Articoli di riviste: "Agroinfiltratio"

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Liu, Pei-Feng, Yanhan Wang, Robert G. Ulrich, et al. "Leaf-Encapsulated Vaccines: Agroinfiltration and Transient Expression of the AntigenStaphylococcal EndotoxinB in Radish Leaves." Journal of Immunology Research 2018 (2018): 1–9. http://dx.doi.org/10.1155/2018/3710961.

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Transgene introgression is a major concern associated with transgenic plant-based vaccines. Agroinfiltration can be used to selectively transform nonreproductive organs and avoid introgression. Here, we introduce a new vaccine modality in which Staphylococcal enterotoxin B (SEB) genes are agroinfiltrated into radishes (Raphanw sativusL.), resulting in transient expression and accumulation of SEBin planta. This approach can simultaneously express multiple antigens in a single leaf. Furthermore, the potential of high-throughput vaccine production was demonstrated by simultaneously agroinfiltrating multiple radish leaves using a multichannel pipette. The expression of SEB was detectable in two leaf cell types (epidermal and guard cells) in agroinfiltrated leaves. ICR mice intranasally immunized with homogenized leaves agroinfiltrated with SEB elicited detectable antibody to SEB and displayed protection against SEB-induced interferon-gamma (IFN-γ) production. The concept of encapsulating antigens in leaves rather than purifying them for immunization may facilitate rapid vaccine production during an epidemic disease.

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Ambrós, Silvia, Choaa El-Mohtar, Susana Ruiz-Ruiz, et al. "Agroinoculation of Citrus tristeza virus Causes Systemic Infection and Symptoms in the Presumed Nonhost Nicotiana benthamiana." Molecular Plant-Microbe Interactions® 24, no. 10 (2011): 1119–31. http://dx.doi.org/10.1094/mpmi-05-11-0110.

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Citrus tristeza virus (CTV) naturally infects only some citrus species and relatives and within these it only invades phloem tissues. Failure to agroinfect citrus plants and the lack of an experimental herbaceous host hindered development of a workable genetic system. A full-genome cDNA of CTV isolate T36 was cloned in binary plasmids and was used to agroinfiltrate Nicotiana benthamiana leaves, with or without coinfiltration with plasmids expressing different silencing-suppressor proteins. A time course analysis in agroinfiltrated leaves indicated that CTV accumulates and moves cell-to-cell for at least three weeks postinoculation (wpi), and then, it moves systemically and infects the upper leaves with symptom expression. Silencing suppressors expedited systemic infection and often increased infectivity. In systemically infected Nicotiana benthamiana plants, CTV invaded first the phloem, but after 7 wpi, it was also found in other tissues and reached a high viral titer in upper leaves, thus allowing efficient transmission to citrus by stem-slash inoculation. Infected citrus plants showed the symptoms, virion morphology, and phloem restriction characteristic of the wild T36 isolate. Therefore, agroinfiltration of Nicotiana benthamiana provided the first experimental herbaceous host for CTV and an easy and efficient genetic system for this closterovirus.

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Wang, Zhiquan, Xiaoyang Xu, Longjie Ni, Jinbo Guo, and Chunsun Gu. "Efficient virus-induced gene silencing in Hibiscus hamabo Sieb. et Zucc. using tobacco rattle virus." PeerJ 7 (August 12, 2019): e7505. http://dx.doi.org/10.7717/peerj.7505.

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Background Hibiscus hamabo Sieb. et Zucc. is a semi-mangrove plant used for the ecological restoration of saline-alkali land, coastal afforestation and urban landscaping. The genetic transformation H. hamabo is currently inefficient and laborious, restricting gene functional studies on this species. In plants, virus-induced gene silencing provides a pathway to rapidly and effectively create targeted gene knockouts for gene functional studies. Methods In this study, we tested the efficiency of a tobacco rattle virus vector in silencing the cloroplastos alterados 1 (CLA1) gene through agroinfiltration. Results The leaves of H. hamabo showed white streaks typical of CLA1 gene silencing three weeks after agroinfiltration. In agroinfiltrated H. hamabo plants, the CLA1 expression levels in leaves with white streaks were all significantly lower than those in leaves from mock-infected and control plants. Conclusions The system presented here can efficiently silence genes in H. hamabo and may be a powerful tool for large-scale reverse-genetic analyses of gene functions in H. hamabo.

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Bridgeland, Aya, Sudip Biswas, Nikolaos Tsakirpaloglou, Michael J. Thomson, and Endang M. Septiningsih. "Optimization of gene editing in cowpea through protoplast transformation and agroinfiltration by targeting the phytoene desaturase gene." PLOS ONE 18, no. 4 (2023): e0283837. http://dx.doi.org/10.1371/journal.pone.0283837.

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Cowpea (Vigna unguiculata) is a legume staple widely grown across Sub-Saharan Africa and other tropical and sub-tropical regions. Considering projected climate change and global population increases, cowpea’s adaptation to hot climates, resistance to drought, and nitrogen-fixing capabilities make it an especially attractive crop for facing future challenges. Despite these beneficial traits, efficient varietal improvement is challenging in cowpea due to its recalcitrance to transformation and long regeneration times. Transient gene expression assays can provide solutions to alleviate these issues as they allow researchers to test gene editing constructs before investing in the time and resource- intensive process of transformation. In this study, we developed an improved cowpea protoplast isolation protocol, a transient protoplast assay, and an agroinfiltration assay to be used for initial testing and validation of gene editing constructs and for gene expression studies. To test these protocols, we assessed the efficacy of a CRISPR-Cas9 construct containing four multiplexed single-guide RNA (sgRNA) sequences using polyethylene glycol (PEG)-mediated transformation and agroinfiltration with phytoene desaturase (PDS) as the target gene. Sanger sequencing of DNA from transformed protoplasts and agroinfiltrated cowpea leaves revealed several large deletions in the target sequences. The protoplast system and agroinfiltration protocol developed in this study provide versatile tools to test gene editing components before initiating plant transformation, thus improving the chance of using active sgRNAs and attaining the desired edits and target phenotype.

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Debler, Johannes W., Bernadette M. Henares, and Robert C. Lee. "Agroinfiltration for transient gene expression and characterisation of fungal pathogen effectors in cool-season grain legume hosts." Plant Cell Reports 40, no. 5 (2021): 805–18. http://dx.doi.org/10.1007/s00299-021-02671-y.

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Abstract Key message Modified pEAQ-HT-DEST1 vectors were used for agroinfiltration in legumes. We demonstrate protein expression and export in pea, lentil, and faba bean; however, the method for chickpea was not successful. Abstract Agroinfiltration is a valuable research method for investigating virulence and avirulence effector proteins from pathogens and pests, where heterologous effector proteins are transiently expressed in plant leaves and hypersensitive necrosis responses and other effector functions can be assessed. Nicotiana benthamiana is widely used for agroinfiltration and the characterisation of broad-spectrum effectors. The method has also been used in other plant species including field pea, but not yet developed for chickpea, lentil, or faba bean. Here, we have modified the pEAQ-HT-DEST1 vector for expression of 6 × histidine-tagged green-fluorescent protein (GFP) and the known necrosis-inducing broad-spectrum effector necrosis and ethylene-inducing peptide (Nep1)-like protein (NLP). Modified pEAQ-based vectors were adapted to encode signal peptide sequences for apoplast targeting of expressed proteins. We used confocal microscopy to assess the level of GFP expression in agroinfiltrated leaves. While at 3 days after infiltration in N. benthamiana, GFP was expressed at a relatively high level, expression in field pea and faba bean at the same time point was relatively low. In lentil, an expression level of GFP similar to field pea and faba bean at 3 days was only observed after 5 days. Chickpea leaf cells were transformed at low frequency and agroinfiltration was concluded to not be successful for chickpea. We concluded that the pEAQ vector is suitable for testing host-specific effectors in field pea, lentil, and faba bean, but low transformation efficiency limits the utility of the method for chickpea.

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Dickinson, Christopher C., Alexandra J. Weisberg, and John G. Jelesko. "Transient Heterologous Gene Expression Methods for Poison Ivy Leaf and Cotyledon Tissues." HortScience 53, no. 2 (2018): 242–46. http://dx.doi.org/10.21273/hortsci12421-17.

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Poison ivy [Toxicodendron radicans (L.) Kuntz] is a widely recognized native plant species because of its production of urushiol, which is responsible for delayed contact dermatitis symptoms in humans. Poison ivy is predicted to become both more prevalent and more noxious in response to projected patterns of climate change. Future studies on poison ivy chemical ecology will require reverse genetics to investigate urushiol metabolism. A prerequisite for reverse genetic procedures is the introduction and expression of recombinant DNA into poison ivy tissues. Poison ivy leaves and cotyledons were marginally susceptible to vacuum- and syringe-agroinfiltration and expression of two firefly luciferase (LUC)–based reporter genes. The efficacy of agroinfiltration and transient LUC expression was dependent on leaf age and plant growth environmental conditions, with young leaves grown in magenta boxes showing highest transient LUC expression levels. Agroinfiltrated leaves showed an Agrobacterium-dependent accumulation of brown–colored pigments. Biolistic transformation of a LUC reporter gene did not show brown pigment accumulation and readily displayed transient LUC bioluminescence in both leaves and cotyledon tissues. These studies establish best practices for introducing and transiently expressing recombinant DNA into poison ivy leaf and cotyledon tissues, on which future reverse genetic procedures can be developed.

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Chong, Xinran, Yue Wang, Xiaoyang Xu, et al. "Efficient Virus-Induced Gene Silencing in Ilex dabieshanensis Using Tobacco Rattle Virus." Forests 14, no. 3 (2023): 488. http://dx.doi.org/10.3390/f14030488.

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Ilex dabieshanensis is not only an important ornamental plant, but can also be used to produce Kuding tea, owing to its lipid-lowering and anti-inflammatory medicinal properties. The genetic transformation of I. dabieshanensis is currently difficult, which restricts functional gene studies and molecular breeding research on this species. Virus-induced gene silencing (VIGS) is a powerful tool for determining gene functions in plants. The present study reports the first application of VIGS mediated by a tobacco rattle virus (TRV) vector in I. dabieshanensis. We tested the efficiency of the VIGS system to silence Mg-chelatase H subunit (ChlH) gene through agroinfiltration. The agroinfiltrated leaves of I. dabieshanensis exhibited a typical yellow-leaf phenotype of ChlH gene silencing at 21 days post infiltration. Endogenous ChlH expression levels in the leaves of yellow-leaf phenotype plants were all significantly lower than that in the leaves of mock-infected and control plants. Overall, our results indicated that the TRV-based VIGS system can efficiently silence genes in I. dabieshanensis, and this system will contribute to efficient functional genomics research in I. dabieshanensis.

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Chiba, Sotaro, Kamal Hleibieh, Alice Delbianco, et al. "The Benyvirus RNA Silencing Suppressor Is Essential for Long-Distance Movement, Requires Both Zinc-Finger and NoLS Basic Residues but Not a Nucleolar Localization for Its Silencing-Suppression Activity." Molecular Plant-Microbe Interactions® 26, no. 2 (2013): 168–81. http://dx.doi.org/10.1094/mpmi-06-12-0142-r.

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The RNA silencing-suppression properties of Beet necrotic yellow vein virus (BNYVV) and Beet soil-borne mosaic virus (BSBMV) cysteine-rich p14 proteins have been investigated. Suppression of RNA silencing activities were made evident using viral infection of silenced Nicotiana benthamiana 16C, N. benthamiana agroinfiltrated with green fluorescent protein (GFP), and GF-FG hairpin triggers supplemented with viral suppressor of RNA silencing (VSR) constructs or using complementation of a silencing-suppressor-defective BNYVV virus in Chenopodium quinoa. Northern blot analyses of small-interfering RNAs (siRNAs) in agroinfiltration tests revealed reduced amounts of siRNA, especially secondary siRNA, suggesting that benyvirus VSR act downstream of the siRNA production. Using confocal laser-scanning microscopy imaging of infected protoplasts expressing functional p14 protein fused to an enhanced GFP reporter, we showed that benyvirus p14 accumulated in the nucleolus and the cytoplasm independently of other viral factors. Site-directed mutagenesis showed the importance of the nucleolar localization signal embedded in a C4 zinc-finger domain in the VSR function and intrinsic stability of the p14 protein. Conversely, RNA silencing suppression appeared independent of the nucleolar localization of the protein, and a correlation between BNYVV VSR expression and long-distance movement was established.

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Tu, Liqin, Shuhua Wu, Danna Gao, Yong Liu, Yuelin Zhu, and Yinghua Ji. "Synthesis and Characterization of a Full-Length Infectious cDNA Clone of Tomato Mottle Mosaic Virus." Viruses 13, no. 6 (2021): 1050. http://dx.doi.org/10.3390/v13061050.

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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. benthamiana leaves. Finally, our results also confirmed that tomato plants inoculated with pToMMV led to a high infection rate of 100% in 4–5 weeks post-infiltration (wpi), while pepper plants inoculated with pToMMV led to an infection rate of 40–47% in 4–5 wpi. This is the first report of the development of a full-length infectious cDNA clone of ToMMV. We believe that this infectious clone will enable further studies of ToMMV genes function, pathogenicity and virus–host interaction.

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Sindarovska, Yana, and Mykola Kuchuk. "Long-Term Potato Virus X (PVX)-Based Transient Expression of Recombinant GFP Protein in Nicotiana benthamiana Culture In Vitro." Plants 10, no. 10 (2021): 2187. http://dx.doi.org/10.3390/plants10102187.

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Plant molecular farming has a great potential to produce valuable proteins. Transient expression technology provides high yields of recombinant proteins in greenhouse-grown plants, but every plant must be artificially agroinfiltrated, and open greenhouse systems are less controlled. Here, we propose to propagate agrobacteria-free plants with high-efficient long-term self-replicated transient gene expression in a well-controlled closed in vitro system. Nicotiana benthamiana plant tissue culture in vitro, with transient expression of recombinant GFP, was obtained through shoot induction from leaf explants infected by a PVX-based vector. The transient expression occurs in new tissues and regenerants due to the natural systemic distribution of viral RNA carrying the target gene. Gene silencing was delayed in plants grown in vitro, and GFP was detected in plants for five to six months. Agrobacteria-free, GFP-expressing plants can be micropropagated in vitro (avoiding an agroinfiltration step), “rejuvenated” through regeneration (maintaining culture for years), or transferred in soil. The mean GFP in the regenerants was 18% of the total soluble proteins (TSP) (0.52 mg/g of fresh leaf weight (FW). The highest value reached 47% TSP (2 mg/g FW). This study proposes a new method for recombinant protein production combining the advantages of transient expression technology and closed cultural systems.

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Fan, Xudong, Zunping Zhang, Fang Ren, et al. "Development of a Full-Length Infectious cDNA Clone of the Grapevine Berry Inner Necrosis Virus." Plants 9, no. 10 (2020): 1340. http://dx.doi.org/10.3390/plants9101340.

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Grapevine berry inner necrosis virus (GINV) belongs to the genus Trichovirus in the family Betaflexiviridae. The GINV isolate LN_BETA_RS was obtained from a “Beta” grapevine (Vitis riparia × Vitis labrusca) exhibiting chlorotic mottling and ring spot in Xingcheng, Liaoning Province, China. To verify the correlation between GINV and grapevine chlorotic mottling and ring spot disease, we constructed an infectious cDNA clone of GINV isolate LN_BETA_RS using the seamless assembly approach. Applied treatments of agroinfiltration infectious cDNA confirmed systemic GINV infection of the Nicotianaoccidentalis 37B by reverse transcription polymerase chain reaction (RT-PCR) and transmission electron microscopy, exhibiting chlorotic mottling symptoms on leaves. Infectious cDNA was also transmitted to new healthy N. occidentalis plants through rub-inoculation. Moreover, the cDNA clone was agroinfiltrated into “Beta” and “Thompson Seedless” grapevine plantlets, and the inoculated grapevines exhibited leaf chlorotic mottling and ringspot during the two years of observation. GINV-inoculated “Beta” grapevines had serious leaf chlorotic mottling and ringspot symptoms on the whole plant, while relatively few symptoms were observed on the leaves of agroinoculated “Thompson Seedless” grapevines in early spring and only weak ring spot gradually appeared later in the top young leaves. Our experiments fulfilled Koch’s postulates and revealed the causative role of GINV in grapevine chlorotic mottling and ring spot disease.

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Malla, Ashwini, Balamurugan Shanmugaraj, Ashutosh Sharma, and Sathishkumar Ramalingam. "Production of Genistein in Amaranthus tricolor var. tristis and Spinacia oleracea by Expression of Glycine max Isoflavone Synthase." Plants 10, no. 11 (2021): 2311. http://dx.doi.org/10.3390/plants10112311.

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Isoflavonoids, the diverse group of secondary metabolites derived from the phenylpropanoid pathway, are distributed predominantly in leguminous plants. It has received considerable attention in recent days due to its health promoting benefits and is known to prevent certain diseases in humans. These isoflavonoids are synthesized from flavonoid intermediates of phenylpropanoid pathway by the enzyme isoflavone synthase. Metabolic engineering of isoflavonoid biosynthesis in non-legume crop plants could offer the health benefits of these compounds in diverse plant species further contributing for crop improvement. The transient expression of heterologous genes in the host is considered as an alternative to stable expression, that can provide a rapid way of studying the pathway engineering for metabolite production and could also act as a production platform for nutraceuticals and biopharmaceuticals. In this study, isoflavone genistein was produced in Amaranthus tricolor var. tristis and Spinacia oleracea by transiently expressing Glycine max isoflavone synthase (GmIFS). The GmIFS gene was cloned in plant expression vector pEarleyGate 102 HA and pEAQ-HT-DEST 3 and transformed into plants by agroinfiltration. The presence of transgene in the agroinfiltrated leaves was confirmed by semiquantitative reverse-transcription polymerase chain reaction. The flavonoid substrate naringenin and isoflavonoid genistein were quantified using high performance liquid chromatography in both wild-type and infiltrated leaf samples of both the plants. The naringenin content varied in the range of 65.5–338.5 nM/g fresh weight, while the accumulation of genistein was observed with varying concentrations from 113 to 182.6 nM/g fresh weight in the agroinfiltrated leaf samples of both A. tricolor var. tristis and S. oleracea. These results indicate that the transient expression of GmIFS gene has led to the synthesis of isoflavonoid genistein in A. tricolor var. tristis and S. oleracea providing an insight that stable expression of this gene could enrich the nutraceutical content in the crop plants. To the best of our knowledge, this is the first report on transient expression of GmIFS gene for the production of genistein in A. tricolor var. tristis and S. oleracea.

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Chen, Qiang, and Huafang Lai. "Gene Delivery into Plant Cells for Recombinant Protein Production." BioMed Research International 2015 (2015): 1–10. http://dx.doi.org/10.1155/2015/932161.

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Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration inNicotianaand non-Nicotianaplant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications.

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Fuhrmann-Aoyagi, Martina Bianca, Saki Igarashi, and Kenji Miura. "Comparative Evaluation of Transient Protein Expression Efficiency in Tissues across Soybean Varieties Using the Tsukuba System." Plants 13, no. 6 (2024): 858. http://dx.doi.org/10.3390/plants13060858.

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Transient protein expression is a versatile tool with diverse applications and can be used in soybeans to study gene function, obtain mutants, and produce proteins for commercial use. However, soybeans are considered recalcitrant for agroinfiltration. Subsequent studies on soybeans have demonstrated a green fluorescent protein (GFP) expression in seedpods, but not in leaves, using syringe agroinfiltration. To evaluate agroinfiltration-based transient protein expression levels in plant cells, we used the transient expression vector pTKB3 harboring the GFP gene. Using Agrobacterium tumefaciens, vacuum agroinfiltration of the leaves and needle agroinfiltration of the seedlings of different soybean varieties were performed. GFP was transiently expressed in all of the samples. However, the Enrei and Williams 82 varieties presented better results than the other varieties in the leaf tissue, with results confirmed by immunoblot analysis, demonstrating that both varieties are good candidates for molecular biological studies. GFP expression in the seedlings was less extensive than that in the leaves, which may be due to the tissue characteristics, with Enrei showing the best results. Based on this observation, we conclude that the Tsukuba system is an effective tool that can be used for different tissues and soybean varieties.

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Primasiwi, Dionysia Heviarie, Yekti Asih Purwestri, and Endang Semiarti. "Improving transient gene expression and agroinfiltration‐based transformation effectiveness in Indonesian orchid Phalaenopsis amabilis (L.) Blume." Indonesian Journal of Biotechnology 29, no. 3 (2024): 111. http://dx.doi.org/10.22146/ijbiotech.80555.

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Transient gene expression is an approach used to study transient genes across various species, with infiltration by Agrobacterium tumefaciens (agroinfiltration) being a commonly used method. Agroinfiltration offers a simple and effective means of delivering transgenes into the plant genome. An alternative method for enhancing the quality and productivity of orchids as ornamental plants is genetic modification through agroinfiltration. Although Agrobacterium‐mediated genetic transformation by immersion has been used on the Phalaenopsis amabilis (L.) Blume species of orchid, transformation efficiency using the immersion technique remains relatively low and the method itself is challenging due to its requirement for aseptic handling. The application of agroinfiltration in P. amabilis has not previously been reported. This study investigates the impact of the injection site, acetosyringone concentration, bacterial density (OD600), and injection volume to determine the optimum conditions for agroinfiltration on P. amabilis. The results demonstrated that injection site had a noticeably distinct impact on transformation effectiveness, with the abaxial position of the leaf being the optimal site for Agrobacterium culture suspension injection. While adjustments in acetosyringone concentration, bacterial density (OD600), and injection volume did not significantly affect transformation efficiency, they did influence the peak time of GFP fluorescence. Acetosyringone at a concentration of 200 µM, an OD600 of 1.0 for Agrobacterium culture, and an injection volume of 500 µL effectively accelerated GFP expression duration.

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Vlot, A. Corina, Aymeric Menard, and John F. Bol. "Role of the Alfalfa Mosaic Virus Methyltransferase-Like Domain in Negative-Strand RNA Synthesis." Journal of Virology 76, no. 22 (2002): 11321–28. http://dx.doi.org/10.1128/jvi.76.22.11321-11328.2002.

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ABSTRACT RNAs 1 and 2 of the tripartite genome of alfalfa mosaic virus (AMV) encode the replicase proteins P1 and P2, respectively. P1 contains a methyltransferase-like domain in its N-terminal half, which has a putative role in capping the viral RNAs. Six residues in this domain that are highly conserved in the methyltransferase domains of alphavirus-like viruses were mutated individually in AMV P1. None of the mutants was infectious to plants. Mutant RNA 1 was coexpressed with wild-type (wt) RNAs 2 and 3 from transferred DNA vectors in Nicotiana benthamiana by agroinfiltration. Mutation of His-100 or Cys-189 in P1 reduced accumulation of negative- and positive-strand RNA in the infiltrated leaves to virtually undetectable levels. Mutation of Asp-154, Arg-157, Cys-182, or Tyr-266 in P1 reduced negative-strand RNA accumulation to levels ranging from 2 to 38% of those for the wt control, whereas positive-strand RNA accumulation by these mutants was 2% or less. The (transiently) expressed replicases of the six mutants were purified from the agroinfiltrated leaves. Polymerase activities of these preparations in vitro ranged from undetectable to wt levels. The data indicate that, in addition to its putative role in RNA capping, the methyltransferase-like domain of P1 has distinct roles in replication-associated functions required for negative-strand RNA synthesis. The defect in negative-strand RNA synthesis of the His-100 and Cys-189 mutants could be complemented in trans by coexpression of wt P1.

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Angel, Carlos A., Yi-Cheng Hsieh, and James E. Schoelz. "Comparative Analysis of the Capacity of Tombusvirus P22 and P19 Proteins to Function as Avirulence Determinants in Nicotiana species." Molecular Plant-Microbe Interactions® 24, no. 1 (2011): 91–99. http://dx.doi.org/10.1094/mpmi-04-10-0089.

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We have used an agroinfiltration assay for a comparative study of the roles of tombusvirus P22 and P19 proteins in elicitation of hypersensitive response (HR)-like necrosis and the role of P19 in silencing suppression in Nicotiana species. The advantage of agroinfiltration rather than expression in plant virus vectors is that putative viral avirulence proteins can be evaluated in isolation, eliminating the possibility of synergistic effects with other viral proteins. We found that tombusvirus P22 and P19 proteins elicited HR-like necrosis in certain Nicotiana species but, also, that Nicotiana species could recognize subtle differences in sequence between these proteins. Furthermore, Nicotiana species that responded with systemic necrosis to virion inoculations responded to agroinfiltration of tombusvirus P19 with a very weak and delayed necrosis, indicating that the rapid HR-like necrosis was associated with putative resistance genes and a plant defense response that limited the spread of the virus. Tombusvirus P19 proteins also appeared to differ in their effectiveness as silencing suppressors; in our assay, the P19 proteins of Cymbidium ringspot virus and Tomato bushy stunt virus were stronger silencing suppressors than Cucumber necrosis virus P20. Finally, we show that agroinfiltration can be used to track the presence of putative plant resistance genes in Nicotiana species that target either tombusvirus P19 or P22.

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Annamalai, Padmanaban, Fady Rofail, Darleen A. DeMason, and A. L. N. Rao. "Replication-Coupled Packaging Mechanism in Positive-Strand RNA Viruses: Synchronized Coexpression of Functional Multigenome RNA Components of an Animal and a Plant Virus in Nicotiana benthamiana Cells by Agroinfiltration." Journal of Virology 82, no. 3 (2007): 1484–95. http://dx.doi.org/10.1128/jvi.01540-07.

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ABSTRACT Flock house virus (FHV), a bipartite RNA virus of insects and a member of the Nodaviridae family, shares viral replication features with the tripartite brome mosaic virus (BMV), an RNA virus that infects plants and is a member of the Bromoviridae family. In BMV and FHV, genome packaging is coupled to replication, a widely conserved mechanism among positive-strand RNA viruses of diverse origin. To unravel the events that modulate the mechanism of replication-coupled packaging, in this study, we have extended the transfer DNA (T-DNA)-based agroinfiltration system to express functional genome components of FHV in plant cells (Nicotiana benthamiana). Replication, intracellular membrane localization, and packaging characteristics in agroinfiltrated plant cells revealed that T-DNA plasmids of FHV were biologically active and faithfully mimicked complete replication and packaging behavior similar to that observed for insect cells. Synchronized coexpression of wild-type BMV and FHV genome components in plant cells resulted in the assembly of virions packaging the respective viral progeny RNA. To further elucidate the link between replication and packaging, coat protein (CP) open reading frames were precisely exchanged between BMV RNA 3 (B3) and FHV RNA 2 (F2), creating chimeric RNAs expressing heterologous CP genes (B3/FCP and F2/BCP). Coinfiltration of each chimera with its corresponding genome counterpart to provide viral replicase (B1+B2+B3/FCP and F1+F2/BCP) resulted in the expected progeny profiles, but virions exhibited a nonspecific packaging phenotype. Complementation with homologous replicase (with respect to CP) failed to enhance packaging specificity. Taken together, we propose that the transcription of CP mRNA from homologous replication and its translation must be synchronized to confer packaging specificity.

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Prudhomme, N., R. Pastora, B. Muselius, M. D. McLean, D. Cossar, and J. Geddes-McAlister. "Exposure of Agrobacterium tumefaciens to agroinfiltration medium demonstrates cellular remodelling and may promote enhanced adaptability for molecular pharming." Canadian Journal of Microbiology 67, no. 1 (2021): 85–97. http://dx.doi.org/10.1139/cjm-2020-0239.

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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. Treatment of large quantities of plants for industrial-scale protein production exposes bacteria (harboring genes of interest) to agroinfiltration medium that is devoid of nutrients and carbon sources for prolonged periods of time (possibly upwards of 24 h). Such conditions may negatively influence bacterial viability, infectivity of plant cells, and target protein production. Here, we explored the role of timing in bacterial culture preparation for agroinfiltration using mass spectrometry-based proteomics to define changes in cellular processes. We observed distinct profiles associated with bacterial treatment conditions and exposure timing, including significant changes in proteins involved in pathogenesis, motility, and nutrient acquisition systems as the bacteria adapt to the new environment. These data suggest a progression towards increased cellular remodelling over time. In addition, we described changes in growth- and environment-specific processes over time, underscoring the interconnectivity of pathogenesis and chemotaxis-associated proteins with transport and metabolism. Overall, our results have important implications for the production of transiently expressed target protein products, as prolonged exposure to agroinfiltration medium suggests remodelling of the bacterial proteins towards enhanced infection of plant cells.

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20

Demone, Jordan, Mariam Maltseva, Maryam Nourimand, et al. "Scalable agroinfiltration-based production of SARS-CoV-2 antigens for use in diagnostic assays and subunit vaccines." PLOS ONE 17, no. 12 (2022): e0277668. http://dx.doi.org/10.1371/journal.pone.0277668.

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Agroinfiltration is a method used in biopharming to support plant-based biosynthesis of therapeutic proteins such as antibodies and viral antigens involved in vaccines. Major advantages of generating proteins in plants is the low cost, massive scalability and the rapid yield of the technology. Herein, we report the agroinfiltration-based production of glycosylated SARS-CoV-2 Spike receptor-binding domain (RBD) protein. We show that it exhibits high-affinity binding to the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) and displays folding similar to antigen produced in mammalian expression systems. Moreover, our plant-expressed RBD was readily detected by IgM, IgA, and IgG antibodies from the serum of SARS-CoV-2 infected and vaccinated individuals. We further demonstrate that binding of plant-expressed RBD to ACE2 is efficiently neutralized by these antibodies. Collectively, these findings demonstrate that recombinant RBD produced via agroinfiltration exhibits suitable biochemical and antigenic features for use in serological and neutralization assays, and in subunit vaccine platforms.

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21

Van der Hoorn, Renier A. L., Franck Laurent, Ronelle Roth, and Pierre J. G. M. De Wit. "Agroinfiltration Is a Versatile Tool That Facilitates Comparative Analyses of Avr9/Cf-9-Induced and Avr4/Cf-4-Induced Necrosis." Molecular Plant-Microbe Interactions® 13, no. 4 (2000): 439–46. http://dx.doi.org/10.1094/mpmi.2000.13.4.439.

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The avirulence genes Avr9 and Avr4 from the fungal tomato pathogen Cladosporium fulvum encode extracellular proteins that elicit a hypersensitive response when injected into leaves of tomato plants carrying the matching resistance genes, Cf-9 and Cf-4, respectively. We successfully expressed both Avr9 and Avr4 genes in tobacco with the Agrobacterium tumefaciens transient transformation assay (agroinfiltration). In addition, we expressed the matching resistance genes, Cf-9 and Cf-4, through agroinfiltration. By combining transient Cf gene expression with either transgenic plants expressing one of the gene partners, Potato virus X (PVX)-mediated Avr gene expression, or elicitor injections, we demonstrated that agroinfiltration is a reliable and versatile tool to study Avr/Cf-mediated recognition. Significantly, agroinfiltration can be used to quantify and compare Avr/Cf-induced responses. Comparison of different Avr/Cf-interactions within one tobacco leaf showed that Avr9/Cf-9-induced necrosis developed slower than necrosis induced by Avr4/Cf-4. Quantitative analysis demonstrated that this temporal difference was due to a difference in Avr gene activities. Transient expression of matching Avr/Cf gene pairs in a number of plant families indicated that the signal transduction pathway required for Avr/Cf-induced responses is conserved within solana-ceous species. Most non-solanaceous species did not develop specific Avr/Cf-induced responses. However, co-expression of the Avr4/Cf-4 gene pair in lettuce resulted in necrosis, providing the first proof that a resistance (R) gene can function in a different plant family.

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22

Xiang, Yu, Kishore Kakani, Ron Reade, Elizabeth Hui, and D'Ann Rochon. "A 38-Amino-Acid Sequence Encompassing the Arm Domain of the Cucumber Necrosis Virus Coat Protein Functions as a Chloroplast Transit Peptide in Infected Plants." Journal of Virology 80, no. 16 (2006): 7952–64. http://dx.doi.org/10.1128/jvi.00153-06.

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ABSTRACT Experiments to determine the subcellular location of the coat protein (CP) of the tombusvirus Cucumber necrosis virus (CNV) have been conducted. By confocal microscopy, it was found that an agroinfiltrated CNV CP-green fluorescent protein (GFP) fusion targets chloroplasts in Nicotiana benthamiana leaves and that a 38-amino-acid (aa) region that includes the complete CP arm region plus the first 4 amino acids of the shell domain are sufficient for targeting. Western blot analyses of purified and fractionated chloroplasts showed that the 38-aa region directs import to the chloroplast stroma, suggesting that the CNV arm can function as a chloroplast transit peptide (TP) in plants. Several features of the 38-aa region are similar to features typical of chloroplast TPs, including (i) the presence of an alanine-rich uncharged region near the N terminus, followed by a short region rich in basic amino acids; (ii) a conserved chloroplast TP phosphorylation motif; (iii) the requirement that the CNV 38-aa sequence be present at the amino terminus of the imported protein; and (iv) specific proteolytic cleavage upon import into the chloroplast stroma. In addition, a region just downstream of the 38-aa sequence contains a 14-3-3 binding motif, suggesting that chloroplast targeting requires 14-3-3 binding, as has been suggested for cellular proteins that are targeted to chloroplasts. Chloroplasts of CNV-infected plants were found to contain CNV CP, but only the shell and protruding domain regions were present, indicating that CNV CP enters chloroplasts during infection and that proteolytic cleavage occurs as predicted from agroinfiltration studies. We also found that particles of a CNV CP mutant deficient in externalization of the arm region have a reduced ability to establish infection. The potential biological significance of these findings is discussed.

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23

Muthusamy, Saraladevi, Ramesh R. Vetukuri, Anneli Lundgren та ін. "Transient expression and purification of β-caryophyllene synthase in Nicotiana benthamiana to produce β-caryophyllene in vitro". PeerJ 8 (28 квітня 2020): e8904. http://dx.doi.org/10.7717/peerj.8904.

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Abstract (sommario):

The sesquiterpene β-caryophyllene is an ubiquitous component in many plants that has commercially been used as an aroma in cosmetics and perfumes. Recent studies have shown its potential use as a therapeutic agent and biofuel. Currently, β-caryophyllene is isolated from large amounts of plant material. Molecular farming based on the Nicotiana benthamiana transient expression system may be used for a more sustainable production of β-caryophyllene. In this study, a full-length cDNA of a new duplicated β-caryophyllene synthase from Artemisia annua (AaCPS1) was isolated and functionally characterized. In order to produce β-caryophyllene in vitro, the AaCPS1 was cloned into a plant viral-based vector pEAQ-HT. Subsequently, the plasmid was transferred into the Agrobacterium and agroinfiltrated into N. benthamiana leaves. The AaCPS1 expression was analyzed by quantitative PCR at different time points after agroinfiltration. The highest level of transcripts was observed at 9 days post infiltration (dpi). The AaCPS1 protein was extracted from the leaves at 9 dpi and purified by cobalt–nitrilotriacetate (Co-NTA) affinity chromatography using histidine tag with a yield of 89 mg kg−1 fresh weight of leaves. The protein expression of AaCPS1 was also confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses. AaCPS1 protein uses farnesyl diphosphate (FPP) as a substrate to produce β-caryophyllene. Product identification and determination of the activity of purified AaCPS1 were done by gas chromatography–mass spectrometry (GC–MS). GC–MS results revealed that the AaCPS1 produced maximum 26.5 ± 1 mg of β-caryophyllene per kilogram fresh weight of leaves after assaying with FPP for 6 h. Using AaCPS1 as a proof of concept, we demonstrate that N. benthamiana can be considered as an expression system for production of plant proteins that catalyze the formation of valuable chemicals for industrial applications.

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Kaur, Maninder, Pooja Manchanda, Anu Kalia, et al. "Agroinfiltration Mediated Scalable Transient Gene Expression in Genome Edited Crop Plants." International Journal of Molecular Sciences 22, no. 19 (2021): 10882. http://dx.doi.org/10.3390/ijms221910882.

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Abstract (sommario):

Agrobacterium-mediated transformation is one of the most commonly used genetic transformation method that involves transfer of foreign genes into target plants. Agroinfiltration, an Agrobacterium-based transient approach and the breakthrough discovery of CRISPR/Cas9 holds trending stature to perform targeted and efficient genome editing (GE). The predominant feature of agroinfiltration is the abolishment of Transfer-DNA (T-DNA) integration event to ensure fewer biosafety and regulatory issues besides showcasing the capability to perform transcription and translation efficiently, hence providing a large picture through pilot-scale experiment via transient approach. The direct delivery of recombinant agrobacteria through this approach carrying CRISPR/Cas cassette to knockout the expression of the target gene in the intercellular tissue spaces by physical or vacuum infiltration can simplify the targeted site modification. This review aims to provide information on Agrobacterium-mediated transformation and implementation of agroinfiltration with GE to widen the horizon of targeted genome editing before a stable genome editing approach. This will ease the screening of numerous functions of genes in different plant species with wider applicability in future.

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Pillay, Priyen, Karl J. Kunert, Stefan van Wyk, Matome Eugene Makgopa, Christopher A. Cullis, and Barend J. Vorster. "Agroinfiltration contributes to VP1 recombinant protein degradation." Bioengineered 7, no. 6 (2016): 459–77. http://dx.doi.org/10.1080/21655979.2016.1208868.

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26

King, Jessica L., John J. Finer, and Leah K. McHale. "Development and optimization of agroinfiltration for soybean." Plant Cell Reports 34, no. 1 (2014): 133–40. http://dx.doi.org/10.1007/s00299-014-1694-4.

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27

Beihaghi, Maria, Hasan Marashi, Abdolreza Bagheri, and Mojtaba Sankian. "Transient Expression of CCL21Chemokine in Tobacco via Agroinfiltration." International Journal of Scientific & Engineering Research 7, no. 10 (2016): 430–35. http://dx.doi.org/10.14299/ijser.2016.10.009.

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28

Suzaki, Takuya, Mai Tsuda, Hiroshi Ezura, Brad Day, and Kenji Miura. "Agroinfiltration-based efficient transient protein expression in leguminous plants." Plant Biotechnology 36, no. 2 (2019): 119–23. http://dx.doi.org/10.5511/plantbiotechnology.19.0220b.

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29

Ma, Zhenguo, Jun-Jun Liu, Arezoo Zamany, and Holly Williams. "Transient gene expression in western white pine using agroinfiltration." Journal of Forestry Research 31, no. 5 (2019): 1823–32. http://dx.doi.org/10.1007/s11676-019-00938-5.

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30

Duc Tien, Nguyen Quang. "Transient Expression of Chi42 Genes from Trichoderma asperellum in Nicotiana benthamiana by Agroinfiltration." International Journal of Agriculture and Biology 26, no. 01 (2021): 177–84. http://dx.doi.org/10.17957/ijab/15.1822.

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Abstract (sommario):

The present study reports the transient expression of chi42 genes encoding 42 kDa chitinase from T. asperellum SH16 in N. benthamiana via agroinfiltration. The efficacy of agroinfiltration for chi42 genes including a wild-type gene (Chi42) and two synthetic genes (syncodChi42-1 and syncodChi42-2) was determined. Accordingly, coinfiltration of two vectors pMYV719 carrying one of three genes chi42 and pMYV508 carrying gene p19 expedited the higher expression of recombinant enzymes whose genes were optimized for codon usage in plant tissues. The highest chitinolytic activity of about 290 U/mL was found in plants containing the gene syncodChi42-2 after 7 days of injection, 1.7 and 2.6 times higher than that of genes syncodChi42-1 and chi42. Recombinant chitinase has also exhibited activity against the pathogenic fungus Sclerotium rolfsii under in vitro condition. A higher expression level of syncodChi42-2 gene in N. benthamiana and its antifungal activity promise potential applications in the field of transgenic crops against phytopathogenic fungi. © 2021 Friends Science Publishers

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31

Do, Van Giap, Youngsuk Lee, Jeong-Hee Kim, et al. "The Synergistic Effects of Environmental and Genetic Factors on the Regulation of Anthocyanin Accumulation in Plant Tissues." International Journal of Molecular Sciences 24, no. 16 (2023): 12946. http://dx.doi.org/10.3390/ijms241612946.

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Abstract (sommario):

Anthocyanin accumulation is responsible for the coloration of apple fruit, and their accumulation depends on the expression of anthocyanin biosynthesis-related genes. Light is an environmental stimulus that induces fruit color by regulating genes involved in the anthocyanin biosynthesis pathway. In this study, the roles of light and genetic factors on fruit coloration and anthocyanin accumulation in apple fruit were investigated. Three genes in the anthocyanin biosynthesis pathway, MdCHS, MdANS, and MdUFGT1, were synthesized and cloned into a viral-based expression vector system for transient expression in ‘Ruby S’ apple fruits. Apple fruits were agroinfiltrated with expression vectors harboring MdCHS, MdANS, and MdUFGT1. Agroinfiltrated apple fruits were then either kept in the dark (bagged fruits) or exposed to light (exposed fruits). The agroinfiltrated fruits showed significantly different coloration patterns, transcript expression levels, and anthocyanin accumulation compared to the control fruits. Moreover, these parameters were higher in exposed fruits than in bagged fruits. For stable expression, MdCHS was introduced into a binary vector under the control of the rice α-amylase 3D (RAmy3D) promoter. The ectopic overexpression of MdCHS in transgenic rice calli showed a high accumulation of anthocyanin content. Taken together, our findings suggest that light, together with the overexpression of anthocyanin biosynthesis genes, induced the coloration and accumulation of anthocyanin content in apple fruits by upregulating the expression of the genes involved in the anthocyanin biosynthesis pathway.

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32

Ghoshal, Kankana, Jane Theilmann, Ron Reade, Ajay Maghodia, and D'Ann Rochon. "Encapsidation of Host RNAs by Cucumber Necrosis Virus Coat Protein during both Agroinfiltration and Infection." Journal of Virology 89, no. 21 (2015): 10748–61. http://dx.doi.org/10.1128/jvi.01466-15.

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Abstract (sommario):

ABSTRACTNext-generation sequence analysis of virus-like particles (VLPs) produced during agroinfiltration of cucumber necrosis virus (CNV) coat protein (CP) and of authentic CNV virions was conducted to assess if host RNAs can be encapsidated by CNV CP. VLPs containing host RNAs were found to be produced during agroinfiltration, accumulating to approximately 1/60 the level that CNV virions accumulated during infection. VLPs contained a variety of host RNA species, including the major rRNAs as well as cytoplasmic, chloroplast, and mitochondrial mRNAs. The most predominant host RNA species encapsidated in VLPs were chloroplast encoded, consistent with the efficient targeting of CNV CP to chloroplasts during agroinfiltration. Interestingly, droplet digital PCR analysis showed that the CNV CP mRNA expressed during agroinfiltration was the most efficiently encapsidated mRNA, suggesting that the CNV CP open reading frame may contain a high-affinity site or sites for CP binding and thus contribute to the specificity of CNV RNA encapsidation. Approximately 0.09% to 0.7% of the RNA derived from authentic CNV virions contained host RNA, with chloroplast RNA again being the most prominent species. This is consistent with our previous finding that a small proportion of CNV CP enters chloroplasts during the infection process and highlights the possibility that chloroplast targeting is a significant aspect of CNV infection. Remarkably, 6 to 8 of the top 10 most efficiently encapsidated nucleus-encoded RNAs in CNV virions correspond to retrotransposon or retrotransposon-like RNA sequences. Thus, CNV could potentially serve as a vehicle for horizontal transmission of retrotransposons to new hosts and thereby significantly influence genome evolution.IMPORTANCEViruses predominantly encapsidate their own virus-related RNA species due to the possession of specific sequences and/or structures on viral RNA which serve as high-affinity binding sites for the coat protein. In this study, we show, using next-generation sequence analysis, that CNV also encapsidates host RNA species, which account for ∼0.1% of the RNA packaged in CNV particles. The encapsidated host RNAs predominantly include chloroplast RNAs, reinforcing previous observations that CNV CP enters chloroplasts during infection. Remarkably, the most abundantly encapsidated cytoplasmic mRNAs consisted of retrotransposon-like RNA sequences, similar to findings recently reported for flock house virus (A. Routh, T. Domitrovic, and J. E. Johnson, Proc Natl Acad Sci U S A 109:1907–1912, 2012). Encapsidation of retrotransposon sequences may contribute to their horizontal transmission should CNV virions carrying retrotransposons infect a new host. Such an event could lead to large-scale genomic changes in a naive plant host, thus facilitating host evolutionary novelty.

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Liu, Lijing, Yiyue Zhang, Sanyuan Tang, et al. "An efficient system to detect protein ubiquitination by agroinfiltration inNicotiana benthamiana." Plant Journal 61, no. 5 (2010): 893–903. http://dx.doi.org/10.1111/j.1365-313x.2009.04109.x.

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34

Heidari-Japelaghi, Reza, Mostafa Valizadeh, Raheem Haddad, Ebrahim Dorani-Uliaie та Mokhtar Jalali-Javaran. "Production of bioactive human IFN-γ protein by agroinfiltration in tobacco". Protein Expression and Purification 173 (вересень 2020): 105616. http://dx.doi.org/10.1016/j.pep.2020.105616.

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35

Deng, Xianbao, Jani Kelloniemi, Tuuli Haikonen, et al. "Modification of Tobacco rattle virus RNA1 to Serve as a VIGS Vector Reveals That the 29K Movement Protein Is an RNA Silencing Suppressor of the Virus." Molecular Plant-Microbe Interactions® 26, no. 5 (2013): 503–14. http://dx.doi.org/10.1094/mpmi-12-12-0280-r.

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Abstract (sommario):

Tobacco rattle virus (TRV) has a bipartite, positive-sense single-stranded RNA genome and is widely used for virus-induced gene silencing (VIGS) in plants. RNA1 of TRV that lacks the gene for the cysteine-rich 16K silencing-suppression protein infects plants systemically in the absence of RNA2. Here, we attempted to engineer RNA1 for use as a VIGS vector by inserting heterologous gene fragments to replace 16K. The RNA1 vector systemically silenced the phytoene desaturase (PDS) gene, although less efficiently than when the original VIGS vector system was used, which consists of wild-type RNA1 and engineered RNA2 carrying the heterologous gene. Infectious RNA1 mutants with a dysfunctional 16K suppressed silencing and enhanced transgene expression in green fluorescent protein-transgenic Nicotiana benthamiana following inoculation by agroinfiltration, unlike mutants that also lacked 29K, a movement protein (MP) gene. The 30K MP gene of Tobacco mosaic virus complemented in cis the movement defect but not the silencing suppression functions of TRV 29K. Silencing suppression by 29K occurred in the context of RNA1 replication but not in an agroinfiltration assay which tested 29K alone for suppression of sense-mediated silencing. Both 29K and 16K were needed to avoid necrotic symptoms in RNA1-infected N. benthamiana. The results shed new light on virulence factors of TRV.

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van Poppel, Pieter M. J. A., Jun Guo, Peter J. I. van de Vondervoort, et al. "The Phytophthora infestans Avirulence Gene Avr4 Encodes an RXLR-dEER Effector." Molecular Plant-Microbe Interactions® 21, no. 11 (2008): 1460–70. http://dx.doi.org/10.1094/mpmi-21-11-1460.

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Abstract (sommario):

Resistance in potato against the oomycete Phytophthora infestans is conditioned by resistance (R) genes that are introgressed from wild Solanum spp. into cultivated potato. According to the gene-for-gene model, proteins encoded by R genes recognize race-specific effectors resulting in a hypersensitive response (HR). We isolated P. infestans avirulence gene PiAvr4 using a combined approach of genetic mapping, transcriptional profiling, and bacterial artificial chromosome marker landing. PiAvr4 encodes a 287-amino-acid-protein that belongs to a superfamily of effectors sharing the putative host-cell-targeting motif RXLR-dEER. Transformation of P. infestans race 4 strains with PiAvr4 resulted in transformants that were avirulent on R4 potato plants, demonstrating that PiAvr4 is responsible for eliciting R4-mediated resistance. Moreover, expression of PiAvr4 in R4 plants using PVX agroinfection and agroinfiltration showed that PiAvr4 itself is the effector that elicits HR on R4 but not r0 plants. The presence of the RXLR-dEER motif suggested intracellular recognition of PiAvr4. This was confirmed in agroinfiltration assays but not with PVX agroinfection. Because there was always recognition of PiAvr4 retaining the signal peptide, extracellular recognition cannot be excluded. Deletion of the RXLR-dEER domain neither stimulated nor prevented elicitor activity of PiAvr4. Race 4 strains have frame shift mutations in PiAvr4 that result in truncated peptides; hence, PiAvr4 is apparently not crucial for virulence.

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37

Wang, Yanfang, Yanzi Zhang, Chongjing Dai, et al. "The Establishment of Two Efficient Transformation Systems to Manipulate and Analyze Gene Functions in Quinoa (Chenopodium quinoa Willd.)." Journal of Advances in Biology & Biotechnology 26, no. 6 (2023): 20–31. http://dx.doi.org/10.9734/jabb/2023/v26i6639.

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Abstract (sommario):

Quinoa (C. quinoa) is considered a gluten-free food with abundant nutrients and high tolerance to multiple abiotic stresses, which is the potential to become a major crop in future. Genetic manipulation will provide powerful tools to investigate the function and mechanism of those important genes in the regulation of quinoa development and stress responses, and further improve the quinoa in the field. However, the efficient plant transformation system for quinoa has not been well developed yet. Here, we established two rapid and efficient transformation systems for quinoa by using hairy roots and agroinfiltration of leaves, which provide useful tools for quick analysis of gene function. Hairy roots were obtained from three types of explants: cotyledon-nod with hypocotyl, cotyledon itself, and hypocotyl pieces. Interestingly, explants of cotyledon-nod with hypocotyl showed the highest transformation efficiency at 67.9%, and cotyledon displayed medium efficiency at 42.2%, while hypocotyl explants with the lowest at 31.6%. We also obtained transgenic quinoa roots successfully in-vivo, which showed low efficiency but provides a potential method to test gene functions in live plants. By using young leaves for agroinfiltration, direct injection showed a better transgenic effect compared with vacuum penetration. In juxtaposition, the transformation systems using both hairy root and leaf infiltration establish an efficient and convenient way to manipulate and analyze gene functions in quinoa, and a potential strategy for transgenic quinoa.

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38

Acanda, Yosvanis, Stacy Welker, Vladimir Orbović, and Amit Levy. "A simple and efficient agroinfiltration method for transient gene expression in Citrus." Plant Cell Reports 40, no. 7 (2021): 1171–79. http://dx.doi.org/10.1007/s00299-021-02700-w.

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39

Kopertekh, Lilya, and Joachim Schiemann. "Agroinfiltration as a Tool for Transient Expression of cre Recombinase in vivo." Transgenic Research 14, no. 5 (2005): 793–98. http://dx.doi.org/10.1007/s11248-005-8293-7.

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40

Matsuo, Kouki, Noriho Fukuzawa, and Takeshi Matsumura. "A simple agroinfiltration method for transient gene expression in plant leaf discs." Journal of Bioscience and Bioengineering 122, no. 3 (2016): 351–56. http://dx.doi.org/10.1016/j.jbiosc.2016.02.001.

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41

Carvalho, Raquel F., Sofia D. Carvalho, Kevin O’Grady, and Kevin M. Folta. "Agroinfiltration of Strawberry Fruit — A Powerful Transient Expression System for Gene Validation." Current Plant Biology 6 (October 2016): 19–37. http://dx.doi.org/10.1016/j.cpb.2016.09.002.

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42

Wang, Yaqin, Yu Song, Yongzhi Wang, Mengji Cao, Tao Hu, and Xueping Zhou. "Discovery and Characterization of a Novel Ampelovirus on Firespike." Viruses 12, no. 12 (2020): 1452. http://dx.doi.org/10.3390/v12121452.

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Abstract (sommario):

A novel RNA virus was identified in firespike (Odontonema tubaeforme) plants exhibiting leaf curling and chlorosis. The molecular features of the viral genomic RNA and proteins resemble those of ampeloviruses. Based on sequence comparisons and phylogenetic analysis, we propose a new species in the genus Ampelovirus, which we have tentatively named Firespike leafroll-associated virus (FLRaV). Bioassays showed that the virus is mechanically transmissible to Nicotiana benthamiana. In addition, a full-length cDNA clone of FLRaV could successfully infect N. benthamiana via agroinfiltration.

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Minh Hang, Nguyen Thi, Ho Thi Thuong, Nguyen Thu Giang, Pham Bich Ngoc, Nguyen Trung Nam, and Chu Hoang Ha. "OPTIMIZATION OF EXPRESSION CONDITIONS OF GENE ENCODING ANTIGEN M OF PRRSV IN LEAVES OF NICOTIANA BENTHAMIANA BY AGROINFILTRATION METHOD." Vietnam Journal of Biotechnology 16, no. 2 (2018): 293–300. http://dx.doi.org/10.15625/1811-4989/16/2/13440.

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Abstract (sommario):

Reproductive disorders and respiratory swine (Porcine Reproductive and Respiratory Syndrome - PRRS) is an infectious disease caused by the PRRS virus (PRRSV), spread fast speeds, causing mass death when infected pigs. M protein is one of the major structural protein of the PRRSV, has a molecular weight of about 19 kDa, encoded by the open reading frame 6 (ORF6), which is used to design a subunit vaccine against back PRRSV. In this study, conditions for transient expression of the gene encoded the M protein of PRRSV in leaves of the Nicotiana benthamiana by agroinfiltration method were determined. The results show that optimal conditions for transient expression of protein M in the leaf are used the simultaneous of the A. tumefaciens strain containing vector carrying the gene encoding the protein M and A. tumefaciens containing vector carrying gene encoding supported protein HC-Pro PVY, concentration of acetosyringone with 450 µM, the bacterial cell density used to infect into leaf with OD600 is 0.5, The physiological age of the leaf suitable for infecting bacteria was young leaf and mature leaf of tobacco plants 4 to 6 weeks of age, and the transient expression time of gene encoding protein M of PRRSV in tobacco leaves is most effective 6 days after infecting. This agroinfiltration method can be used to express large amounts of protein - antigen M of PRRSV in tobacco plants to produce vaccines against this virus.

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Fischer, Rainer, Carmen Vaquero‐Martin, Markus Sack, Jürgen Drossard, Neil Emans, and Ulrich Commandeur. "Towards molecular farming in the future: transient protein expression in plants." Biotechnology and Applied Biochemistry 30, no. 2 (1999): 113–16. http://dx.doi.org/10.1111/j.1470-8744.1999.tb00900.x.

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Abstract (sommario):

Molecular farming in plants can be achieved by stable or transient expression of a recombinant protein. Transient expression of recombinant proteins in plants can rapidly provide large amounts of the proteins for detailed characterization. It is fast, flexible and can be carried out at field scale using viral vectors, but it lacks the increases in production volume that can be achieved easily with stable transgenic crops. This review article focuses on discussing the applications of transient expression using viral vectors, biolistic methods or agroinfiltration.

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45

SONG, Guo-qing, and Ken-ichi YAMAGUCHI. "Efficient Agroinfiltration-mediated Transient GUS Expression System for Assaying Different Promoters in Rice." Plant Biotechnology 20, no. 3 (2003): 235–39. http://dx.doi.org/10.5511/plantbiotechnology.20.235.

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46

Park, Sang-Ho, and Kook-Hyung Kim. "Agroinfiltration-based Potato Virus X Replicons to Dissect the Requirements of Viral Infection." Plant Pathology Journal 22, no. 4 (2006): 386–90. http://dx.doi.org/10.5423/ppj.2006.22.4.386.

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47

Xiong, Yongao, Qiongyu Li, Muchena Kailemia, Carlito Lebrilla, Somen Nandi, and Karen McDonald. "Glycoform Modification of Secreted Recombinant Glycoproteins through Kifunensine Addition during Transient Vacuum Agroinfiltration." International Journal of Molecular Sciences 19, no. 3 (2018): 890. http://dx.doi.org/10.3390/ijms19030890.

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48

Palanichelvam, Karuppaiah, Anthony B. Cole, Monir Shababi, and James E. Schoelz. "Agroinfiltration of Cauliflower mosaic virus Gene VI Elicits Hypersensitive Response in Nicotiana Species." Molecular Plant-Microbe Interactions® 13, no. 11 (2000): 1275–79. http://dx.doi.org/10.1094/mpmi.2000.13.11.1275.

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Abstract (sommario):

Cauliflower mosaic virus strain W260 induces hypersensitive response (HR) in Nicotiana edwardsonii and systemic cell death in N. clevelandii. In contrast, the D4 strain of Cauliflower mosaic virus evades the host defenses in Nicotiana species; it induces chlorotic primary lesions and a systemic mosaic in both hosts. Previous studies with chimeric viruses had indicated that gene VI of W260 was responsible for elicitation of HR or cell death. To prove conclusively that W260 gene VI is responsible, we inserted gene VI of W260 and D4 into the Agrobacterium tumefaciens binary vector pKYLX7. Agroinfiltration of these constructs into the leaves of N. edwardsonii and N. clevelandii revealed that gene VI of W260 elicited HR in N. edwardsonii 4 to 5 days after infiltration and cell death in N. clevelandii approximately 9 to 12 days after infiltration. In contrast, gene VI of D4 did not elicit HR or cell death in either Nicotiana species. A frameshift mutation introduced into gene VI of W260 abolished its ability to elicit HR or cell death in both Nicotiana species, demonstrating that the elicitor is the gene VI protein.

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49

Wieczorek, Przemysław, Marta Budziszewska, and Aleksandra Obrępalska-Stęplowska. "Construction of infectious clones of tomato torrado virus and their delivery by agroinfiltration." Archives of Virology 160, no. 2 (2014): 517–21. http://dx.doi.org/10.1007/s00705-014-2266-1.

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50

Natorajan, D., HY Yong, and I. Zainal. "Feasibility analysis of leaf disc samples produced via agroinfiltration for promoter trapping studies." Emirates Journal of Food and Agriculture 22, no. 6 (2010): 448. http://dx.doi.org/10.9755/ejfa.v22i6.4662.

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