Dissecting dynamic plant virus synergism in mixed infections of poleroviruses, umbraviruses, and tombusvirus-like associated RNAs.

Mixed infections of a plant infecting polerovirus, umbravirus, and/or tombusvirus-like associated RNAs (tlaRNAs) produce unique virus disease complexes that exemplify "helper-dependence" interactions, a type of viral synergism that occurs when a "dependent" virus that lacks genes encoding for certain protein products necessary for it to complete its infection cycle can utilize complementary proteins encoded by a co-infecting "helper" virus. While much research has focused on polerovirus-umbravirus or polerovirus-tlaRNA interactions, only recently have umbravirus-tlaRNA interactions begun to be explored. To expand on the limited understanding of umbravirus-tlaRNA interactions in such disease complexes, we established various co-infection pairings of the polerovirus turnip yellows virus (TuYV), the umbravirus carrot mottle virus (CMoV), and three different tlaRNAs-carrot red leaf virus aRNAs (CRLVaRNAs) gamma and sigma, and the TuYVaRNA ST9-in the model plant Nicotiana benthamiana, then investigated the effects of these different co-infections on tlaRNA systemic movement within the host, and on virus accumulation, and aphid and mechanical transmission of each of these viruses. We found that CMoV alone could support systemic movement of each of the tlaRNAs, making this the second report to demonstrate such an interaction between an umbravirus and tlaRNAs. We also report for the first time that CMoV could also impart mechanical transmissibility to the tlaRNAs sigma and ST9, and that co-infections of either of these tlaRNAs with both TuYV and CMoV increased the efficiency with which TuYV could be mechanically co-transmitted with CMoV.

Construction and use of an infectious cDNA clone to identify aphid vectors and susceptible monocot hosts of the polerovirus barley virus G.

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.

High-quality, chromosome-scale genome assemblies: comparisons of three Diaphorina citri (Asian citrus psyllid) geographic populations.

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.

Citrus sudden death-associated virus as a new expression vector for rapid in planta production of heterologous proteins, chimeric virions, and virus-like particles.

The more we understand the strategies used by viruses for protein expression, the more possibilities we have to exploit viruses as expression vectors for heterologous protein production. Advances in the development of virus-based expression systems have been possible due to generation of many virus infectious clones, especially those derived from plant viruses, which have the capability for rapid and high-level transient expression of proteins in plant cells, a robust and low-cost bioreactor. In this work, we generated new replicative virus expression vectors based on a previously constructed citrus sudden death-associated virus (CSDaV) infectious cDNA clone. These vectors were generated to express the reporter green fluorescent protein (GFP) in Nicotiana benthamiana leaves by taking advantage of the expression strategies used by CSDaV to produce its structural proteins. We show that higher amounts of GFP can be produced from a coat protein (CP)-independent CSDaV-based vector, compared to levels of GFP expressed from a widely used non-replicative vector (pEAQ series); or GFP can be produced in fusion with the major CSDaV CP (CPp21) to be incorporated into chimeric virions. However, GFP-recombinant CSDaV virions do not appear uniformly assembled, but more likely as mosaic particles. Cryo-electron microscopy analysis from this work revealed the structures of the wild-type and the GFP-recombinant CSDaV virions, but it was not able to reveal where exactly the GFP is displayed in the chimeric virions. We show though that the incorporation of GFP-CPp21 fusion protein into virions occurs solely due to its interaction with free/non-fused CPp21, independent of other viral proteins. Therefore, individual co-expression of GFP-CPp21 and CPp21 in the same plant cells leads to the production of chimeric virus-like particles (VLPs), while GFP-CPp21 fusion protein itself is not able to self-assemble into VLPs. The new CSDaV-based expression vectors may provide an alternative platform for use in molecular farming, either for production of heterologous proteins or as scaffold for heterologous protein display.

Development of Agrobacterium tumefaciens Infiltration of Infectious Clones of Grapevine Geminivirus A Directly into Greenhouse-Grown Grapevine and Nicotiana benthamiana Plants.

Grapevine virus infectious clones are important tools for fundamental studies, but also because of their potential for translational applications for grapevine improvement. Although several grapevine virus infectious clones have been developed, there has been difficulty in directly infecting mature grapevine plants, and many of the viruses used still cause disease symptoms in grapevine plants, making them less likely candidates for biotechnological applications in grapes. Here, we developed an improved Agrobacterium tumefaciens infiltration method that can be used to deliver DNA plasmids and viral infectious clones directly into approximately 20- to 40-cm-high (above soil) greenhouse-grown grapevine plants. We also developed infectious clones for two isolates of grapevine geminivirus A (GGVA): Longyan (China; GenBank accession KX570611; GGVA-76) and Super Hamburg (Japan; GenBank accession KX570610; GGVA-93). Neither virus caused any obvious symptoms when inoculated to plants of grapevine varieties Colombard, Salt Creek, Cabernet Sauvignon, and Vaccarèse. However, the two GGVA isolates induced different symptom severity and viral titer in Nicotiana benthamiana plants. The two GGVA isolates used here were found to accumulate to different titers in different parts/branches of the infected grapevine plants. The GGVA infectious clones and the improved grapevine infiltration technique developed here provide new, valuable tools that can be applied to grapevine plants, possibly even for translational applications such as disease management and desired trait improvements.

Artificial microRNA guide strand selection from duplexes with no mismatches shows a purine-rich preference for virus- and non-virus-based expression vectors in plants.

Artificial microRNA (amiRNA) technology has allowed researchers to direct efficient silencing of specific transcripts using as few as 21 nucleotides (nt). However, not all the artificially designed amiRNA constructs result in selection of the intended ~21-nt guide strand amiRNA. Selection of the miRNA guide strand from the mature miRNA duplex has been studied in detail in human and insect systems, but not so much for plants. Here, we compared a nuclear-replicating DNA viral vector (tomato mottle virus, ToMoV, based), a cytoplasmic-replicating RNA viral vector (tobacco mosaic virus, TMV, based), and a non-viral binary vector to express amiRNAs in plants. We then used deep sequencing and mutational analysis and show that when the structural factors caused by base mismatches in the mature amiRNA duplex were excluded, the nucleotide composition of the mature amiRNA region determined the guide strand selection. We found that the strand with excess purines was preferentially selected as the guide strand and the artificial miRNAs that had no mismatches in the amiRNA duplex were predominantly loaded into AGO2 instead of loading into AGO1 like the majority of the plant endogenous miRNAs. By performing assays for target effects, we also showed that only when the intended strand was selected as the guide strand and showed AGO loading, the amiRNA could provide the expected RNAi effects. Thus, by removing mismatches in the mature amiRNA duplex and designing the intended guide strand to contain excess purines provide better control of the guide strand selection of amiRNAs for functional RNAi effects.

Completion of Maize Stripe Virus Genome Sequence and Analysis of Diverse Isolates.

Maize stripe virus is a pathogen of corn and sorghum in subtropical and tropical regions worldwide. We used high-throughput sequencing to obtain the complete nucleotide sequence for the reference genome of maize stripe virus and to sequence the genomes of ten additional isolates collected from the United States or Papua New Guinea. Genetically, maize stripe virus is most closely related to rice stripe virus. We completed and characterized the RNA1 sequence for maize stripe virus, which revealed a large open reading frame encoding a putative protein with ovarian tumor-like cysteine protease, endonuclease, and RNA-dependent RNA polymerase domains. Phylogenetic and amino acid identity analyses among geographically diverse isolates revealed evidence for reassortment in RNA3 that was correlated with the absence of RNA5. This study yielded a complete and updated genetic description of the tenuivirus maize stripe virus and provided insight into potential mechanisms underpinning its diversity.

Carrot mottle virus ORF4 movement protein targets plasmodesmata by interacting with the host cell SUMOylation system.

Plant virus movement proteins (MPs) facilitate virus spread in their plant hosts, and some of them are known to target plasmodesmata (PD). However, how the MPs target PD is still largely unknown. Carrot mottle virus (CMoV) encodes the ORF3 and ORF4 proteins, which are involved in CMoV movement. In this study, we used CMoV as a model to study the PD targeting of a plant virus MP. We showed that the CMoV ORF4 protein, but not the ORF3 protein, modified PD and led to the virus movement. We found that the CMoV ORF4 protein interacts with the host cell small ubiquitin-like modifier (SUMO) 1, 2 and the SUMO-conjugating enzyme SCE1, resulting in the ORF4 protein SUMOylation. Downregulation of mRNAs for NbSCE1 and NbSUMO impaired CMoV infection. The SUMO-interacting motifs (SIMs) LVIVF, VIWV, and a lysine residue at position 78 (K78) are required for the ORF4 protein SUMOylation. The mutation of these motifs prevented the protein to efficiently target PD, and further slowed or completely abolished CMoV systemic movement. Finally, we found that some of these motifs are highly conserved among umbraviruses. Our data suggest that the CMoV ORF4 protein targets PD by interacting with the host cell SUMOylation system.

RNA interference approaches for plant disease control.

Plant diseases caused by a variety of pathogens can have severe effects on crop plants and even plants in natural ecosystems. Despite many effective conventional approaches to control plant diseases, new, efficacious, environmentally sound and cost-effective approaches are needed, particularly with our increasing human population and the effects on crop production and plant health caused by climate change. RNA interference (RNAi) is a gene regulation and antiviral response mechanism in eukaryotes; transgenic and non transgenic plant-based RNAi approaches have shown great effectiveness and potential to target specific plant pathogens and help control plant diseases, especially when no alternatives are available. Here we discuss ways in which RNAi has been used against different plant pathogens, and some new potential applications for plant disease control.

Endogenous Viral Element-Derived Piwi-Interacting RNAs (piRNAs) Are Not Required for Production of Ping-Pong-Dependent piRNAs from Diaphorina citri Densovirus.

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.

Special Issue: "Plant Virus Pathogenesis and Disease Control".

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 [...].

Plant Immune System Activation Upon Citrus Leprosis Virus C Infection Is Mimicked by the Ectopic Expression of the P61 Viral Protein.

Citrus leprosis virus C (CiLV-C, genus Cilevirus, family Kitaviridae) is an atypical virus that does not spread systemically in its plant hosts. Upon its inoculation by Brevipalpus mites, only localized lesions occur, and the infection remains limited to cells around mite feeding sites. Here, we aimed to gain insights into the putative causes of viral unfitness in plants by expanding the limited knowledge of the molecular mechanisms underlying plant/kitavirid interactions. Firstly, we quantified the CiLV-C viral RNAs during the infection in Arabidopsis thaliana plants using RT-qPCR and systematized it by defining three stages of distinguishing subgenomic and genomic RNA accumulation: i) 0-24 h after infestation, ii) 2-4 days after infestation (dai), and iii) 6-10 dai. Accordingly, the global plant response to CiLV-C infection was assessed by RNA-Seq at each period. Results indicated a progressive reprogramming of the plant transcriptome in parallel to the increasing viral loads. Gene ontology enrichment analysis revealed the induction of cell growth-related processes at the early stages of the infection and the triggering of the SA-mediated pathway, ROS burst and hypersensitive response (HR) at the presymptomatic stage. Conversely, infected plants downregulated JA/ET-mediated pathways and processes involved in the primary metabolism including photosynthesis. Marker genes of unfolded protein response were also induced, suggesting a contribution of the endoplasmic reticulum stress to the cell death caused by the viral infection. Finally, we transiently expressed CiLV-C proteins in Nicotiana benthamiana plants to undertake their roles in the elicited plant responses. Expression of the CiLV-C P61 protein consistently triggered ROS burst, upregulated SA- and HR-related genes, increased SA levels, reduced JA levels, and caused cell death. Mimicry of responses typically observed during CiLV-C-plant interaction indicates P61 as a putative viral effector causing the HR-like symptoms associated with the infection. Our data strengthen the hypothesis that symptoms of CiLV-C infection might be the outcome of a hypersensitive-like response during an incompatible interaction. Consequently, the locally restricted infection of CiLV-C, commonly observed across infections by kitavirids, supports the thesis that these viruses, likely arising from an ancestral arthropod-infecting virus, are unable to fully circumvent plant defenses.

Diaphorina citri reovirus is most closely related to fijiviruses.

The Asian citrus psyllid, Diaphorina citri Kuwayama, is an important insect vector of Candidatus Liberibacter asiaticus, the causal agent of Huanglongbing, which is the most destructive disease of citrus worldwide. Sequences for putative Diaphorina citri reovirus (DcRV) were identified from some worldwide populations of D. citri. Here, field surveys indicated that the virus was common in D. citri populations from Hawaii and Fuzhou of PR China. Electron microscopy showed that DcRV virions possessed a typical reovirus-like morphology. The U. S. and Chinese DcRV isolates both showed 10 segments of double-stranded RNA sharing >96% nucleotide sequence identity, and encoding 11 deduced proteins. All genome segments contained conserved 5' and 3' terminal nucleotide sequences and inverted repeats that are hallmarks of reovirus sequence. Phylogenetic analysis showed that DcRV may be considered a new species of the genus Fijivirus sharing a most recent common ancestor with the insect-specific fijivirus Nilaparvata lugens reovirus.

Diaphorina citri densovirus is a persistently infecting virus with a hybrid genome organization and unique transcription strategy.

Diaphorina citri densovirus (DcDV) is an ambisense densovirus with a 5071 nt genome. Phylogenetic analysis places DcDV in an intermediate position between those in the Ambidensovirus and Iteradensovirus genera, a finding that is consistent with the observation that DcDV possesses an Iteradensoviris-like non-structural (NS) protein-gene cassette, but a capsid-protein (VP) gene cassette resembling those of other ambisense densoviruses. DcDV is maternally transmitted to 100 % of the progeny of infected female Diaphorina citri, and the progeny of infected females carry DcDV as a persistent infection without outward phenotypic effects. We were unable to infect naïve individuals by oral inoculation, however low levels of transient viral replication are detected following intrathoracic injection of DcDV virions into uninfected D. citri insects. Transcript mapping indicates that DcDV produces one transcript each from the NS and VP gene cassettes and that these transcripts are polyadenylated at internal sites to produce a ~2.2 kb transcript encoding the NS proteins and a ~2.4 kb transcript encoding the VP proteins. Additionally, we found that transcriptional readthrough leads to the production of longer non-canonical transcripts from both genomic strands.

Phylogenetic classification of a group of self-replicating RNAs that are common in co-infections with poleroviruses.

Tombusvirus-like associated RNAs (tlaRNAs) are positive-sense single-stranded RNAs found in plants co-infected with viruses of the genus Polerovirus. TlaRNAs depend upon capsid proteins supplied in trans by the co-infecting polerovirus vector for transmission and intra-host systemic movement. Here, the full-length genomes of five tlaRNAs were determined using a combination of RT-PCR and next-generation sequencing, and evidence is provided for an additional tlaRNA associated with potato leafroll virus. Phylogenetic analyses based on conserved domains of the RdRp placed tlaRNAs as a monophyletic clade clustering with members of the family Tombusviridae and comprising three different subclades. Full-length clones of tlaRNAs from two of three subclades were confirmed to replicate autonomously, and each produces a subgenomic RNA during infection.

Two Novel Negative-Sense RNA Viruses Infecting Grapevine Are Members of a Newly Proposed Genus within the Family Phenuiviridae.

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.

A Nonstructural Protein Responsible for Viral Spread of a Novel Insect Reovirus Provides a Safe Channel for Biparental Virus Transmission to Progeny.

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.

Endogenous Viral Elements Are Widespread in Arthropod Genomes and Commonly Give Rise to PIWI-Interacting RNAs.

Arthropod genomes contain sequences derived from integrations of DNA and nonretroviral RNA viruses. These sequences, known as endogenous viral elements (EVEs), have been acquired over the course of evolution and have been proposed to serve as a record of past viral infections. Recent evidence indicates that EVEs can function as templates for the biogenesis of PIWI-interacting RNAs (piRNAs) in some mosquito species and cell lines, raising the possibility that EVEs may serve as a source of immunological memory in these organisms. However, whether piRNAs are derived from EVEs or serve an antiviral function in other arthropod species is unknown. Here, we used publicly available genome assemblies and small RNA sequencing data sets to characterize the repertoire and function of EVEs across 48 arthropod genomes. We found that EVEs are widespread in arthropod genomes and primarily correspond to unclassified single-stranded RNA (ssRNA) viruses and viruses belonging to the Rhabdoviridae and Parvoviridae families. Additionally, EVEs were enriched in piRNA clusters in a majority of species, and we found that production of primary piRNAs from EVEs is common, particularly for EVEs located within piRNA clusters. While the abundance of EVEs within arthropod genomes and the frequency with which EVEs give rise to primary piRNAs generally support the hypothesis that EVEs contribute to an antiviral response via the piRNA pathway, limited nucleotide identity between currently described viruses and EVEs identified here likely limits the extent to which this process plays a role during infection with known viruses in the arthropod species analyzed.IMPORTANCE Our results greatly expand the knowledge of EVE abundance, diversity, and function in an exceptionally wide range of arthropod species. We found that while previous findings in mosquitoes regarding the potential of EVEs to serve as sources of immunological memory via the piRNA pathway may be generalized to other arthropod species, speculation regarding the antiviral function of EVE-derived piRNAs should take into context the fact that EVEs are, in the vast majority of cases, not similar enough to currently described viruses at the nucleotide level to serve as sources of antiviral piRNAs against them.

Rescue of Citrus sudden death-associated virus in Nicotiana benthamiana plants from cloned cDNA: insights into mechanisms of expression of the three capsid proteins.

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.

A Distinct, Non-Virion Plant Virus Movement Protein Encoded by a Crinivirus Essential for Systemic Infection.

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.