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Since its discovery in 2016, the Polerovirus Barley virus G has been reported in at least nine countries and multiple species of monocot plants. All of these reports have used PCR and/or sequencing based assays to identify BVG, however none have investigated the biology of BVG. In this study we detail the generation of the first infectious cDNA clone of BVG from archived RNA, thereby producing a valuable experimental tool and system for studying BVG biology. Using this system we identified two compatible aphid vectors and confirmed the susceptibility of several monocot plants, and the dicotyledonous plant host Nicotiana benthamiana, to BVG.

The Asian citrus psyllid, Diaphorina citri, is the insect vector of the causal agent of huanglongbing (HLB), a devastating bacterial disease of commercial citrus. Presently, few genomic resources exist for D. citri. In this study, we utilized PacBio HiFi and chromatin confirmation contact (Hi-C) sequencing to sequence, assemble, and compare three high-quality, chromosome-scale genome assemblies of D. citri collected from California, Taiwan, and Uruguay. Our assemblies had final sizes of 282.67 Mb (California), 282.89 Mb (Taiwan), and 266.67 Mb (Uruguay) assembled into 13 pseudomolecules-a reduction in assembly size of 41-45% compared with previous assemblies which we validated using flow cytometry. We identified the X chromosome in D. citri and annotated each assembly for repetitive elements, protein-coding genes, transfer RNAs, ribosomal RNAs, piwi-interacting RNA clusters, and endogenous viral elements. Between 19,083 and 20,357 protein-coding genes were predicted. Repetitive DNA accounts for 36.87-38.26% of each assembly. Comparative analyses and mitochondrial haplotype networks suggest that Taiwan and Uruguay D. citri are more closely related, while California D. citri are closely related to Florida D. citri. These high-quality, chromosome-scale assemblies provide new genomic resources to researchers to further D. citri and HLB research.

Piwi-interacting RNAs (piRNAs) are a class of small RNAs primarily responsible for silencing transposons in the animal germ line. The ping-pong cycle, the posttranscriptional silencing branch of the piRNA pathway, relies on piRNAs produced from endogenous transposon remnants to direct cleavage of transposon RNA via association with Piwi-family Argonaute proteins. In some mosquito species and mosquito-derived cell lines expressing a functionally expanded group of Piwi-family Argonaute proteins, both RNA and DNA viruses are targeted by piRNAs in a manner thought to involve direct processing of exogenous viral RNA into piRNAs. Whether viruses are targeted by piRNAs in nonmosquito species is unknown. Partial integrations of DNA and nonretroviral RNA virus genomes, termed endogenous viral elements (EVEs), are abundant in arthropod genomes and often produce piRNAs that are speculated to target cognate viruses through the ping-pong cycle. Here, we describe a Diaphorina citri densovirus (DcDV)-derived EVE in the genome of Diaphorina citri We found that this EVE gives rise to DcDV-specific primary piRNAs and is unevenly distributed among D. citri populations. Unexpectedly, we found that DcDV is targeted by ping-pong-dependent virus-derived piRNAs (vpiRNAs) in D. citri lacking the DcDV-derived EVE, while four naturally infecting RNA viruses of D. citri are not targeted by vpiRNAs. Furthermore, a recombinant Cricket paralysis virus containing a portion of the DcDV genome corresponding to the DcDV-derived EVE was not targeted by vpiRNAs during infection in D. citri harboring the EVE. These results demonstrate that viruses can be targeted by piRNAs in a nonmosquito species independently of endogenous piRNAs.IMPORTANCE Small RNAs serve as specificity determinants of antiviral responses in insects. Piwi-interacting RNAs (piRNAs) are a class of small RNAs found in animals, and their primary role is to direct antitransposon responses. These responses require endogenous piRNAs complementary to transposon RNA. Additionally, piRNAs have been shown to target RNA and DNA viruses in some mosquito species. In contrast to transposons, targeting of viruses by the piRNA pathway in these mosquito species does not require endogenous piRNAs. Here, we show that piRNAs target a DNA virus, but not RNA viruses, in an agricultural insect pest. We found that targeting of this DNA virus did not require endogenous piRNAs and that endogenous piRNAs did not mediate targeting of an RNA virus with which they shared complementary sequence. Our results highlight differences between mosquitoes and our experimental system and raise the possibility that DNA viruses may be targeted by piRNAs in other species.

Plant viruses are emerging and re-emerging to cause important diseases in many plants that humans grow for food and/or fiber, and sustainable, effective strategies for controlling many plant virus diseases remain unavailable [...].

Diaphorina citri reovirus (DcRV) was previously identified based on metagenomics surveys for virus discovery. Here, we demonstrated that DcRV induces persistent infection in its psyllid host, Diaphorina citri DcRV was efficiently vertically passed to offspring in a biparental manner. Transmission electron microscopic and immunological analyses showed that the DcRV-encoded nonstructural protein P10 assembled into a virion-packaging tubular structure which is associated with the spread of DcRV throughout the bodies of D. citri insects. P10 tubules containing virions were associated with oocytes of female and sperm of male D. citri insects, suggesting a role in the highly efficient biparental transmission of DcRV. Knocking down P10 by RNA interference for males reduced the percentage of DcRV-infected progeny and for females reduced the viral accumulation in progeny. These results, for the first time, show that a nonstructural protein of a novel insect reovirus provides a safe and pivotal channel for virus spread and biparental transmission to progeny.IMPORTANCE The Asian citrus psyllid, Diaphorina citri Kuwayama, is an important pest in the worldwide citrus industry. It is the vector of "Candidatus Liberibacter asiaticus," the bacterial pathogen of Huanglongbing, which is currently considered the most destructive disease of citrus worldwide. DcRV was previously identified based on metagenomics surveys for virus discovery. Here, we found that this novel and persistent insect reovirus took advantage of a virus-encoded nonstructural protein, P10, for efficient vertical transmission from parents to progeny. P10 assembled into a virion-packaging tubular structure and was associated with oocytes of female D. citri and sperm of males. Consistent with this, knockdown of P10 for either male or female D. citri insects inhibited DcRV transmission to offspring. This tubular strategy for viral spread and biparental transmission might serve as a target for controlling viral vertical transmission and population expansion.

Citrus sudden death-associated virus (CSDaV) is a member of the genus Marafivirus in the family Tymoviridae, and has been associated with citrus sudden death (CSD) disease in Brazil. Difficulties in the purification of CSDaV from infected citrus plants have prevented progress in the investigation of the role of this virus in CSD and an understanding of its molecular biology. In this work, we have constructed a full-length cDNA clone of CSDaV driven by the 35S promoter (35SRbz-CSDaV). Agrobacterium tumefaciens-mediated inoculation of 35SRbz-CSDaV in Nicotiana benthamiana plants enabled a fast recovery of large amounts of virions from the agroinfiltrated leaves, which allowed a better molecular characterization of CSDaV. In vivo analyses of mutant versions of 35SRbz-CSDaV revealed the expression strategies used by CSDaV for production of the capsid proteins (CPs). We showed that CSDaV virions contain three forms of CP, each of which is generated from the same coding sequence, but by different mechanisms. The major CPp21 is a product of direct translation by leaky scanning from the second start codon in the subgenomic RNA (sgRNA), whereas the minor CPs, p25 and p23, are produced by direct translation from the first start codon in the sgRNA and by trans-proteolytic cleavage processing derived from the p25 precursor, respectively. Together, these findings contribute to advance our understanding of CSDaV genome expression strategies. In addition, the construction and characterization of the CSDaV infectious clone represent important steps towards the investigation of the role of this virus in CSD and of its use as a tool for citrus biotechnology.

Two novel negative-stranded (ns)RNA viruses were identified by high throughput sequencing in grapevine. The genomes of both viruses, named grapevine Muscat rose virus (GMRV) and grapevine Garan dmak virus (GGDV), comprise three segments with each containing a unique gene. Based on sequence identity and presence of typical domains/motifs, the proteins encoded by the two viruses were predicted to be: RNA-dependent RNA polymerase (RdRp), nucleocapsid protein (NP), and putative movement protein (MP). These proteins showed the highest identities with orthologs in the recently discovered apple rubbery wood viruses 1 and 2, members of a tentative genus (Rubodvirus) within the family Phenuiviridae. The three segments of GMRV and GGDV share almost identical sequences at their 5' and 3' termini, which are also complementary to each other and may form a panhandle structure. Phylogenetics based on RdRp, NP and MP placed GMRV and GGDV in the same cluster with rubodviruses. Grapevine collections were screened for the presence of both novel viruses via RT-PCR, identifying infected plants. GMRV and GGDV were successfully graft-transmitted, thus, they are the first nsRNA viruses identified and transmitted in grapevine. Lastly, different evolutionary scenarios of nsRNA viruses are discussed.

Plant-infecting viruses utilize various strategies involving multiple viral and host factors to achieve successful systemic infections of their compatible hosts. Lettuce infectious yellows virus (LIYV), genus Crinivirus, family Closteroviridae, has long, filamentous flexuous virions and causes phloem-limited infections in its plant hosts. The LIYV-encoded P26 is a distinct non-virion protein that shows no similarities to proteins in current databases: it induces plasmalemma deposits over plasmadesmata (PD) pit fields and is speculated to have roles in LIYV virion transport within infected plants. In this study, P26 was demonstrated to be a PD-localized protein, and its biological significance was tested in planta by mutagenesis analysis. An LIYV P26 knockout mutant (P26X) showed viral RNA replication and virion formation in inoculated leaves of Nicotiana benthamiana plants, but failed to give systemic infection. Confirmation by using a modified green fluorescent protein (GFP)-tagged LIYV P26X showed GFP accumulation only in infiltrated leaf tissues, while wild-type LIYV GFP readily spread systemically in the phloem. Attempts to rescue P26X by complementation in trans were negative. However a translocated LIYV P26 gene in the LIYV genome rescued systemic infection, but P26 orthologs from other criniviruses did not. Mutagenesis in planta assays showed that deletions in P26, as well as 2 of 11 specific alanine-scanning mutants, abolished the ability to systemically infect N. benthamianaIMPORTANCE Plant viruses encode specific proteins that facilitate their ability to establish multicellular/systemic infections in their host plants. Relatively little is known of the transport mechanisms for plant viruses whose infections are phloem limited, including those of the family Closteroviridae. These viruses have complex, long filamentous virions that spread through the phloem. Lettuce infectious yellows virus (LIYV) encodes a non-virion protein, P26, which forms plasmalemma deposits over plasmodesmata pit fields, and LIYV virions are consistently found attached to those deposits. Here we demonstrate that P26 is a unique movement protein required for LIYV systemic infection in plants. LIYV P26 shows no sequence similarities to other proteins, but other criniviruses encode P26 orthologs. However, these failed to complement movement of LIYV P26 mutants.

RNA silencing is a conserved antiviral defence mechanism that has been used to develop robust resistance against plant virus infections. Previous efforts have been made to develop RNA silencing-mediated resistance to criniviruses, yet none have given immunity. In this study, transgenic Nicotiana benthamiana plants harbouring a hairpin construct of the Lettuce infectious yellows virus (LIYV) RNA-dependent RNA polymerase (RdRp) sequence exhibited immunity to systemic LIYV infection. Deep sequencing analysis was performed to characterize virus-derived small interfering RNAs (vsiRNAs) generated on systemic LIYV infection in non-transgenic N. benthamiana plants as well as transgene-derived siRNAs (t-siRNAs) derived from the immune-transgenic plants before and after LIYV inoculation. Interestingly, a similar sequence distribution pattern was obtained with t-siRNAs and vsiRNAs mapped to the transgene region in both immune and susceptible plants, except for a significant increase in t-siRNAs of 24 nucleotides in length, which was consistent with small RNA northern blot results that showed the abundance of t-siRNAs of 21, 22 and 24 nucleotides in length. The accumulated 24-nucleotide sequences have not yet been reported in transgenic plants partially resistant to criniviruses, and thus may indicate their correlation with crinivirus immunity. To further test this hypothesis, we developed transgenic melon (Cucumis melo) plants immune to systemic infection of another crinivirus, Cucurbit yellow stunting disorder virus (CYSDV). As predicted, the accumulation of 24-nucleotide t-siRNAs was detected in transgenic melon plants by northern blot. Together with our findings and previous studies on crinivirus resistance, we propose that the accumulation of 24-nucleotide t-siRNAs is associated with crinivirus immunity in transgenic plants.

Genomic analysis of Lettuce infectious yellows virus (LIYV) has revealed two short open reading frames (ORFs) on LIYV RNA2, that are predicted to encode a 5-kDa (P5) and a 9-kDa (P9) protein. The P5 ORF is part of the conserved quintuple gene block in the family Closteroviridae, while P9 orthologs are found in all Criniviruses. In this study, the expression of LIYV P5 and P9 proteins was confirmed; P5 is further characterized as an endoplasmic reticulum (ER)-localized integral transmembrane protein and P9 is a soluble protein. The knockout LIYV mutants presented reduced symptom severity and virus accumulation in Nicotiana benthamiana or lettuce plants, indicating their importance in efficient virus infection. The P5 mutant was successfully complemented by a dislocated P5 in the LIYV genome. The structural regions of P5 were tested and all were found to be required for the appropriate functions of P5. In addition, P5, as well as its ortholog P6, encoded by Citrus tristeza virus (CTV) and another ER-localized protein encoded by LIYV RNA1, were found to cause cell death when expressed in N. benthamiana plants from a TMV vector, and induce ER stress and the unfolded protein response (UPR).

We have previously demonstrated that the inducible plant viral vector (CMViva) in transgenic plant cell cultures can significantly improve the productivity of extracellular functional recombinant human alpha-1-antiryspin (rAAT) compared with either a common plant constitutive promoter (Cauliflower mosaic virus (CaMV) 35S) or a chemically inducible promoter (estrogen receptor-based XVE) system. For a transgenic plant host system, however, viral or transgene-induced post-transcriptional gene silencing (PTGS) has been identified as a host response mechanism that may dramatically reduce the expression of a foreign gene. Previous studies have suggested that viral gene silencing suppressors encoded by a virus can block or interfere with the pathways of transgene-induced PTGS in plant cells. In this study, the capability of nine different viral gene silencing suppressors were evaluated for improving the production of rAAT protein in transgenic plant cell cultures (CMViva, XVE or 35S system) using an Agrobacterium-mediated transient expression co-cultivation process in which transgenic plant cells and recombinant Agrobacterium carrying the viral gene silencing suppressor were grown together in suspension cultures. Through the co-cultivation process, the impacts of gene silencing suppressors on the rAAT production were elucidated, and promising gene silencing suppressors were identified. Furthermore, the combinations of gene silencing suppressors were optimized using design of experiments methodology. The results have shown that in transgenic CMViva cell cultures, the functional rAAT as a percentage of total soluble protein is increased 5.7 fold with the expression of P19, and 17.2 fold with the co-expression of CP, P19 and P24.

Plant virus-based vectors are valuable tools for recombinant gene expression and functional genomics for both basic and applied research. In this study, Lettuce infectious yellows virus (LIYV) of the genus Crinivirus was engineered into a virus vector that is applicable for efficient protein expression and virus-induced gene silencing (VIGS) in plants. We examined gene replacement and “add a gene” strategies to develop LIYV-derived vectors for transient expression of the green fluorescent protein (GFP) reporter in Nicotiana benthamiana plants. The latter yielded higher GFP expression and was further examined by testing the effects of heterologous controller elements (CEs). A series of five vector constructs with progressively extended LIYV CP sgRNA CEs were tested, the longest CE gave the highest GFP expression but lower virus accumulation. The whitefly transmissibility of the optimized vector construct to other host plants, and the capability to accommodate and express a larger gene, a 1.8 kb β-glucuronidase (GUS) gene, were confirmed. Furthermore, the LIYV vector was also validated VIGS by silencing the endogenous gene, phytoene desaturase (PDS) in N. benthamiana plants, and the transgene GFP in N. benthamiana line 16c plants. Therefore, LIYV-derived vectors could provide a technical reference for developing vectors of other economically important criniviruses.

Citrus sudden death (CSD) has caused the death of approximately four million orange trees in a very important citrus region in Brazil. Although its etiology is still not completely clear, symptoms and distribution of affected plants indicate a viral disease. In a search for viruses associated with CSD, we have performed a comparative high-throughput sequencing analysis of the transcriptome and small RNAs from CSD-symptomatic and -asymptomatic plants using the Illumina platform. The data revealed mixed infections that included Citrus tristeza virus (CTV) as the most predominant virus, followed by the Citrus sudden death-associated virus (CSDaV), Citrus endogenous pararetrovirus (CitPRV) and two putative novel viruses tentatively named Citrus jingmen-like virus (CJLV), and Citrus virga-like virus (CVLV). The deep sequencing analyses were sensitive enough to differentiate two genotypes of both viruses previously associated with CSD-affected plants: CTV and CSDaV. Our data also showed a putative association of the CSD-symptomatic plants with a specific CSDaV genotype and a likely association with CitPRV as well, whereas the two putative novel viruses showed to be more associated with CSD-asymptomatic plants. This is the first high-throughput sequencing-based study of the viral sequences present in CSD-affected citrus plants, and generated valuable information for further CSD studies.

It is known that plant viruses can change the performance of their vectors. However, there have been no reports on whether or how a semipersistent plant virus manipulates the feeding behaviors of its whitefly vectors. Cucurbit chlorotic yellows virus (CCYV) (genus Crinivirus, family Closteroviridae) is an emergent plant virus in many Asian countries and is transmitted specifically by B and Q biotypes of tobacco whitefly, Bemisia tabaci (Gennadius), in a semipersistent manner. In the present study, we used electrical penetration graph (EPG) technique to investigate the effect of CCYV on the feeding behaviors of B. tabaci. The results showed that CCYV altered feeding behaviors of both biotypes and sexes of B. tabaci with different degrees. CCYV had stronger effects on feeding behaviors of Q biotype than those of B biotype, by increasing duration of phloem salivation and sap ingestion, and could differentially manipulate feeding behaviors of males and females in both biotype whiteflies, with more phloem ingestion in Q biotype males and more non-phloem probing in B biotype males than their respective females. With regard to feeding behaviors related to virus transmission, these results indicated that, when carrying CCYV, B. tabaci Q biotype plays more roles than B biotype, and males make greater contribution than females.

Carrot motley dwarf (CMD) is a viral disease complex caused by co-infection of the polerovirus carrot red leaf virus with the umbraviruses carrot mottle virus or carrot mottle mimic virus, and/or a tombusvirus like associated RNA (tlaRNA), which depend on co-infection with a helper polerovirus to gain aphid transmissibility. In 2020 and 2021 carrot samples from Washington, United States (U.S.), and parsley and cilantro samples from California, U.S., exhibiting typical symptoms of CMD were submitted for diagnosis. Initial RT-PCR diagnostic assays identified the typical CMD viruses in the carrot samples, however only the umbraviruses and tlaRNAs were detected in the parsley and cilantro samples; as such, these samples were retested with another RT-PCR assay for generic polerovirus detection. Unexpectedly, the poleroviruses Torilis crimson leaf virus (TorCLV) and fennel motley virus were identified. Subsequent RNA sequencing analysis was conducted to confirm these results and look for other emergent viruses. In addition to confirming the diagnostic results, the recently described polerovirus Foeniculum vulgare polerovirus, the umbraviruses Pastinaca umbravirus 1 and wild carrot mottle virus, and the tlaRNA Arracacha latent virus E associated RNA were identified, making this the first report of these viruses and tlaRNA in the U.S. Using phylogenetic and pairwise identity comparisons and RDP4 recombination analyses, we also identified a putative novel polerovirus, for which we propose the name parsley polerovirus, that appears to be a recombinant between carrot polerovirus 1, sharing 92% amino acid (aa) identity with the RNA dependent RNA polymerase in the 5' gene block, and TorCLV, sharing >98% aa identity with the capsid protein in the 3 gene block. This work adds to the growing list of polerovirus species exhibiting recombination between the 5' and 3' gene blocks, and highlights the unique, variable, and dynamic associations that can occur in polerovirus, umbravirus, and tlaRNA disease complexes. [ABSTRACT FROM AUTHOR]

We present here the complete nucleotide sequence and genome organization of a novel putative RNA virus identified in field populations of the Asian citrus psyllid, Diaphorina citri, through sequencing of the transcriptome followed by reverse transcription-PCR (RT-PCR). We tentatively named this virus Diaphorina citri-associated C virus (DcACV). DcACV is an unclassified positive-sense RNA virus.

Here, we report the complete genome sequence of a putative densovirus of the Asian citrus psyllid, Diaphorina citri Diaphorina citri densovirus (DcDNV) was originally identified through metagenomics, and here, we obtained the complete nucleotide sequence using PCR-based approaches. Phylogenetic analysis places DcDNV between viruses of the Ambidensovirus and Iteradensovirus genera.

UnlabelledThe Asian citrus psyllid, Diaphorina citri, is the natural vector of the causal agent of Huanglongbing (HLB), or citrus greening disease. Together; HLB and D. citri represent a major threat to world citrus production. As there is no cure for HLB, insect vector management is considered one strategy to help control the disease, and D. citri viruses might be useful. In this study, we used a metagenomic approach to analyze viral sequences associated with the global population of D. citri. By sequencing small RNAs and the transcriptome coupled with bioinformatics analysis, we showed that the virus-like sequences of D. citri are diverse. We identified novel viral sequences belonging to the picornavirus superfamily, the Reoviridae, Parvoviridae, and Bunyaviridae families, and an unclassified positive-sense single-stranded RNA virus. Moreover, a Wolbachia prophage-related sequence was identified. This is the first comprehensive survey to assess the viral community from worldwide populations of an agricultural insect pest. Our results provide valuable information on new putative viruses, some of which may have the potential to be used as biocontrol agents.ImportanceInsects have the most species of all animals, and are hosts to, and vectors of, a great variety of known and unknown viruses. Some of these most likely have the potential to be important fundamental and/or practical resources. In this study, we used high-throughput next-generation sequencing (NGS) technology and bioinformatics analysis to identify putative viruses associated with Diaphorina citri, the Asian citrus psyllid. D. citri is the vector of the bacterium causing Huanglongbing (HLB), currently the most serious threat to citrus worldwide. Here, we report several novel viral sequences associated with D. citri.

To optimize the expression, extraction and purification of plant-derived tetrameric recombinant human butyrylcholinesterase (prBChE), we describe the development and use of plant viral amplicon-based gene expression system; Tobacco Mosaic Virus (TMV) RNA-based overexpression vector (TRBO) to express enzymatically active FLAG-tagged plant made recombinant butyrylcholinesterase (rBChE) in Nicotiana benthamiana leaves using transient agroinfiltration. Two gene expression cassettes were designed to express the recombinant protein in either the ER or to the apoplastic compartment. Leaf homogenization was used to isolate ER-retained recombinant butyrylcholinesterase (prBChE-ER) while apoplast-targeted rBChE was isolated by either leaf homogenization (prBChE) or vacuum-extraction of apoplastic wash fluid (prBChE-AWF). rBChE from apoplast wash fluid had a higher specific activity but lower enzyme yield than leaf homogenate. To optimize the isolation and purification of total recombinant protein from leaf homogenates, an acidic extraction buffer was used. The acidic extraction buffer yielded >95% enzymatically active tetrameric rBChE as verified by Coomassie stained and native gel electrophoresis. Furthermore, when compared to human butyrylcholinesterase, the prBChE was found to be similar in terms of tetramerization and enzyme kinetics. The N-linked glycan profile of purified prBChE-ER was found to be mostly high mannose structures while the N-linked glycans on prBChE-AWF were primarily complex. The glycan profile of the prBChE leaf homogenates showed a mixture of high mannose, complex and paucimannose type N-glycans. These findings demonstrate the ability of plants to produce rBChE that is enzymatically active and whose oligomeric state is comparable to mammalian butyrylcholinesterase. The process of plant made rBChE tetramerization and strategies for improving its pharmacokinetics properties are also discussed.

Abstract Background Using plant viruses to produce desirable proteins in plants allows for using non-transgenic plant hosts and if necessary, the ability to make rapid changes in the virus construct for increased or modified protein product yields. The objective of this work was the development of advanced CMV-based protein production systems to produce Acidothermus cellulolyticus endo-1, 4-β-glucanase (E1) in non-transgenic plants. Results We used two new Cucumber mosaic virus (CMV)-based vector systems for producing the green fluorescent protein (GFP) and more importantly, the Acidothermus cellulolyticus endo-1, 4-β-glucanase (E1) in non-transgenic Nicotiana benthamiana plants. These are the inducible CMVin (CMV-based inducible) and the autonomously replicating CMVar (CMV-based advanced replicating) systems. We modified a binary plasmid containing the complete CMV RNA 3 cDNA to facilitate insertion of desired sequences, and to give modifications of the subgenomic mRNA 4 leader sequence yielding several variants. Quantitative RT-PCR and immunoblot analysis showed good levels of CMV RNA and coat protein accumulation for some variants of both CMVin and CMVar. When genes for E1 or GFP were inserted in place of the CMV coat protein, both were produced in plants as shown by fluorescence (GFP) and immunoblot analysis. Enzymatic activity assays showed that active E1 was produced in plants with yields up to ~ 11 μg/g fresh weight (FW) for specific variant constructs. We also compared in vitro CMV genomic RNA reassortants, and CMV RNA 3 mutants which lacked the C’ terminal 33 amino acids of the 3A movement protein in attempts to further increase E1 yield. Taken together specific variant constructs yielded up to ~21 μg/g FW of E1 in non-transgenic plants. Conclusions Intact, active E1 was rapidly produced in non-transgenic plants by using agroinfiltration with the CMV-based systems. This reduces the time and cost compared to that required to generate transgenic plants and still gives the comparable yields of active E1. Our modifications described here, including manipulating cloning sites for foreign gene introduction, enhance the ease of use. Also, N. benthamiana, which is particularly suitable for agroinfiltration, is a very good plant for transient protein production.