Nicotiana benthamiana protein, NbPCIP1, interacting with Potato virus X coat protein plays a role as susceptible factor for viral infection.
The interactions of viral coat protein (CP) and host factors play an important role in viral replication and/or host defense mechanism. In this study, we constructed Nicotiana benthamiana cDNA library to find host factors interacting with Potato virus X (PVX) CP. Using yeast two-hybrid assay, we screened 3.3 x 10(6) independent yeast transformants from N. benthamiana cDNA library and identified six positive clones. One positive clone, named PVX CP-interacting protein 1 (NbPCIP1), is a plant-specific protein with homologue in N. tabacum (GenBank accession no. AB04049). We confirmed the PVX CP-NbPCIP1 interaction using yeast-two hybrid assay in yeast, protein-protein binding assay in vitro, and bimolecular fluorescent complementation assay in planta. Quantitative real-time RT-PCR analysis showed that the mRNA level of NbPCIP1 increased in PVX-infected N. benthamiana plants as compared to that of healthy plants. The green fluorescent protein (sGFP)-fused NbPCIP1 (NbPCIP1-sGFP) was localized in ER or ER-associated granular-like structure of cells. When we co-express NbPCIP1-sGFP and red fluorescent protein (RFP)-fused PVX CP (PVX CP-RFP), which were introduced by transiently expressing these proteins in N. benthamiana protoplasts and epidermal cells, however, we observed the co-localization of these proteins in the inclusion body-like complex in areas surrounding nucleus. Transient over-expression and transgene silencing of NbPCIP1 assay analysis indicated that NbPCIP1 plays a critical role in viral replication during PVX infection in host plant.
Park
Mi-Ri
MR
Department of Agricultural Biotechnology and Plant Genomics and Breeding Institute, Seoul National University, Seoul 151-921, Republic of Korea.
Park
Sang-Ho
SH
Cho
Sang-Yun
SY
Kim
Kook-Hyung
KH
D000595:Amino Acid Sequence; D036022:Capsid Proteins / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D015723:Gene Library; D020868:Gene Silencing; D017343:Genes, Plant; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000821:virology; D019441:Plant Epidermis / Q000378:metabolism / Q000821:virology; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism / Q000821:virology; D017863:Potexvirus / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D018749:RNA, Plant / Q000378:metabolism; D016415:Sequence Alignment; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism / Q000821:virology*; D020798:Two-Hybrid System Techniques
D036022:Capsid Proteins; D010940:Plant Proteins; D012333:RNA, Messenger; D018749:RNA, Plant; C058750:coat protein, Potato virus X; D049452:Green Fluorescent Proteins