Engineering maize rayado fino virus for virus-induced gene silencing

Authors: MLOTSHWA, SIZOLWENKOSI - The Ohio State University; XU, JUNHUAN - The Ohio State University; Willie, Kristen; KHATRI, NITIKA - The Ohio State University; Marty, Deemarie - Dee; Stewart, Lucy

Submitted to: Plant Direct
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/14/2020
Publication Date: 8/1/2020
Citation: Mlotshwa, S., Xu, J., Willie, K.J., Khatri, N., Marty, D., Stewart, L.R. 2020. Engineering maize rayado fino virus for virus-induced gene silencing. Plant Direct. 4(8). Article e00224. https://doi.org/10.1002/pld3.224.
DOI: https://doi.org/10.1002/pld3.224

Interpretive Summary: Viruses can be developed as tools to better understand the functions of plant genes by selectively reducing expression of target genes and testing phenotypes in research. This approach, virus-induced gene silencing (VIGS) is powerful for identification of gene function in plants, but few such virus tools with this capability are available for important grass crops such as corn. The viruses that currently have been adapted to examine plant gene function using VIGS are disadvantaged by insert instability (loss of plant-gene targeting sequences by the virus) that limit gene silencing in plants as well as the length of time in which plant phenotypes due to VIGS can be assessed. Using maize rayado fino virus (MRFV), we demonstrate development of a virus-derived tool that bypasses these challenges with unprecedented stability of insertion sequences over up to months post-inoculation. The impact of this research is a vastly improved high-throughput tool to assess corn gene functions using VIGS.

Technical Abstract: Maize rayado fino virus (MRFV) is the type species of the genus Marafivirus in the family Tymoviridae. It infects maize (Zea mays), its natural host, to which it is transmitted by leafhoppers including Dalbulus maidis and Graminella nigrifrons in a persistent-propagative manner. The MRFV monopartite RNA genome encodes a precursor polyprotein that is processed into replication-associated proteins. The genome is encapsidated by two carboxy co-terminal coat proteins, CP1 and CP2. Cloned MRFV can be readily transmitted to maize by vascular puncture inoculation, and virus systems that can be used in maize are valuable to examine plant gene function by gene silencing. However, the efficacy of marafiviruses for virus-induced gene silencing (VIGS) has not been investigated to date. To this end, MRFV genomic loci were tested for their potential to host foreign insertions without attenuating virus viability. This was done using infectious MRFV clones engineered to carry maize phytoene desaturase (PDS) gene fragments (ZmPDS) at various genomic regions. Several MRFV constructs were generated and tested for infectivity and VIGS in maize. This culminated in identification of the helicase/polymerase (HEL/POL) junction as a viable insertion site that preserved virus infectivity, as well as several sites at which sequence insertion caused loss of virus infectivity. Transcripts of the viable constructs induced stable local and systemic MRFV symptoms similar to wild type infections, and triggered PDS VIGS initiating in veins and spreading into both inoculated and uninoculated leaves. The constructs were remarkably stable, retaining inserted sequences for at least four vascular puncture inoculation (VPI) passages while maintaining transmissibility by both D. maidis and N. nigrifrons. Our data thus identify the MRFV HEL/POL junction as an insertion site useful for gene silencing in maize. This new tool will benefit researchers seeking improved approaches to study the functions of maize genes over an extended time.