Potato Virus X (PVX) is the type member of Potexvirus genus, a group of plant viruses with a positive-strand RNA genome (~6.4 kb). PVX is able to establish compatible infections in Nicotiana benthamiana, a commonly used host in plant virology, leading to mild symptoms, such as chlorotic mosaic and mottling. PVX has been widely used as a viral vector for more than two decades (Chapman et al., 1992; Baulcombe et al., 1995; Aguilar et al., 2015). It provides a feasible means for the systemic expression in plants of heterologous proteins, such as avirulence factors, proteins with pharmacological properties, etc., (Hammond-Kosack et al., 1995; Gleba et al., 2014), and also as a tool to help decipher the function of genes in plants by virus-induced gene silencing (VIGS) (Lacomme and Chapman, 2008). Two different protocols, i.e., rubbing (A) and agroinfiltration (B), that allow efficient multiplication and propagation of PVX in N. benthamiana are described here in detail. The rubbing method requires previous infected sap, and infection is achieved by inducing mechanical damages to leaf tissue, allowing viral particles to penetrate the plant surface. Agroinfiltration needs previously modified agrobacterium to carry and deliver T-DNA with PVX sequences into the plant cell. Agrobacterium is grown until saturation and infection is established by infiltrating it into plant tissue with a syringe. Any of these two methods can be successfully applied, and the choice should be based mainly on the availability of material and time, but it is recommended to use agroinfiltration when chimeric viruses are being used.
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