TERMINATOR-LESS TRANSGENE CONSTRUCTS FOR EFFICIENT GENE SILENCING IN CROP PLANTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: OTHER GRANTS
• Reporting Frequency: Annual
• Accession No.: 0223055
• Grant No.: 2010-38821-21540
• Cumulative Award Amt.: $500,000.00
• Proposal No.: 2010-02496
• Project Start Date: Sep 1, 2010
• Project End Date: Aug 31, 2015
• Grant Year: 2010
• Program Code: [EQ]- Research Project

Recipient Organization
UNIV OF ARKANSAS
(N/A)
PINE BLUFF,AR 71601

Performing Department
Department of Agriculture

Non Technical Summary
The proposal is designed to (a) improve grain yield and biomass in rice, (b) modify lignin biosynthesis for biofuel production in rice, and (c) understand the mechanism of disease resistance in common bean. We will use the novel terminator-less (TL)sense transgene constructs for silencing/down-regulating genes such as HOG1 (homologydependent gene silencing 1) that exhibit high affinity cytokinin binding activity, for improving grain yield and biomass in rice. In Arabidopsis, silencing of HOG1 has resulted in significant increase in yield and biomass. We will also use TL constructs for down-regulation of lignin biosynthetic genes, cinnamate 4-hydroxylase (C4H), hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) and coumarate 3-hydroxylase (C3'H) to decrease lignin content in rice. Further, we will use TL constructs to understand the mechanism of rust resistance in common bean. Two loci, Ur-3 and Crg, are known to provide resistance to rust. Silencing of Ur-3 and Crg sequences through TL construct will shed light on the much needed mechanism of rust resistance in common bean. The project will lead to: 1) improved grain yield and biomass in rice, 2) reduced lignin in rice straw for biofuel, 3) better understanding of rust resistance in common bean, 4) trained undergraduate/graduate minority students in biotechnology, 4) establishment of collaboration between UAPB and other institutions, and 5) strengthened research and teaching capacity of UAPB.

Animal Health Component

60%

Research Effort Categories

Basic

40%

Applied

60%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1530	1080	60%
206	1410	1040	40%

Knowledge Area
206 - Basic Plant Biology; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1410 - Beans (dry); 1530 - Rice;

Field Of Science
1040 - Molecular biology; 1080 - Genetics;

Keywords

gene silencing

biomass

biofuel

disease resistance

rice

common bean

Goals / Objectives
The main objectives of this project are: a. Evaluating the efficiency of TL constructs in down-regulating target genes in crop plants, rice and common bean. We will use a novel design that induces silencing of specific target at a high rate, and is simpler to construct.It has been found that transgene constructs lacking the transcription termination signal efficiently induce silencing of homologous targets. b. Enhancing yield and biomass through silencing of HOG1 orthog in rice. Experimental evidence strongly indicated that in Arabidopsis, down-regulation of cytokinin binding protein,HOG1 leads to profuse branching, delayed flowering, increased leaf size and higher seed yield. We propose to down-regulate rice HOG1 to enhance grain yield and biomass in rice. c. Modification of lignin biosynthesis for biofuel in rice. Currently, rice farmers leave the rice straw in the fields, burn it or thrown away. But, if it is converted into a biofuel, it could eventually help provide an affordable, renewable energy resource. We propose to down regulate genes involved in lignin biosynthesis such as cinnamate 4-hydroxylase (C4H), hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) and coumarate 3-hydroxylase (C3'H) in rice to reduce lignin for efficient conversion to cellulosic biofuel. d. Validation of candidate Ur-3- and Crg-mediated resistance in common bean. Although genetic and mutant analyses suggest that the loci Ur-3 and Crg may be involved in resistance to rust pathogen in common bean, no conclusive evidence is available to link Ur-3 and Crg with resistance. We propose to silence Ur-3 and Crg from resistance line Sierra for better understanding of resistance in common bean against rust. e. Integrate the knowledge and resources of this project into teaching/training of students at all levels. Undergraduate and graduate students will be trained in research each year. Students will be involved in tissue culture, cloning and vector construction, transformation, molecular analyses of transgenic plants etc.

Project Methods
1. Evaluating the efficiency of TL constructs in down regulating target genes in crop plants, rice and bean- About 0.5 kb fragment corresponding to the cDNA will be amplified using primers tagged with appropriate restriction enzyme sites at 5' end, and cloned into pSJN15 or p35S vector. If Agrobacterium mediated transformation is planned, pSJN14 will be used, which carries a kanamycin resistance gene for selection of transformants. However, if particle bombardment mediated transformation is planned, p35S vector will be used for cloning the amplified TL fragment. The p35S based TL vector will be co-bombarded with pKAN that contains a kanamycin resistance gene for selection of transformants. The rice TL vector will contain hygromycin resistance gene to facilitate selection of stable transformants. These vectors will be verified by sequencing and used for rice or common bean transformation. 2. Enhancing yield and biomass through silencing of rice HOG- PCR will be used to clone full length HOG1 ortholog cDNA in rice. The amplified products will be ligated into pGEM -T Easy vector and sequenced. The complete open reading frame of rice HOG1 cDNA will be used for the over expression construct. TL constructs will also be prepared for HOG1 ortholog for silencing. Both silencing and overexpression constructs will be used for transformation of rice cv. Nipponbare through Agrobacterium tumefaciencs. Homozygous lines will be analyzed for biomass and yield characteristics such as plant height, root length and size, number of tillers, leaf and panicle size, size and number of seeds per panicle, fresh and dry weight of biomass after harvest. 3. Modification of lignin biosynthesis for biofuel in rice - PCR will be used to clone full length sequences of cinnamate 4-hydroxylase (C4H), hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) and coumarate 3-hydroxylase (C3'H) from rice based on primers synthesized from existing sequences. The amplified PCR fragments will be cloned into pGEM-T Easy (Promega) and sequenced. Binary vectors for silencing and overexpression will be prepared and constructs will be used for transformation of rice cv. Nipponbare through Agrobacterium-tumefaciencs. Molecular, progeny and phenotypic analyses will be done on over-expressed and silenced lines. Lignin and cellulose contents will be analyzed in both silencing and over-expressed lines. 4. Validation of candidate Ur-3-mediated resistance in common bean - Binary vector for silencing and over-expression of Ur-3 and Crg will be prepared and used for particle bombardment of embryonic axes. After bombardment, the embryonic axes will be cultured on MS medium containing 44.3 micromole 6- benzylaminopurine to induce multiple shoot development. After 3 weeks in culture, the bombarded apical meristems that produced elongated shoots and roots will be transferred to peat pellets first and subsequently to greenhouse. Molecular and progeny analyses will be carried out to confirm transgenic nature of the regenerated plants. T1 plants from over-expressed and silenced lines will be screened with U. appendiculatus race 53 to determine the effect of Ur-3 and Crg in restoring resistance.

Progress 09/01/10 to 08/31/15

Outputs
Target Audience:Rice researchers and growers in Arkansas Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Post-docs, graduate and undergraduate students has been successfully trained in various plant transformation and molecular biology techniques. Post-docs and students were also presented their research in meetings and conferences. How have the results been disseminated to communities of interest?The results were presented during the Field Day for Farmers conducted by the University of Arkansas at Pine Bluff. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Rice straw is one of the largest biomass in the world that can potentially be exploited for bio-fuel. Nevertheless, the association of lignin with cellulose and hemicellulose has hindered the efficient utilization of rice straw for cellulosic bio-fuel. The objective of this study was, therefore, to down-regulate genes involved in lignin biosynthesis pathway such as cinnamate 4-hydroxylase (C4H), hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT), coumarate 3-hydroxylase (C3'H), cinnamoyl CoA reductase (CCR), and cinnamyl alcohol dehydrogenase (CAD) through terminator-less construct to reduce lignin in transgenic rice straw for its use in cellulosic bio-fuel. Real-time qPCR analyses of T1 transgenic rice plants indicated at least 30-50% transcript reduction in CAD lines, 90% in C4H lines, 10-60% in C3H lines, 50-90% in HCT lines, and 50% in CCR lines. Of the 10 silenced lines tested for lignin, syringyl (S) lignin was decreased in CCR-16, whereas seven lines, CAD-1, C4H-3, C4H-13, C3H-11, HCT-3, CCR-12 and CCR-16, showed decreased guaiacyl (G) lignin. In addition, five lines, CAD-1, C4H-3, HCT-3, CCR-12 and CCR-16, showed decreased p-hydroxy phenyl (H) lignin and two lines, CAD-7 and HCT-4, showed reduced S/G ratio. Overall, four of the ten silenced lines tested, CAD-1, C4H-3, CCR-12, CCR-16, showed reduced lignin content ranging 3.2-5.6%. The results from this study indicated that the rice straw from transgenic lines containing reduced lignin could be used as feedstock for cellulosic biofuel.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Nandy S, Zhao S, Pathak BP, Manoharan M, Srivastava V. 2015. Gene stacking in plant cell using recombinases for gene integration and nucleases for marker gene deletion. BMC Biotechnology, 15:93.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Ayyappan V, Kalavacharla V, Thimmapuram J, Bhide KP, Sripathi VR, Smolinski TG, Manoharan M, Thurston Y, Todd A, Kingham B. 2015. Genome-Wide Profiling of Histone Modifications (H3K9me2 and H4K12ac) and Gene Expression in Rust (Uromyces appendiculatus) Inoculated Common Bean (Phaseolus vulgaris L.). PLOS One, DOI:10.1371/journal.pone.0132176.
• Type: Journal Articles Status: Submitted Year Published: 2015 Citation: Ponniah SK, Shang Z, Akbudak MA, Srivastava V, Manoharan M. 2015. Down-regulation of lignin biosynthetic genes and effects on lignin content and composition in rice (Oryza sativa L. ssp. Japonica cv. Nipponbare). Plant Cell Reports (submitted for publication).

Progress 09/01/13 to 08/31/14

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? 1 Graduate student was trained in plant biotechnology and molecular biology. How have the results been disseminated to communities of interest? The results were presented (Z. Shang, S. Ponniah, V. Srivastava, and M. Manoharan. 2014. Down-regulation of Genes Involved in Lignin Biosynthesis in Rice) at 2014 World Forum on Biology, Savannah, GA May 31-June 4; P-3018 What do you plan to do during the next reporting period to accomplish the goals? Real-time PCR analyses of transgenic lines (C4H, CAD HCT, CCR and C3H). Analysis of lignin content in transgenic lines. Project Report and Manuscript preparation for publication.

Impacts
What was accomplished under these goals? Six terminator-less (TL) binary vectors (pPZP211 backbone) for the following genes were constructed: Cinnamoyl-CoA reductase (CCR), Cinnamyl Alcohol Dehydrogenase (CAD), Hydroxy Cinnamoyl Transferase (HCT), p-Coumarate 3- Hydroxylase (C3H), and Cinnamic Acid 4-Hydroxylase (C4H) and homology-dependent gene silencing 1 (HOG1). All the vectors were sequenced and confirmed for the presence of inserts. The vectors were transformed into Agrobacterium (EHA105) through freeze thaw method. Plasmids were isolated and PCR was done to confirm the presence of individual genes with respective primers. Transformation of calli with the agrobacterium containing all five genes was carried out (callus induction, co-cultivation, selection and regeneration of plants). Several putative transgenic lines were transferred to growth chamber. Transgenic nature of the regenerated plants was confirmed by PCR and segregation analyses. At least 15 independent transgenic lines were successfully generated for each of the transgenes (C4H, CAD HCT, CCR and C3H). Real-time PCR analyses of some of the transgenic lines containing CAD and HCT showed 50% gene silencing.

Publications

Progress 09/01/12 to 08/31/13

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Post-doc and graduate student were trained in vector construction and transformation. Undergraduate student was trained in tissue culture. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? 1. Expression studies will be done for all ofthe transgenic lines. 2. Lignin analysis will be done to find out the level of lignin in transgenic lines.

Impacts
What was accomplished under these goals? Transgenic lines were successfully generated using the TL vectors containing Cinnamoyl CoA Reductase (CCR), Cinnamate 4-Hydroxylase (C4H), Cinnamate 3-Hydroxylase (C3H, Cinnamyl Alcohol Dehydrogenase (CAD)and Hydroxy Cinnamoyl Transferase (HCT) genes. At least 10 transgenic lines were generated for each of the genes. The transgenic nature of these plants were tested by PCR and the seeds were collected. Further, the collected seeds were germinated on MS medium with selection (kanamycin). The segregating plants were further tested by PCR to confirm the transgenic nature of these progenies. RNA was isolated from CAD lines and the results indicated 50% reduction of CAD expression in some of the transgenic plants, confirming that the TL vectors can be used to down-regulate genes in rice.

Publications

Progress 09/01/11 to 08/31/12

Outputs
OUTPUTS: Terminator-less vectors for the following genes were made: Cinnamoyl CoA Reductase (CCR1), Cinnamate 4-Hydroxylase (C4H), Cinnamate 3-Hydroxylase (C3H, Cinnamyl Alcohol Dehydrogenase (CAD)and Hydroxy Cinnamoyl Transferase (HCT). Genetic transformation of Cinnamoyl CoA Reductase gene fragment (CCR1-TL) into rice calli derived from scutellum was carried out as per published protocol. A number of transformants were identified using PCR based techniques. Southern analysis is underway to confirm the transgenic nature of the regenerated plants. Similarly Cinnamate 4-Hydroxylase (C4H-TL) and Cinnamate 3-Hydroxylase (C3H-TL) transformation procedures were carried out and transgenic plants were identified by PCR. Regeneration of Cinnamyl Alcohol Dehydrogenase (CAD-TL) transgenic plants are under progress with putative plantlets emerging from the regeneration medium. Transformation of Hydroxy Cinnamoyl Transferase (HCT-TL) into rice is in progress. PARTICIPANTS: Manoharan Muthusamy (PI)- working on rice transformation with TL vectors. Vibha Srivastava (Co-PI; University of Arkansas- Fayetteville) has prepared TL vectors. Venugopal Kalavacharla (Co-PI; Delaware state University)- working on bean transformation with TL vectors. TARGET AUDIENCES: Rice farmers in Arkansas and other states. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Construction of TL vectors containing Cinnamoyl CoA Reductase (CCR1), Cinnamate 4-Hydroxylase (C4H), Cinnamate 3-Hydroxylase (C3H, Cinnamyl Alcohol Dehydrogenase (CAD)and Hydroxy Cinnamoyl Transferase (HCT) genes. These vectors can also be tested in other plants for reducing lignin.

Publications

• No publications reported this period

Progress 09/01/10 to 08/31/11

Outputs
OUTPUTS: cDNA clones for the rice cytokinin binding protein, HOG1, cinnamate 4-hydroxylase (C4H), hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT), and coumarate 3-hydroxylase (C3'H) were amplified using primers tagged with appropriate restriction enzyme sites at 5' end, and cloned into pSJN15 vector, which carries a kanamycin resistance gene for selection of transformants. Callus was initiated from the mature seeds of japonica cultivar Nipponbare. Dehusked seeds were surface sterilized in 70% (v/v) ethanol for 1 min. and subsequently treated by 1.0% (w/v) sodium hypochlorite for 60 minutes. They were rinsed three times with sterilized water and cultured on callus induction medium (CIM) containing macro and micro elements and vitamins of N6 medium, casaminoacids 1.0g/L, proline 500 mg/L, 2,4-D 2.0 mg/L and sucrose 30 g/L. One week old calli were used for agrobacterium transformation. After co-cultivation for three days, calli were transferred to CIM containing 50 mg/L Kanamycin. After four weeks, the resistant calli were transferred to the regeneration medium. Putative transgenic shoots appeared after 2-3 weeks in culture. The shoots were transferred to half strength MS medium for shoot elongation and rooting. The rooted plants were transferred to peat pellets and kept for one week. The plants were transferred to pots containing soil and peat mass in 3:1 ratio and kept under green house. We have also generated additional TL-PDS (phytoenedesaturase) and TP-PDS lines in order to study the mechanistic aspects of TL-mediated gene silencing in rice. PARTICIPANTS: Muthusamy Manoharan- University of Arkansas at Pine Bluff- prepared cDNA for vector construction; Vibha Srivastava- University of Arkansas at Fayetteville- prepared binary vector pSJN15 for transformation; Venugopal Kalavacharla- Delaware State University- involved in the preparation of Ur-3 and Crg genes. TARGET AUDIENCES: Rice farmers in Arkansas. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Binary vector, PSJN15 containg genes from rice cytokinin binding protein, HOG1, cinnamate 4-hydroxylase (C4H), hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT), and coumarate 3-hydroxylase (C3'H) were prepared for transformation.

Publications

• No publications reported this period