Browsing by Author "Wang, Xu"
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Item Facilitating viral vector movement enhances heterologous protein production in an established plant system(Plant Biotechnology Journal, 2023-01-04) Wang, Xu; Prokhnevsky, Alexei I.; Skarjinskaia, Marina; Razzak, Md Abdur; Streatfield, Stephen J.; Lee, Jung-YounMolecular farming technology using transiently transformed Nicotiana plants offers an economical approach to the pharmaceutical industry to produce an array of protein targets including vaccine antigens and therapeutics. It can serve as a desirable alternative approach for those proteins that are challenging or too costly to produce in large quantities using other heterologous protein expression systems. However, since cost metrics are such a critical factor in selecting a production host, any system-wide modifications that can increase recombinant protein yields are key to further improving the platform and making it applicable for a wider range of target molecules. Here, we report on the development of a new approach to improve target accumulation in an established plant-based expression system that utilizes viral-based vectors to mediate transient expression in Nicotiana benthamiana. We show that by engineering the host plant to support viral vectors to spread more effectively between host cells through plasmodesmata, protein target accumulation can be increased by up to approximately 60%.Item Targeting of plasmodesmal proteins requires unconventional signals(The Plant Cell, 2023-08-02) Luna, Gabriel Robles; Li, Jiefu; Wang, Xu; Liao, Li; Lee, Jung-YounEffective cellular signaling relies on precise spatial localization and dynamic interactions among proteins in specific subcellular compartments or niches, such as cell-to-cell contact sites and junctions. In plants, endogenous and pathogenic proteins gained the ability to target plasmodesmata, membrane-lined cytoplasmic connections, through evolution to regulate or exploit cellular signaling across cell wall boundaries. For example, the receptor-like membrane protein PLASMODESMATA-LOCATED PROTEIN 5 (PDLP5), a potent regulator of plasmodesmal permeability, generates feed-forward or feed-back signals important for plant immunity and root development. However, the molecular features that determine the plasmodesmal association of PDLP5 or other proteins remain largely unknown, and no protein motifs have been identified as plasmodesmal targeting signals. Here, we developed an approach combining custom-built machine-learning algorithms and targeted mutagenesis to examine PDLP5 in Arabidopsis thaliana and Nicotiana benthamiana. We report that PDLP5 and its closely related proteins carry unconventional targeting signals consisting of short stretches of amino acids. PDLP5 contains 2 divergent, tandemly arranged signals, either of which is sufficient for localization and biological function in regulating viral movement through plasmodesmata. Notably, plasmodesmal targeting signals exhibit little sequence conservation but are located similarly proximal to the membrane. These features appear to be a common theme in plasmodesmal targeting.Item Towards understanding of plasmodesmal association and regulation using Plasmodesmata-Located Protein 5 as a model(University of Delaware, 2017) Wang, XuPlasmodesmata (PDs) mediate intercellular communication by facilitating cell- to-cell exchange of soluble molecules in plants. Despite a growing number of proteins are identified to associate with PD and affect PD permeability, not much is known about how they are targeted to PD and regulate PD function. Arabidopsis Plasmodesmata-Located Protein 5 (PDLP5), a receptor-like type I transmembrane protein associated with PD and restricts PD permeability. PDLP5 expression is controlled by a salicylic acid (SA)-dependent signaling pathway, and functional PDLP5 is required for the immunity. However, it is not understood how PDLP5 associates with PD and how it restricts PD permeability during defense responses. To gain insight into the mechanisms underlying PDLP5 targeting and function, a mutagenesis approach in combination with various functional analyses was taken. Subcellular localization studies of the mutant constructs fused to fluorescent proteins revealed that while the presence of transmembrane domain (TMD) is important for plasmodesmal membrane association, no specific sequence within the TMD is required for its targeting to PD. Instead, we found that other domains differentially contribute to PD localization and that the TMD is responsible for homo- or heteromeric interactions with other PDLP members. Functional analyses using fluorescent dye and GFP movement assays indicated that the extracellular domain is essential for PDLP5 function. Analyzing Arabidopsis mutants that are impaired in SA biosynthetic or signaling pathways such as eds1, ics1 or npr1, using PD permeability assays and PD callose quantifications showed that, SA accumulation is critical for PDLP5-mediated PD-callose deposition and -closure during microbial pathogen infection.