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Analyzing the Localization of SAUL1 and AtPUB43 Proteins in Nicotiana benthamiana
Author(s) -
Alcantara Michelle A.,
Tyler Brett
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.07451
Subject(s) - nicotiana benthamiana , microbiology and biotechnology , microvesicles , biology , arabidopsis , tetraspanin , fusion protein , secretion , ubiquitin ligase , agroinfiltration , lipid bilayer fusion , transport protein , snare complex , bimolecular fluorescence complementation , ubiquitin , biochemistry , exocytosis , membrane , yeast , cell , microrna , gene , mutant , recombinant dna
Plants have evolved elaborate immune systems to resist infection, and thus are resistant to most microbes. In response to this resistance, pathogens have evolved in order to successfully overcome the immune systems of their specific hosts. Over the last few years, a new mechanism used both by plants for defense and by microbes for attack has been uncovered. This mechanism involves the secretion of membranous vesicles called exosomes. Exosomes transport small RNAs, proteins, and toxic chemicals from one organism to another during infection. However, little is known about the mechanisms by which plants and pathogens produce and release exosomes. Multivesicular bodies have been identified as a potential source of exosomes through fusion with the plasma membrane, but the mechanism of fusion is unknown. Recently the Tyler lab documented that, in plants, MVBs could be tethered to the plasma membrane by artificial protein fusions and by certain E3 ubiquitin ligase proteins that contain ARM (armadillo) domains, namely the Arabidopsis proteins SAUL1 and AtPUB43. They showed that the ARM domains of SAUL1 and AtPUB43 could trigger tethering of MVBs to the plasma membrane. Furthermore, the full‐length proteins could bind the plasma membrane and could trigger tethering during infection of plants by Phytophthora. This research project aims to test which parts of the SAUL1 and AtPUB43 proteins are involved in tethering using agroinfiltration. Different pieces of the proteins will be fused to Venus fluorescent protein and then expressed in Nicotiana benthamiana leaves. The leaves, examined by confocal microscopy, will observe whether tethering has occurred. Proteins will be expressed independently in Nicotiana benthamiana leaves to verify their localization. Once verified, proteins will then be coexpressed and analyzed to see if any tethering had occurred. In this study, we have found that the artificial infusion of the proteins, Vam7p and ACBP1 coexpressed, triggered the fusion of MVBs to the plasma membrane. The tethering of MVBs seen from these proteins in Nicotiana benthamiana leaves, can further test the hypothesis that Vam7p and ACBP1 can trigger the tethering of MVBs to the plasma membrane in the pathogen Phytopthora sojae. This can further explore the hypothesis that tethering might be involved in the release of exosomes of Phytophthora during infections.