Engineering Maize rayado fino virus for virus‐induced gene silencing

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 (VPI), and such 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‐PDS 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 viable constructs, carrying PDS inserts in the HEL/POL junction, 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 noninoculated leaves. These constructs were remarkably stable, retaining inserted sequences for at least four VPI passages while maintaining transmissibility by D. maidis. Our data thus identify the MRFV HEL/POL junction as an insertion site useful for gene silencing in maize.

Simultaneous gene expression and multi-gene silencing in Zea mays using maize dwarf mosaic virus

“…1.4 kb) is much greater than that reported for maize-infecting tools based on viruses with spherical virions, which are expected to have physical constraints on insertion size and carry silencing target sequences ca. 300 nt or smaller (BMV [ 20 , 21 ], CMV [ 13 ], MRFV [ 22 ]). pWX56 sequence cargo size is comparable or slightly smaller than those reported for other viruses that also have rod-shaped virions, which may increase capsid length corresponding with increased sequence insertion.…”

“…For insertion stability, pWX56 ranks among the most insert-stable virus-based tools for maize, such as the single protein expression tool based on the related virus SCMV [ 27 ], with insert retention and associated phenotypes observed systemically up to the last observation time points and topmost leaves, and up to three passages. However, its relative stability with progressive insert loss is not as impressive as the nearly complete insert retention over 60 days of the single-gene silencing tool based on MRFV [ 22 ]. Silencing efficacy of each of the target genes for pWX56, with gene expressions reduced to 39–46% of control expression levels, is within ranges expected for single-target VIGS BMV constructs (ca.…”

“…Silencing efficacy of each of the target genes for pWX56, with gene expressions reduced to 39–46% of control expression levels, is within ranges expected for single-target VIGS BMV constructs (ca. 30–60% of control expression) [ 21 ], but has lower apparent silencing penetrance than some single-target VIGS constructs (13–33% of control expression in silenced tissue for BSMV [ 23 ], and 16–33% of control expression for MRFV [ 22 ])--noting again that differences in methodology and conditions make such comparisons imprecise. Protein expression of GFP from pWX56 is also visually robust, but as no quantitative assessments were performed here beyond insertion and expression stability, we cannot compare protein expression quantity among available reported tools.…”

“…In one case, a plant virus vector derived from bean pod mottle virus (BPMV) was even developed for simultaneous single gene expression and single gene silencing [ 19 ]. For maize, several viruses have been developed as tools for gene expression or silencing, including brome mosaic virus (BMV) [ 20 , 21 ], cucumber mosaic virus (CMV) [ 13 ], and maize rayado fino virus (MRFV) [ 22 ] for gene silencing [ 23 ], wheat streak mosaic virus (WSMV) [ 24 – 26 ] and sugarcane mosaic virus [ 27 ] for protein expression, foxtail mosaic virus (FoMV) for gene silencing [ 28 ] or protein expression [ 29 ] and guide RNA delivery for CRISPR/Cas9 gene editing [ 30 ], barley stripe mosaic virus for gene silencing [ 23 ] or protein expression [ 31 ] or for guide RNA delivery [ 32 ] and barley yellow striate mosaic virus (BYSMV) for simultaneous guide RNA and high cargo-capacity protein expression [ 33 ].…”

Assessing the Functionality of RNA Interference (RNAi) in the Phloem-feeding Maize pestDalbulus maidis

“…At least seven vector-borne diseases are known in maize, five of which are viral and two bacterial (Carloni et al, 2013;Hammond & Bedendo, 2005;Liu & Wang, 2018;Meyer & Pataky, 2010;Orlovskis et al, 2017;Zhang et al, 2011). Dalbulus maidis is the insect vector responsible for transmitting three of the most important maize disease agents, namely Maize rayado fino virus (MRFV), Maize bushy stunt phytoplasma, and Spiroplasma kunkelii (CSS), which it can acquire and transmit together or individually (Galvão et al, 2020;Mlotshwa et al, 2020;Sabato et al, 2020). Pathogens vectored by D. maidis are transmitted in a persistent, propagative manner, and as such D. maidis remains a vector throughout its life cycle, which facilitates disease spread across maize fields (Canale et al, 2018;Luft Albarracin et al, 2009;Ramos et al, 2020).…”