Direct Interaction of Ligand–Receptor Pairs Specifying Stomatal Patterning
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Research Overview Research Goals
10/21/2008 Research Overview • Plant biotechnology/bioprocess engineering with applications to – Bioppp(gharmaceutical production (e.g. human α1- antitrypsin) • Ting-Kuo Huang (transgenic tobacco cell suspension cultures in stirred tank bioreactors) • Kittipong Rattanaporn (development of improved viral amplicon expression vectors for transient agroinfiltration) – Biofuels applications (in-planta production of cellulase enzymes) • Ben Lindenmuth (transient production in tobacco leaves) • Sang-KJKyu Jung (trans ien t pro duc tion in sw itc hgrass ) – Biopolymers (human gelatin and collagen) • Corey Dodge (rice cell suspension cultures in bioreactors) • Lucas Arzola (maize cell suspension cultures in bioreactors) Research Goals • Efficient production of recombinant proteins in plants and plant cell cultures Genetic Instructions Host Factors Bioprocess Approaches - Amino acid sequence - Choice of plant - Environmental conditions - Transient vs stable production - Stability - Operational strategies - Inducible promoter - Byproducts - Scale-up - Secretion signal - Optimized genes (codon usage/introns) 1 10/21/2008 Collaborations University: • Abhaya Dandekar, Plant Sciences • Bryce Falk, Plant Pathology • Jean VanderGheynst, Bio & Ag Engr. Industry: • Ventria Biosciences (Applied Phytologics) •FibroGen • Planet Biotechnology • Chevron •BioRad Ben Lindenmuth • PhD candidate in Chemical Engineering with a Designated Emphasis in Biotechnology • B.S. in ChE from Penn State 2 10/21/2008 Transient in planta expression of cellulose-degrading enzymes: -
Plant Molecular Farming: a Viable Platform for Recombinant Biopharmaceutical Production
plants Review Plant Molecular Farming: A Viable Platform for Recombinant Biopharmaceutical Production Balamurugan Shanmugaraj 1,2, Christine Joy I. Bulaon 2 and Waranyoo Phoolcharoen 1,2,* 1 Research Unit for Plant-Produced Pharmaceuticals, Chulalongkorn University, Bangkok 10330, Thailand; [email protected] 2 Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences Chulalongkorn University, Bangkok 10330, Thailand; [email protected] * Correspondence: [email protected]; Tel.: +66-2-218-8359; Fax: +66-2-218-8357 Received: 1 May 2020; Accepted: 30 June 2020; Published: 4 July 2020 Abstract: The demand for recombinant proteins in terms of quality, quantity, and diversity is increasing steadily, which is attracting global attention for the development of new recombinant protein production technologies and the engineering of conventional established expression systems based on bacteria or mammalian cell cultures. Since the advancements of plant genetic engineering in the 1980s, plants have been used for the production of economically valuable, biologically active non-native proteins or biopharmaceuticals, the concept termed as plant molecular farming (PMF). PMF is considered as a cost-effective technology that has grown and advanced tremendously over the past two decades. The development and improvement of the transient expression system has significantly reduced the protein production timeline and greatly improved the protein yield in plants. The major factors that drive the plant-based platform towards potential competitors for the conventional expression system are cost-effectiveness, scalability, flexibility, versatility, and robustness of the system. Many biopharmaceuticals including recombinant vaccine antigens, monoclonal antibodies, and other commercially viable proteins are produced in plants, some of which are in the pre-clinical and clinical pipeline. -
Transcriptome Profiling of Agrobacterium‑Mediated Infiltration of Transcription Factors to Discover Gene Function and Expression Networks in Plants Donna M
Bond et al. Plant Methods (2016) 12:41 DOI 10.1186/s13007-016-0141-7 Plant Methods RESEARCH Open Access Infiltration‑RNAseq: transcriptome profiling of Agrobacterium‑mediated infiltration of transcription factors to discover gene function and expression networks in plants Donna M. Bond1*, Nick W. Albert2, Robyn H. Lee1, Gareth B. Gillard1,4, Chris M. Brown1, Roger P. Hellens3 and Richard C. Macknight1,2* Abstract Background: Transcription factors (TFs) coordinate precise gene expression patterns that give rise to distinct pheno- typic outputs. The identification of genes and transcriptional networks regulated by a TF often requires stable trans- formation and expression changes in plant cells. However, the production of stable transformants can be slow and laborious with no guarantee of success. Furthermore, transgenic plants overexpressing a TF of interest can present pleiotropic phenotypes and/or result in a high number of indirect gene expression changes. Therefore, fast, efficient, high-throughput methods for assaying TF function are needed. Results: Agroinfiltration is a simple plant biology method that allows transient gene expression. It is a rapid and powerful tool for the functional characterisation of TF genes in planta. High throughput RNA sequencing is now a widely used method for analysing gene expression profiles (transcriptomes). By coupling TF agroinfiltration with RNA sequencing (named here as Infiltration-RNAseq), gene expression networks and gene function can be identified within a few weeks rather than many months. As a proof of concept, we agroinfiltrated Medicago truncatula leaves with M. truncatula LEGUME ANTHOCYANIN PRODUCITION 1 (MtLAP1), a MYB transcription factor involved in the regula- tion of the anthocyanin pathway, and assessed the resulting transcriptome. -
Agroinfiltration As an Effective and Scalable Strategy of Gene Delivery for Production of Pharmaceutical Proteins
s in que Bio ni lo h g c y e T & d M Chen et al., Adv Tech Biol Med 2013, 1:1 e e c Advanced Techniques in d n i c a i DOI: 10.4172/2379-1764.1000103 v n d e A ISSN: 2379-1764 Biology & Medicine ReviewResearch Article Article OpenOpen Access Access Agroinfiltration as an Effective and Scalable Strategy of Gene Delivery for Production of Pharmaceutical Proteins Qiang Chen1,2*, Huafang Lai1, Jonathan Hurtado2, Jake Stahnke1, Kahlin Leuzinger2 and Matthew Dent1 1The Biodesign Institute, Center for Infectious Disease and Vaccinology, Arizona State University, USA 2College of Technology and Innovation, Arizona State University, USA Abstract Current human biologics are most commonly produced by mammalian cell culture-based fermentation technologies. However, its limited scalability and high cost prevent this platform from meeting the ever increasing global demand. Plants offer a novel alternative system for the production of pharmaceutical proteins that is more scalable, cost-effective, and safer than current expression paradigms. The recent development of deconstructed virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins, and in turn, provided a preferred plant based production platform. One of the remaining challenges for the commercial application of this platform was the lack of a scalable technology to deliver the transgene into plant cells. Therefore, this review focuses on the development of an effective and scalable technology for gene delivery in plants. Direct and indirect gene delivery strategies for plant cells are first presented, and the two major gene delivery technologies based on agroinfiltration are subsequently discussed. -
Transcriptome Analysis of the Eggplant Fruits Overexpressing a Gene of Chlorogenic Acid Pathway
bioRxiv preprint doi: https://doi.org/10.1101/2020.10.09.332791; this version posted October 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Transcriptome Analysis of the Eggplant Fruits Overexpressing a Gene of Chlorogenic Acid Pathway Prashant Kaushik1, 2,* 1 Instituto de Conservación y Mejora de la Agrodiversidad Valenciana, Universitat Politècnica de València, 46022 Valencia, Spain 2 Nagano University, 1088 Komaki, Ueda, 386-0031 Nagano, Japan * Correspondence: [email protected] Received: date; Accepted: date; Published: date Abstract: Chlorogenic acid is the primary phenolic acids present in Eggplant fruits. For the generation of chlorogenic acid in the eggplant hydroxycinnamoyl CoA-quinate transferase (SmHQT), is a central enzyme that catalyzes the reaction to the chlorogenic acid production. Although a precise function of SmHQT is not well defined in the eggplant fruit. In this study, the overexpression of SmHQT in the eggplant fruit´s flesh was studied using the agroinfiltration technique. Furthermore, to determine the differences at the genomic level RNA-seq analysis was performed, and its results showed that 415 genes of the phenylpropanoid pathway were upregulated in the transgenic fruit. Also, it was determined that the differentially expressed genes were dominantly related to phenylpropanoid pathway along with cell expansion, and cytokinin. The agroinfiltrated fruit exhibited more than twice the chlorogenic content in the normal fruit. Overall, the result provides new insight into the eggplant chlorogenic content increment at the molecular level and unseal the opportunities to design new strategies for the improvement of chlorogenic content as nutrition in eggplant. -
Synthesis and Characterization of a Full-Length Infectious Cdna Clone of Tomato Mottle Mosaic Virus
viruses Article Synthesis and Characterization of a Full-Length Infectious cDNA Clone of Tomato Mottle Mosaic Virus Liqin Tu 1,2 , Shuhua Wu 2, Danna Gao 1, Yong Liu 3, Yuelin Zhu 1,* and Yinghua Ji 2,* 1 College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China; [email protected] (L.T.); [email protected] (D.G.) 2 Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences/Key Lab of Food Quality and Safety of Jiangsu Province-State Key Laboratory Breeding Base, Nanjing 210014, China; [email protected] 3 Institute of Plant Protection, Hunan Academy of Agricultural Sciences, Changsha 410125, China; [email protected] * Correspondence: [email protected] (Y.Z.); [email protected] (Y.J.); Tel.: +86-25-84396472 (Y.Z.); +86-25-84390394 (Y.J.) Abstract: Tomato mottle mosaic virus (ToMMV) is a noteworthy virus which belongs to the Virgaviridae family and causes serious economic losses in tomato. Here, we isolated and cloned the full-length genome of a ToMMV Chinese isolate (ToMMV-LN) from a naturally infected tomato (Solanum lycopersicum L.). Sequence analysis showed that ToMMV-LN contains 6399 nucleotides (nts) and is most closely related to a ToMMV Mexican isolate with a sequence identity of 99.48%. Next, an infectious cDNA clone of ToMMV was constructed by a homologous recombination approach. Both the model host N. benthamiana and the natural hosts tomato and pepper developed severe symptoms upon agroinfiltration with pToMMV, which had a strong infectivity. Electron micrographs indicated that a large number of rigid rod-shaped ToMMV virions were observed from the agroinfiltrated N. -
An Improved Syringe Agroinfiltration Protocol to Enhance Transformation Efficiency by Combinative Use of 5-Azacytidine, Ascorbat
plants Article An Improved Syringe Agroinfiltration Protocol to Enhance Transformation Efficiency by Combinative Use of 5-Azacytidine, Ascorbate Acid and Tween-20 Huimin Zhao 1, Zilong Tan 2, Xuejing Wen 2 and Yucheng Wang 1,2,* 1 State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin 150040, China; [email protected] 2 Key Laboratory of Biogeography and Bioresource in Arid Land, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi 830011, China; [email protected] (Z.T.); [email protected] (X.W.) * Correspondence: [email protected]; Tel.: +86-451-82190607 Academic Editor: Milan S. Stankovic Received: 1 January 2017; Accepted: 9 February 2017; Published: 14 February 2017 Abstract: Syringe infiltration is an important transient transformation method that is widely used in many molecular studies. Owing to the wide use of syringe agroinfiltration, it is important and necessary to improve its transformation efficiency. Here, we studied the factors influencing the transformation efficiency of syringe agroinfiltration. The pCAMBIA1301 was transformed into Nicotiana benthamiana leaves for investigation. The effects of 5-azacytidine (AzaC), Ascorbate acid (ASC) and Tween-20 on transformation were studied. The β-glucuronidase (GUS) expression and GUS activity were respectively measured to determine the transformation efficiency. AzaC, ASC and Tween-20 all significantly affected the transformation efficiency of agroinfiltration, and the optimal concentrations of AzaC, ASC and Tween-20 for the transgene expression were identified. Our results showed that 20 µM AzaC, 0.56 mM ASC and 0.03% (v/v) Tween-20 is the optimal concentration that could significantly improve the transformation efficiency of agroinfiltration. -
Rescue of Tomato Spotted Wilt Virus Entirely from Complementary DNA Clones
Rescue of tomato spotted wilt virus entirely from complementary DNA clones Mingfeng Fenga, Ruixiang Chenga, Minglong Chena, Rong Guoa, Luyao Lia, Zhike Fenga, Jianyan Wua, Li Xieb, Jian Hongb, Zhongkai Zhangc, Richard Kormelinkd, and Xiaorong Taoa,1 aDepartment of Plant Pathology, Nanjing Agricultural University, 210095 Nanjing, People’s Republic of China; bAnalysis Center of Agrobiology and Environmental Sciences, Zhejiang University, 317502 Hangzhou, People’s Republic of China; cYunnan Provincial Key Laboratory of Agri-Biotechnology, Institute of Biotechnology and Genetic Resources, Yunnan Academy of Agricultural Sciences, 650223 Kunming, People’s Republic of China; and dLaboratory of Virology, Department of Plant Sciences, Wageningen University, 6708PB Wageningen, The Netherlands Edited by David C. Baulcombe, University of Cambridge, Cambridge, United Kingdom, and approved December 2, 2019 (received for review June 26, 2019) Negative-stranded/ambisense RNA viruses (NSVs) include not only losses due to this virus have been estimated at more than $1 dangerous pathogens of medical importance but also serious plant billion annually (7, 17). pathogens of agronomic importance. Tomato spotted wilt virus TSWV consists of spherical, enveloped virus particles (80 to (TSWV) is one of the most important plant NSVs, infecting more 120 nm) that contain a tripartite genome consisting of large- (L), than 1,000 plant species, and poses major threats to global food medium- (M), and small-sized (S) RNA segments (14). The L security. The segmented negative-stranded/ambisense RNA ge- RNA segment is negative sense and encodes a single large RNA- nomes of TSWV, however, have been a major obstacle to molecular dependent RNA polymerase (RdRp, ∼330 kDa) that is required genetic manipulation. -
Cjm-2020-0239.Pdf
Canadian Journal of Microbiology Exposure of Agrobacterium tumefaciens to agroinfiltration medium demonstrates cellular remodeling and may promote enhanced adaptability for molecular pharming Journal: Canadian Journal of Microbiology Manuscript ID cjm-2020-0239.R2 Manuscript Type: Article Date Submitted by the 15-Jul-2020 Author: Complete List of Authors: Prudhomme, Nicholas; University of Guelph Pastora, Rebecca; PlantForm Inc. Muselius, Benjamin; University of Guelph McLean, Mike;Draft PlantForm Corporation Canada Cossar, Doug; PlantForm Corporation Canada Geddes-McAlister, Jennifer; University of Guelph, Molecular and Cellular Biology Agrobacterium tumefaciens, agroinfiltration medium, quantitative Keyword: proteomics, molecular pharming Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? : https://mc06.manuscriptcentral.com/cjm-pubs Page 1 of 39 Canadian Journal of Microbiology Title: Exposure of Agrobacterium tumefaciens to agroinfiltration medium demonstrates cellular remodeling and may promote enhanced adaptability for molecular pharming. Authors: Prudhomme, N.1, Pastora, R.2, Muselius, B.,1 McLean, M.D.2, Cossar, D.2, Geddes- McAlister, J.1* Affiliations: 1Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, N1G 2W1 2PlantForm Corporation Canada, Toronto, Ontario, M4S 3E2 Draft Corresponding author: Jennifer Geddes-McAlister, Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, N1G 2W1. Phone: 519-824-4120 ext. 52129. Email: [email protected] https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 2 of 39 Abstract Agroinfiltration is used to treat plants with modified strains of Agrobacterium tumefaciens for the purpose of transient in planta expression of genes transferred from the bacterium. These genes encode valuable recombinant proteins for therapeutic or industrial applications. -
AGRIVITA Journal of Agricultural Science
313 AGRIVITA Journal of Agricultural Science. 2018. 40(2): 313-319 AGRIVITA Journal of Agricultural Science Transient Transformation of Potato Plant (Solanum tuberosum L.) Granola Cultivar Using Syringe Agroinfiltration Yesy John Mba’u, Iriawati and Ahmad Faizal*) Plant Science and Biotechnology Research Group, School of Life Sciences and Technology, Institut Teknologi Bandung, Indonesia ARTICLE INFO ABSTRACT Keywords: Genetic transformation has been used as an alternative approach to Agroinfiltration improve the quality and the productivity of potato plant. In this study, Green Fluorescence Protein different conditions have been set up to optimize transient GFP (Green Nuclear Localization Signal Fluorescence Protein) expression in potato cv. Granola. Leaves of potato Potato were infiltrated with Agrobacterium tumefaciens strain C58C1 harboring Transient expression pK7FWGF2 vector with a nuclear-targeted GFP by simple pressure. GFP signals allowed simple evaluation of transformation efficiency which Article History: were indicated by GFP expression in nucleus of leaf cells in infiltrated Received: June 9, 2017 areas. The results showed that leaf position, co-cultivation time, optical Accepted: April 3, 2018 density and the presence of acetosyringone significantly affected the *) transformation efficiency. The fourth terminal leaves from four-week Corresponding author: old plants were the optimum age for transformation. Furthermore, the E-mail: [email protected] highest transient transformation efficiency was obtained upon 48 h post- -
Agroinfiltration and PVX Agroinfection in Potato and Nicotiana Benthamiana
Journal of Visualized Experiments www.jove.com Video Article Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana Juan Du1,2, Hendrik Rietman1, Vivianne G. A. A. Vleeshouwers1 1 Wageningen UR Plant Breeding, Wageningen University 2 Key Laboratory of Horticultural Plant Biology, Huazhong Agricultural University, Ministry of Education, National Centre for Vegetable Improvement (Central China), Potato Engineering and Technology Research Centre of Hubei Province, Huazhong Agricultural University Correspondence to: Vivianne G. A. A. Vleeshouwers at [email protected] URL: https://www.jove.com/video/50971 DOI: doi:10.3791/50971 Keywords: Plant Biology, Issue 83, Genetics, Bioengineering, Plants, Genetically Modified, DNA, Plant Immunity, Plant Diseases, Genes, Genome, Plant Pathology, Effectoromics, Agroinfiltration, PVX agroinfection, potato, Nicotiana benthamiana, high-throughput, functional genomics Date Published: 1/3/2014 Citation: Du, J., Rietman, H., Vleeshouwers, V.G. Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana. J. Vis. Exp. (83), e50971, doi:10.3791/50971 (2014). Abstract Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. -
A Virus-Targeted Plant Receptor-Like Kinase Promotes Cell-To-Cell Spread of Rnai
A virus-targeted plant receptor-like kinase promotes cell-to-cell spread of RNAi Tabata Rosas-Diaza, Dan Zhanga,b, Pengfei Fana,b, Liping Wanga,b, Xue Dinga,b, Yuli Jianga,b, Tamara Jimenez-Gongoraa,b, Laura Medina-Puchea, Xinyan Zhaoa,b, Zhengyan Fenga,b, Guiping Zhanga,b, Xiaokun Liuc, Eduardo R. Bejaranod, Li Tana, Heng Zhanga, Jian-Kang Zhua,e, Weiman Xinga, Christine Faulknerc,f, Shingo Nagawaa,1, and Rosa Lozano-Durana,f,2 aShanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences (CAS), Shanghai 201602 China; bUniversity of Chinese Academy of Sciences, 100049 Beijing, China; cDepartment of Crop Genetics, John Innes Centre, Norwich NR4 7UH, United Kingdom; dInstituto de Hortofruticultura Subtropical y Mediterránea Mayora” (IHSM-UMA-CSIC), Area de Genética, Facultad de Ciencias, Universidad de Málaga, E-29071 Málaga, Spain; eDepartment of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907; and fChinese Academy of Sciences–John Innes Centre Center of Excellence for Plant and Microbial Science, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 200032Shanghai, China Edited by Xuemei Chen, University of California, Riverside, CA, and approved December 27, 2017 (received for review September 2, 2017) RNA interference (RNAi) in plants can move from cell to cell, protein (Fig. 1A and SI Appendix, Fig. S1). A mutant virus allowing for systemic spread of an antiviral immune response. expressing a nonmyristoylable version of C4 (C4G2A), which lo- How this cell-to-cell spread of silencing is regulated is currently calizes to chloroplasts exclusively, displays severely compromised Tomato unknown.