Expression of prune dwarf Ilarvirus coat protein sequences in Nicotiana benthamiana plants interferes with PDV systemic proliferation

Abstract

Prune dwarf virus (PDV) is an Ilarvirus systemically infecting almond trees and other Prunus species and spreading through pollen, among other means. We have studied strategies based on coat protein (cp) gene to block PDV replication in host plant cells. A Portuguese isolate of PDV was obtained from infected almond leaves and used to produce the cDNA of the cp gene. Various constructs were prepared based on this sequence, aiming for the transgenic expression of the original or modified PDV coat protein (cpPDVSense and cpPDVMutated) or for the expression of cpPDV RNA (cpPDVAntisense and cpPDVwithout start codon). All constructs were tested in a PDV host model, Nicotiana benthamiana, and extensive molecular characterization and controlled infections were performed on transformants and their progenies. Transgenic plants expressing the coat protein RNA were able to block the proliferation of a PDV isolate sharing only 91% homology with the isolate used for cpPDV cloning, as evaluated by DAS-ELISA on newly developed leaves. With cp expression, the blockage of PDV proliferation in newly developed leaves was only achieved with the construct cpPDV Mutated, where the coat protein has a substitution in the 14th aa residue, with arginine replaced by alanine. This result points to a possible role of the mutated amino acid in the virus ability to replicate and proliferate. This work reveals the possibility of achieving protection against PDV through either coat protein RNA or mutated cp sequence.

Stable Plastid Transformation in Nicotiana benthamiana

“…It is a close relative of cultivated tobacco (Nicotiana tabacum) and a member of the Solanaceae that includes the important crops of potatoes and tomatoes. This unique Australian native is the most popular experimental host for evaluating plant virology mainly because of its susceptibility to diverse viruses (Raquel et al 2008). N. benthamiana is the best known model plant for virus-induced gene silencing (VIGS), a tool for functional analyses of genes and studies of plant disease (Baulcombe 1999).…”

Transgenic Resistance

“…Nontranslatable forms of the capsid protein gene or 25-kDa movement protein gene of PVX expressed in transgenic tobacco plants showed excellent resistance in some lines to PVX infection, with more lines expressing nontranslatable movement protein gene (39.7%) than capsid protein gene (13.3%) showing resistance (Liu, Zhang, et al, 2005). Transgenic N. benthamiana expressing nontranslatable sequences of Prune dwarf virus (PDV) capsid protein gene showed only a recovery form of resistance in one line (Raquel et al, 2008). Transgenic rice expressing a nontranslatable form of the Rice yellow mottle virus (RYMV) capsid protein gene showed only a delay in virus accumulation (Kouassi et al, 2006).…”

“…Expression of antisense RNA to the 505-nt 3 0 -NTR of APMoV in transgenic tobacco plants resulted in segregating susceptibility, resistance, or partial protection in several lines (Vaskin et al, 2001). Transgenic N. benthamiana expressing antisense RNA to capsid protein gene of PDV showed either resistance or a recovery form of resistance (Raquel et al, 2008). Transgenic rice expressing antisense RNA of the RYMV capsid protein gene showed only a delay in virus accumulation (Kouassi et al, 2006).…”

CRISPR/Cas9‐mediated knockout of a prolyl‐4‐hydroxylase subfamily in Nicotiana benthamiana using DsRed2 for plant selection

“…This species has been used extensively as a model for the functional analysis of plant genes and interactions with pathogens because it has a short lifecycle and carries a mutation that makes it hyper susceptible to many viruses. [16] This prompted its development as an expression host for viral vectors, and more recently it has emerged as the key industrial platform for transient expression, particularly for the production of antibodies [12,17,18] and vaccine candidates. [19] For example, N. benthamiana was used to manufacture ZMapp, which is a cocktail of three IgG antibodies for the treatment of Ebola virus infections, [20] as well as clinical vaccine candidates against hepatitis B virus, [21] influenza virus, [22] HIV [23] and, most recently, SARS-CoV-2.…”