Engineering partial resistance to cucumber mosaic virus in tobacco using intrabodies specific for the viral polymerase. (June 2019)

• Record Type: Journal Article
• Authors: Matić, Slavica; Noris, Emanuela; Contin, Roberta; Marian, Daniele; Thompson, Jeremy R.
• Is Part Of: Phytochemistry. Volume 162(2019)
• Page Start: 99
• Page End: 108
• Publication Date: 2019-06
• Subjects: Nicotiana tabacum -- Solanaceae -- Tobacco -- Cucumber mosaic virus (CMV) -- Yeast two-hybrid system -- Transgenic plants -- Virus resistance -- Intrabodies -- scFv -- RNA-Dependent RNA polymerase (RdRp); Botanical chemistry -- Periodicals; Biochemistry -- Periodicals; Botany -- Periodicals; Chimie végétale -- Périodiques; 572.2
• DOI: 10.1016/j.phytochem.2019.03.006
• Languages: English
• ISSNs: 0031-9422

Abstract

Abstract: A single-chain variable antibody fragment (scFv) library tested against the non-structural NSP5 protein of human rotavirus A was screened by a yeast two-hybrid system against three proteins derived from the RNA-dependent RNA polymerase (RdRp) of cucumber mosaic virus (CMV), with the aim of blocking their function and preventing viral infection once expressed in planta . The constructs tested were (i) '2a' consisting of the full-length 2a gene (839 amino acids, aa), (ii) 'Motifs' covering the conserved RdRp motifs (IV-VII) (132 aa) and (iii) 'GDD' located within the conserved RdRp motif VI (GDD, 22 aa). In yeast two-hybrid (Y2H) selection assays the '2a' and 'Motifs' constructs interacted with 96 and 25 library constructs, respectively, while the 'GDD' construct caused transactivation. Y2H-interacting scFvs were analyzed in vivo for their interaction with the 2a and Motifs proteins in a mammalian transient expression system. Eighteen tobacco lines stably transformed with four selected scFvs were produced and screened for resistance against two different CMV isolates. Different levels of resistance and rate of recovery were observed with CMV of both groups I and II, particularly in lines expressing intrabodies against the full-length 2a protein. This work describes for the first time the use of intrabodies against the RdRp of CMV to obtain plants that reduce infection of a pandemic virus, showing that the selected scFvs can modulate virus infection and induce premature recovery in tobacco plants. Graphical abstract: Image 1 Highlights: A scFv library previously screened in a Y2H system against a human rotavirus was tested against CMV 2a protein derivatives. Positive scFvs were analyzed for their interaction with CMV 2a protein derivatives in mammalian transient expression system. Tobacco plants transformed with four selected scFvs had reduced levels of CMV and showed premature recovery from infection.