Transient Protein Expression by Agroinfiltration in Lettuce

Chen, Qiang; Dent, Matthew; Hurtado, Jonathan; Stahnke, Jake; McNulty, Alyssa; Leuzinger, Kahlin; Lai, Huafang

doi:10.1007/978-1-4939-3289-4_4

Cited by 28 publications

(21 citation statements)

References 36 publications

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“…The technique has been developed in N. benthamiana using a disarmed strain where the virulence factors encoded by the Ti plasmid were deleted ( Goodin et al, 2008 ). Then, the technique was optimized for different plants such as potato ( Bhaskar et al, 2009 ), lettuce ( Chen et al, 2016 ), grapevine ( Santos-Rosa et al, 2008 ), Medicago truncatula ( Picard et al, 2013 ) and recently in soybeans ( King et al, 2015 ). However, the technique is not established in the legume plants, which are hosts for A. pisum .…”

Section: Introductionmentioning

confidence: 99%

Optimization of Agroinfiltration in Pisum sativum Provides a New Tool for Studying the Salivary Protein Functions in the Pea Aphid Complex

et al. 2016

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Aphids are piercing-sucking insect pests and feed on phloem sap. During feeding, aphids inject a battery of salivary proteins into host plant. Some of these proteins function like effectors of microbial pathogens and influence the outcome of plant–aphid interactions. The pea aphid (Acyrthosiphon pisum) is the model aphid and encompasses multiple biotypes each specialized to one or a few legume species, providing an opportunity to investigate the underlying mechanisms of the compatibility between plants and aphid biotypes. We aim to identify the aphid factors that determine the compatibility with host plants, hence involved in the host plant specialization process, and hypothesize that salivary proteins are one of those factors. Agrobacterium-mediated transient gene expression is a powerful tool to perform functional analyses of effector (salivary) proteins in plants. However, the tool was not established for the legume species that A. pisum feeds on. Thus, we decided to optimize the method for legume plants to facilitate the functional analyses of A. pisum salivary proteins. We screened a range of cultivars of pea (Pisum sativum) and alfalfa (Medicago sativa). None of the M. sativa cultivars was suitable for agroinfiltration under the tested conditions; however, we established a protocol for efficient transient gene expression in two cultivars of P. sativum, ZP1109 and ZP1130, using A. tumefaciens AGL-1 strain and the pEAQ-HT-DEST1 vector. We confirmed that the genes are expressed from 3 to 10 days post-infiltration and that aphid lines of the pea adapted biotype fed and reproduced on these two cultivars while lines of alfalfa and clover biotypes did not. Thus, the pea biotype recognizes these two cultivars as typical pea plants. By using a combination of ZP1109 and an A. pisum line, we defined an agroinfiltration procedure to examine the effect of in planta expression of selected salivary proteins on A. pisum fitness and demonstrated that transient expression of one candidate salivary gene increased the fecundity of the aphids. This result confirms that the agroinfiltration can be used to perform functional analyses of salivary proteins in P. sativum and consequently to study the molecular mechanisms underlying host specialization in the pea aphid complex.

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Section: Introductionmentioning

confidence: 99%

Optimization of Agroinfiltration in Pisum sativum Provides a New Tool for Studying the Salivary Protein Functions in the Pea Aphid Complex

et al. 2016

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“…Lettuce has been shown to be a promising plant system capable of rapidly producing recombinant proteins (Chen et al ., ; Lai et al ., ). To further investigate the generality of our results, we also tested a variety of 3’ UTRs in tobacco and lettuce.…”

Section: Discussionmentioning

confidence: 99%

Chimeric 3’ flanking regions strongly enhance gene expression in plants

Diamos

Mason

2018

Plant Biotechnology Journal

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SummaryPlants represent a promising platform for the highly scalable production of recombinant proteins. Previously, we identified the tobacco extensin terminator lacking its intron as an element that reduced transcript read‐through and improved recombinant protein production in a plant‐based system. In this study, we systematically compared nonreplicating plant expression vectors containing over 20 commonly used or newly identified terminators from diverse sources. We found that eight gene terminators enhance reporter gene expression significantly more than the commonly used 35S and NOS terminators. The intronless extensin terminator provided a 13.6‐fold increase compared with the NOS terminator. Combining terminators in tandem produced large synergistic effects, with many combinations providing a >25‐fold increase in expression. Addition of the tobacco Rb7 or TM6 matrix attachment region (MAR) strongly enhanced protein production when added to most terminators, with the Rb7 MAR providing the greatest enhancement. Using deletion analysis, the full activity of the 1193 bp Rb7 MAR was found to require only a 463‐bp region at its 3’ end. Combined terminators and MAR together provided a >60‐fold increase compared with the NOS terminator alone. These combinations were then placed in a replicating geminiviral vector, providing a total of >150‐fold enhancement over the original NOS vector, corresponding to an estimated yield of 3–5 g recombinant protein per kg leaf fresh weight or around 50% of the leaf total soluble protein. These results demonstrate the importance of 3’ flanking regions in optimizing gene expression and show great potential for 3’ flanking regions to improve DNA‐based recombinant protein production systems.

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“…Furthermore, VP1 in lyophilized plant tissue maintained long‐term stability and antigenicity at ambient temperature, effectively eliminating the requirement for cold chain (Chan et al ., ). Edible plants such as lettuce may offer a more palatable choice for the production of oral vaccines (Chen et al ., ; Lai et al ., ). Notably, a very recent publication demonstrated that oral administration of lettuce‐derived hepatitis C virus E1E2 dimers following an intramuscular priming elicited both systemic and mucosal immune responses (Clarke et al ., ).…”

Section: Discussionmentioning

confidence: 99%

Plant‐produced Zika virus envelope protein elicits neutralizing immune responses that correlate with protective immunity against Zika virus in mice

Yang

Sun

Lai

et al. 2017

Plant Biotechnology Journal

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Summary The global Zika virus (ZIKV) outbreak and its link to fetal and newborn microcephaly and severe neurological complications in adults call for the urgent development of ZIKV vaccines. In response, we developed a subunit vaccine based on the ZIKV envelope (E) protein and investigated its immunogenicity in mice. Transient expression of ZIKV E (zE) resulted in its rapid accumulation in leaves of Nicotiana benthamiana plants. Biochemical analysis revealed that plant-produced ZIKV E (PzE) exhibited specific binding to a panel of monoclonal antibodies that recognize various zE conformational epitopes. Furthermore, PzE can be purified to >90% homogeneity with a one-step Ni2+ affinity chromatography process. PzE are found to be highly immunogenic, as two doses of PzE elicited both potent zE-specific antibody and cellular immune responses in mice. The delivery of PzE with alum induced a mixed Th1/Th2 immune response, as the antigen-specific IgG isotypes were a mixture of high levels of IgG1/IgG2c and splenocyte cultures from immunized mice secreted significant levels of IFN-gamma, IL-4 and IL-6. Most importantly, the titers of zE-specific and neutralizing antibodies exceeded the threshold that correlates with protective immunity against multiple strains of ZIKV. Thus, our results demonstrated the feasibility of plant-produced ZIKV protein antigen as effective, safe and affordable vaccines against ZIKV.

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Transient Protein Expression by Agroinfiltration in Lettuce

Cited by 28 publications

References 36 publications

Optimization of Agroinfiltration in Pisum sativum Provides a New Tool for Studying the Salivary Protein Functions in the Pea Aphid Complex

Optimization of Agroinfiltration in Pisum sativum Provides a New Tool for Studying the Salivary Protein Functions in the Pea Aphid Complex

Chimeric 3’ flanking regions strongly enhance gene expression in plants

Plant‐produced Zika virus envelope protein elicits neutralizing immune responses that correlate with protective immunity against Zika virus in mice

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