Rescue of the first alphanucleorhabdovirus entirely from cloned complementary DNA: An efficient vector for systemic expression of foreign genes in maize and insect vectors

Abstract

Recent reverse genetics technologies have enabled genetic manipulation of plant negative‐strand RNA virus (NSR) genomes. Here, we report construction of an infectious clone for the maize‐infecting Alphanucleorhabdovirus maydis, the first efficient NSR vector for maize. The full‐length infectious clone was established using agrobacterium‐mediated delivery of full‐length maize mosaic virus (MMV) antigenomic RNA and the viral core proteins (nucleoprotein N, phosphoprotein P, and RNA‐directed RNA polymerase L) required for viral transcription and replication into Nicotiana benthamiana. Insertion of intron 2 ST‐LS1 into the viral L gene increased stability of the infectious clone in Escherichia coli and Agrobacterium tumefaciens. To monitor virus infection in vivo, a green fluorescent protein (GFP) gene was inserted in between the N and P gene junctions to generate recombinant MMV‐GFP. Complementary DNA (cDNA) clones of MMV‐wild type (WT) and MMV‐GFP replicated in single cells of agroinfiltrated N. benthamiana. Uniform systemic infection and high GFP expression were observed in maize inoculated with extracts of the infiltrated N. benthamiana leaves. Insect vectors supported virus infection when inoculated via feeding on infected maize or microinjection. Both MMV‐WT and MMV‐GFP were efficiently transmitted to maize by planthopper vectors. The GFP reporter gene was stable in the virus genome and expression remained high over three cycles of transmission in plants and insects. The MMV infectious clone will be a versatile tool for expression of proteins of interest in maize and cross‐kingdom studies of virus replication in plant and insect hosts.

Exportin 1 is required for the reproduction and maize mosaic virus accumulation in its insect vectorPeregrinus maidis

“…To quantify PmXPO1 knockdown efficiency at protein level, immunoblot analysis was performed according to Kanakala et al, 2023. For total protein extraction, three pooled insects were homogenized with a disposable pestle in a microcentrifuge tube containing 320 µl of 2x Laemmli Sample Buffer (BioRad) and 5% of 2-mercaptoethanol. The homogenate was incubated at 100 o C for 10 minutes and spun down for 5 minutes at 5,000 rpm in a benchtop microcentrifuge.…”

Leveraging insect viruses and genetic manipulation for sustainable agricultural pest control

“…Advances in nucleic acid delivery technology are gradually overcoming this limitation. Another strategy involves rescuing viruses in plants and then reintroducing the modified virus into insect hosts through injection 7,8 . Subsequent sections discuss the progress and challenges in developing reverse genetics systems for ISV viruses represented by Iflaviridae and Nodaviridae families, as well as insect‐borne viruses represented by Rhabdoviridae and Tospoviridae families.…”

“…This technology furnishes a robust platform for the creation and modification of insect viruses as Virus-Induced Gene Silencing (VIGS) or heterologous protein expression vectors, thereby augmenting their virulence or curbing the proliferation of insectborne viruses. [6][7][8] Moreover, it shows promise for adaptation as a gene-editing tool, facilitating precise genomic alterations in insects using techniques like Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR associated protein 9 (CRISPR/ Cas9), thus opening avenues for targeted genetic modifications of pest species and potent new strategies for pest management within agricultural ecosystems. 9,10 This paper embarks on an exhaustive exploration of methodologies for insect virus identification, with a specific focus on HTS technology.…”

Foxtail mosaic virus: A tool for gene function analysis in maize and other monocots

“…The viruses mentioned so far have single‐stranded, positive‐sense RNA genomes, and for these kinds of viruses the technology to produce infectious clones and manipulate them to accept the insertion of foreign sequences has been available since the 1980s (e.g., Ahlquist et al, 1984 ; French et al, 1986 ). Maize mosaic virus (MMV) and barley yellow striate mosaic virus (BYSMV) in contrast are negative (−)‐strand RNA viruses, and only recently has the ability to engineer infectious clones derived from them been demonstrated (Gao et al, 2019 ; Kanakala et al, 2022 ). These (−)‐strand RNA viruses are interesting because they have more stable insertions that are less susceptible to homologous recombination and spontaneous deletions, and they independently express multiple sequences, including ORFs, gene fragments, and guide RNA.…”