labs on Phytophthora sojae. The project also provided the opportunity to collaborate with the following two groups. Dr. Zhanyuan Zhang Plant Transformation Core Facility University of Missouri Columbia, MO 65211 Dr. Wenbo Ma Department of Plant Pathology and Microbiology University of California Riverside, CA 92521 How have the results been disseminated to communities of interest? Results from this project were discussed at the following seminars. Seminar at Hunan Agricultural University, Huanan, China, June 18, 2012. Seminar at Shenzhen University, Shenzhen, China, June 17, 2012. Seminar at Center for Advanced Agricultural Genomics, Barcelona, Spain. July 13, 2012. What do you plan to do during the next reporting period to accomplish the goals? The most important goal to achieve in the next reporting period is to generate transgenic soybean lines with reduced expression of Pol IV or Pol V. The constructs have been generated and soybean transformation is being conducted at the Plant Transformation Center at University of Missouri. But they have run into difficulties generating transgenic lines. If necesary, we will initiate new collaborations to produce these lines.
Impacts
What was accomplished under these goals?
Objective 1 is nearly accomplished. We profiled small RNAs (both microRNAs and endogenous siRNAs) using high throughput sequencing during seed germination at different time points. The dynamic changes in microRNA levels have been determined. Endogenous siRNAs were identified and mapped to the genome in 500bp windows. The flux of siRNAs in each window during germination has been determined. The changes in microRNA and siRNA levels during germination have been validated by northern blotting. RNA-seq is currently being carried out to examine changes in gene expression. Objective 2 is in progress. We have profiled two histone marks, H3K9me2 and H3K27me3, and DNA methylation using ChIP-seq and Bisulfite-seq, respectively. The libraries have been made and are awaiting sequencing. Analysis of the datasets will provide information on the dynamics of epigenetic changes in soybean germination. For objective 3, the functional dissection of the role of small RNAs in gene expression relies on genetic material in which the small RNAs are abrogated. We constructed RNAi plasmids that separately target the largest subunit of RNA polymerase IV (Pol IV), the largest subunit of Pol V, and the common second largest subunit in Pol IV and Pol V. From work in Arabidopsis and maize, Pol IV and Pol V are known to be major players in siRNA biogenesis and siRNA-mediated transcriptional gene silencing, respectively. Each subunit is encoded by two genes in soybean. The RNAi constructs were designed such that both genes are targeted by the same construct. Soybean transformation using these constructs is currently being conducted at the University of Missouri. Unfortunately, the facility has run into difficulties in obtaining transgenic soybean material and is currently switching to another soybean cultivar. This has become a major obstacle to the progress of the project. Finally, this NIFA funding has made it possible for us to form collaboration with Dr. Wenbo Ma’s lab in the department of plant pathology and microbiology at UC Riverside to work on soybean’s defense against Phytophthora sojae. Root and stem rot caused by the oomycete pathogen P. sojae is one of the most destructive diseases of soybean. In this collaborative project, we showed that two effector proteins from P. sojae, PSR1 and PSR2, are suppressors of RNA silencing. They inhibit small RNA biogenesis via distinct mechanisms. Knocking down PSR2 in P. sojae greatly compromised the ability of the pathogen to cause symptoms in soybean. This study is the first to establish RNAi as a host defense mechanism against a eukaryotic pathogen.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Yongli Qiao, Lin Liu, Cristina Flores, James Wong, Xianbing Wang, Qijun Xiang, Xigang Liu, Shushu Jiang, Jinxia Shi, Fuchun Zhang, Howard S. Judelson, Xuemei Chen, Wenbo Ma*. (2013). Oomycete Pathogens Encode RNA Silencing Suppressors. Nature Genetics 45, 330333.
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Xurong Tang, Shaomin Bian, Mingjuan Tang, Qing Lu, Shengben Li, Xigang Liu, Gang Tian, Vi Nguyen, Edward W.T. Tsang, Aiming Wang, Steven J. Rothstein, Xuemei Chen*, and Yuhai Cui*. (2012). MicroRNA-mediated repression of the seed maturation program during vegetative development in Arabidopsis. Plos Genetics 8(11): e1003091. doi:10.1371/journal.pgen.1003091
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