A sugarcane mosaic virus vector for rapid in planta screening of proteins that inhibit the growth of insect herbivores

Abstract

Summary Spodoptera frugiperda (fall armyworm) is a notorious pest that threatens maize production worldwide. Current control measures involve the use of chemical insecticides and transgenic maize expressing Bacillus thuringiensis (Bt) toxins. Although additional transgenes have confirmed insecticidal activity, limited research has been conducted in maize, at least partially due to the technical difficulty of maize transformation. Here, we describe implementation of a sugarcane mosaic virus (SCMV) vector for rapidly testing the efficacy of both endogenous maize genes and heterologous genes from other organisms for the control of S. frugiperda in maize. Four categories of proteins were tested using the SCMV vector: (i) maize defence signalling proteins: peptide elicitors (Pep1 and Pep3) and jasmonate acid conjugating enzymes (JAR1a and JAR1b); (ii) maize defensive proteins: the previously identified ribosome‐inactivating protein (RIP2) and maize proteinase inhibitor (MPI), and two proteins with predicted but unconfirmed anti‐insect activities, an antimicrobial peptide (AMP) and a lectin (JAC1); (iii) lectins from other plant species: Allium cepa agglutinin (ACA) and Galanthus nivalis agglutinin (GNA); and (iv) scorpion and spider toxins: peptides from Urodacus yaschenkoi (UyCT3 and UyCT5) and Hadronyche versuta (Hvt). In most cases, S. frugiperda larval growth was reduced by transient SCMV‐mediated overexpression of genes encoding these proteins. Additionally, experiments with a subset of the SCMV‐expressed genes showed effectiveness against two aphid species, Rhopalosiphum maidis (corn leaf aphid) and Myzus persicae (green peach aphid). Together, these results demonstrate that SCMV vectors are a rapid screening method for testing the efficacy and insecticidal activity of candidate genes in maize.

Use of sugarcane mosaic virus for virus-induced gene silencing in maize

“…Both genes were expressed at a higher level in ZmCYP94B1-silenced plants in response to S. frugiperda feeding than in the corresponding SCMV-GFP controls (Figure 6B,C), indicating that defense induction is enhanced. Spodoptera frugiperda (Figure 6D), S. americana (Figure 6E), and R. maidis (Figure 6F) grew less well on ZmCYP94B1-silenced maize than on plants infected with SCMV-GFP, consistent with previous reports showing that MPI and RIP2 inhibit insect growth (Vila et al, 2005; Chuang et al, 2014; Quilis et al, 2014; Chung et al, 2021). However, it is likely that, in addition to these two proteins, other jasmonate-regulated maize defenses are increased in response to ZmCYP94B1 expression silencing.…”

“…A 363 bp fragment for simultaneous silencing of CYP94B1 and JIH1 (Table S2) was synthesized by Twist Bioscience (www.twistbioscience.com). The pSCMV-CS3 vector (Chung et al, 2021), which expresses full-length SCMV RNA from the cauliflower mosaic virus 35S promoter, was cut with the restriction enzymes PspOMI and PmeI (New England Biolabs, www.neb.com), the amplified gene fragments were cloned into the cut site such that they were in-frame with the viral RNA, and the constructs were transformed into Escherichia coli Top10 competent cells (www.thermofisher.com). A fragment of GFP (Table S2) was cloned into SCMV-CS3 as a control For biolistic plant transformation (as described by Chung et al, 2021), plasmid DNA carrying the SCMV constructs was coated onto 3 mg 1.0 µm diameter gold particles.…”

“…Raw data underlying the graphs in Figure 8 Table S7. Raw data underlying the graphs in Figure 9 3 Results and Discussion We used the previously described SCMV-CS3 vector (Mohr, 2019;Beernink et al, 2021;Chung et al, 2021) to determine whether SCMV can be used for gene expression silencing in maize. As SCMV is translated as a single polycistronic protein prior to being cleaved by virus-encoded proteases (Mei et al, 2019), sense gene fragments must be cloned such that they are in-frame with the viral coding sequence.…”

“…An SCMV infectious clone was engineered as a vector for transient gene overexpression in maize (Mei et al, 2019). We have used a version of this vector, SCMV-CS3 (Mohr, 2019; Beernink et al, 2021), which allows cloning of transgenes between the SCMV P1 and HC-Pro cistrons, to enhance maize pest tolerance by overexpressing endogenous maize resistance genes, spider and scorpion toxins, and lectins from other plant species (Chung et al, 2021). Additionally, we used SCMV-VIGS for targeted reduction of gene expression in Rhopalosiphum maidis (corn leaf aphids) feeding on maize (Chung and Jander, 2022).…”

“…Expression of JAR1a and JAR1b , two of the five maize genes predicted to encode JA-Ile conjugating enzymes (Borrego and Kolomiets, 2016), was strongly induced by caterpillar feeding (Tzin et al, 2017). Transient overexpression of these genes in maize using SCMV caused reduced growth of Spodoptera frugiperda (fall armyworm) larvae (Chung et al, 2021). Conversely, RNA interference targeting JIH1 in N. attenuata made these plants more resistant to both Manduca sexta (tobacco hornworm) and Spodoptera littoralis (Egyptian cotton leafworms) (Woldemariam et al, 2012), and silencing expression of N. attenuata JA hydroxylases increased resistance to Spodoptera litura (Tang et al, 2020).…”