Metagenomic identification of novel viruses of maize and teosinte in North America.

BACKGROUND: Maize-infecting viruses are known to inflict significant agronomic yield loss throughout the world annually. Identification of known or novel causal agents of disease prior to outbreak is imperative to preserve food security via future crop protection efforts. Toward this goal, a large-scale metagenomic approach utilizing high throughput sequencing (HTS) was employed to identify novel viruses with the potential to contribute to yield loss of graminaceous species, particularly maize, in North America. RESULTS: Here we present four novel viruses discovered by HTS and individually validated by Sanger sequencing. Three of these viruses are RNA viruses belonging to either the Betaflexiviridae or Tombusviridae families. Additionally, a novel DNA virus belonging to the Geminiviridae family was discovered, the first Mastrevirus identified in North American maize. CONCLUSIONS: Metagenomic studies of crop and crop-related species such as this may be useful for the identification and surveillance of known and novel viral pathogens of crops. Monitoring related species may prove useful in identifying viruses capable of infecting crops due to overlapping insect vectors and viral host-range to protect food security.

Impacts of RNA Mobility Signals on Virus Induced Somatic and Germline Gene Editing.

Viral vectors are being engineered to deliver CRISPR/Cas9 components systemically in plants to induce somatic or heritable site-specific mutations. It is hypothesized that RNA mobility signals facilitate entry of viruses or single guide RNAs (sgRNAs) into the shoot apical meristem where germline mutations can occur. Our objective was to understand the impact of RNA mobility signals on virus-induced somatic and germline gene editing in Nicotiana benthamiana and Zea mays. Previously, we showed that foxtail mosaic virus (FoMV) expressing sgRNA induced somatic mutations in N. benthamiana and Z. mays expressing Cas9. Here, we fused RNA mobility signals to sgRNAs targeting the genes encoding either N. benthamiana phytoene desaturase (PDS) or Z. mays high affinity potassium transporter 1 (HKT1). Addition of Arabidopsis thaliana Flowering Locus T (AtFT) and A. thaliana tRNA-Isoleucine (AttRNAIle) did not improve FoMV-induced somatic editing, and neither were sufficient to facilitate germline mutations in N. benthamiana. Maize FT homologs, Centroradialus 16 (ZCN16) and ZCN19, as well as AttRNAIle were found to aid somatic editing in maize but did not enable sgRNAs delivered by FoMV to induce germline mutations. Additional viral guide RNA delivery systems were assessed for somatic and germline mutations in N. benthamiana with the intention of gaining a better understanding of the specificity of mobile signal-facilitated germline editing. Potato virus X (PVX), barley stripe mosaic virus (BSMV), and tobacco rattle virus (TRV) were included in this comparative study, and all three of these viruses delivering sgRNA were able to induce somatic and germline mutations. Unexpectedly, PVX, a potexvirus closely related to FoMV, expressing sgRNA alone induced biallelic edited progeny, indicating that mobility signals are dispensable in virus-induced germline editing. These results show that PVX, BSMV, and TRV expressing sgRNA all have an innate ability to induce mutations in the germline. Our results indicate that mobility signals alone may not be sufficient to enable virus-based delivery of sgRNAs using the viruses, FoMV, PVX, BSMV, and TRV into cell types that result in germline mutations.

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

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.

Direct Agroinoculation of Maize Seedlings by Injection with Recombinant Foxtail Mosaic Virus and Sugarcane Mosaic Virus Infectious Clones.

Agrobacterium-based inoculation approaches are widely used for introducing viral vectors into plant tissues. This study details a protocol for the injection of maize seedlings near meristematic tissue with Agrobacterium carrying a viral vector. Recombinant foxtail mosaic virus (FoMV) clones engineered for gene silencing and gene expression were used to optimize this method, and its use was expanded to include a recombinant sugarcane mosaic virus (SCMV) engineered for gene expression. Gene fragments or coding sequences of interest are inserted into a modified, infectious viral genome that has been cloned into the binary T-DNA plasmid vector pCAMBIA1380. The resulting plasmid constructs are transformed into Agrobacterium tumefaciens strain GV3101. Maize seedlings as young as 4 days old can be injected near the coleoptilar node with bacteria resuspended in MgSO4 solution. During infection with Agrobacterium, the T-DNA carrying the viral genome is transferred to maize cells, allowing for the transcription of the viral RNA genome. As the recombinant virus replicates and systemically spreads throughout the plant, viral symptoms and phenotypic changes resulting from the silencing of the target genes lesion mimic 22 (les22) or phytoene desaturase (pds) can be observed on the leaves, or expression of green fluorescent protein (GFP) can be detected upon illumination with UV light or fluorescence microscopy. To detect the virus and assess the integrity of the insert simultaneously, RNA is extracted from the leaves of the injected plant and RT-PCR is conducted using primers flanking the multiple cloning site (MCS) carrying the inserted sequence. This protocol has been used effectively in several maize genotypes and can readily be expanded to other viral vectors, thereby offering an accessible tool for viral vector introduction in maize.

Functions of maize genes encoding pyruvate phosphate dikinase in developing endosperm.

Maize opaque2 (o2) mutations are beneficial for endosperm nutritional quality but cause negative pleiotropic effects for reasons that are not fully understood. Direct targets of the bZIP transcriptional regulator encoded by o2 include pdk1 and pdk2 that specify pyruvate phosphate dikinase (PPDK). This enzyme reversibly converts AMP, pyrophosphate, and phosphoenolpyruvate to ATP, orthophosphate, and pyruvate and provides diverse functions in plants. This study addressed PPDK function in maize starchy endosperm where it is highly abundant during grain fill. pdk1 and pdk2 were inactivated individually by transposon insertions, and both genes were simultaneously targeted by endosperm-specific RNAi. pdk2 accounts for the large majority of endosperm PPDK, whereas pdk1 specifies the abundant mesophyll form. The pdk1- mutation is seedling-lethal, indicating that C4 photosynthesis is essential in maize. RNAi expression in transgenic endosperm eliminated detectable PPDK protein and enzyme activity. Transgenic kernels weighed the same on average as nontransgenic siblings, with normal endosperm starch and total N contents, indicating that PPDK is not required for net storage compound synthesis. An opaque phenotype resulted from complete PPDK knockout, including loss of vitreous endosperm character similar to the phenotype conditioned by o2-. Concentrations of multiple glycolytic intermediates were elevated in transgenic endosperm, energy charge was altered, and starch granules were more numerous but smaller on average than normal. The data indicate that PPDK modulates endosperm metabolism, potentially through reversible adjustments to energy charge, and reveal that o2- mutations can affect the opaque phenotype through regulation of PPDK in addition to their previously demonstrated effects on storage protein gene expression.

Functions of heteromeric and homomeric isoamylase-type starch-debranching enzymes in developing maize endosperm.

Functions of isoamylase-type starch-debranching enzyme (ISA) proteins and complexes in maize (Zea mays) endosperm were characterized. Wild-type endosperm contained three high molecular mass ISA complexes resolved by gel permeation chromatography and native-polyacrylamide gel electrophoresis. Two complexes of approximately 400 kD contained both ISA1 and ISA2, and an approximately 300-kD complex contained ISA1 but not ISA2. Novel mutations of sugary1 (su1) and isa2, coding for ISA1 and ISA2, respectively, were used to develop one maize line with ISA1 homomer but lacking heteromeric ISA and a second line with one form of ISA1/ISA2 heteromer but no homomeric enzyme. The mutations were su1-P, which caused an amino acid substitution in ISA1, and isa2-339, which was caused by transposon insertion and conditioned loss of ISA2. In agreement with the protein compositions, all three ISA complexes were missing in an ISA1-null line, whereas only the two higher molecular mass forms were absent in the ISA2-null line. Both su1-P and isa2-339 conditioned near-normal starch characteristics, in contrast to ISA-null lines, indicating that either homomeric or heteromeric ISA is competent for starch biosynthesis. The homomer-only line had smaller, more numerous granules. Thus, a function of heteromeric ISA not compensated for by homomeric enzyme affects granule initiation or growth, which may explain evolutionary selection for ISA2. ISA1 was required for the accumulation of ISA2, which is regulated posttranscriptionally. Quantitative polymerase chain reaction showed that the ISA1 transcript level was elevated in tissues where starch is synthesized and low during starch degradation, whereas ISA2 transcript was relatively abundant during periods of either starch biosynthesis or catabolism.