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. 2020 Nov 4;11:571807. doi: 10.3389/fmicb.2020.571807

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Copyright © 2020 Leastro, Freitas-Astúa, Kitajima, Pallás and Sánchez-Navarro.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Membrane association analysis of dichorhavirus MP and N proteins. (A) Separation into membranous and soluble fraction of CiLV-N and OFV-citrus MP and N proteins expressed in planta. Respective proteins targeted with HA were expressed in Nicotiana benthamiana leaves by agroinfiltration. As controls, we used leaf protein extracts containing unfused expressed eGFP (non-membrane) and the HA-tagged Lep (leader peptidase) (integral membrane) proteins, respectively. The supernatant from ultracentrifugation after membrane fractioning (S30), and comparable pellet (P30), untreated and alkaline or urea (2M, 4M, and 8M) treatments were analyzed by Western blot method using an anti-NtGFP antibody (Sigma-Aldrich, Steinheim, Germany) or anti-HA antibody (Thermo Fisher Scientific, Waltham, MA, United States). Relative quantification values are presented. (B) Triton X-114 partitioning assay of CiLV-N and OFV-citrus MPs and Ns. The P30 fractions subjected to treatment with Triton X-114 were separated in aqueous (AP) and organic (OP) phases. Equivalent amounts of fractions were analyzed by Western blot, and the same controls mentioned above were used.