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.

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.

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.

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.

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.

Enhancement of Recombinant Protein Production in Transgenic Nicotiana benthamiana Plant Cell Suspension Cultures with Co-Cultivation of Agrobacterium Containing Silencing Suppressors.

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.

The Impact of "Coat Protein-Mediated Virus Resistance" in Applied Plant Pathology and Basic Research.

Worldwide, plant viruses cause serious reductions in marketable crop yield and in some cases even plant death. In most cases, the most effective way to control virus diseases is through genetically controlled resistance. However, developing virus-resistant (VR) crops through traditional breeding can take many years, and in some cases is not even possible. Because of this, the demonstration of the first VR transgenic plants in 1985 generated much attention. This seminal report served as an inflection point for research in both basic and applied plant pathology, the results of which have dramatically changed both basic research and in a few cases, commercial crop production. The typical review article on this topic has focused on only basic or only applied research results stemming from this seminal discovery. This can make it difficult for the reader to appreciate the full impact of research on transgenic virus resistance, and the contributions from fundamental research that led to translational applications of this technology. In this review, we take a global view of this topic highlighting the significant changes to both basic and applied plant pathology research and commercial food production that have accumulated in the last 30 plus years. We present these milestones in the historical context of some of the scientific, economic, and environmental drivers for developing specific VR crops. The intent of this review is to provide a single document that adequately records the significant accomplishments of researchers in both basic and applied plant pathology research on this topic and how they relate to each other. We hope this review therefore serves as both an instructional tool for students new to the topic, as well as a source of conversation and discussion for how the technology of engineered virus resistance could be applied in the future.

Diverse Array of New Viral Sequences Identified in Worldwide Populations of the Asian Citrus Psyllid (Diaphorina citri) Using Viral Metagenomics.

UNLABELLED: The 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. IMPORTANCE: Insects 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.

Complete sequence of three different biotypes of tomato spotted wilt virus (wild type, tomato Sw-5 resistance-breaking and pepper Tsw resistance-breaking) from Spain.

Tomato spotted wilt virus (TSWV) occurs worldwide and causes production losses in many important horticultural crops such as tomato and pepper. Breeding resistant cultivars has been the most successful method so far for TSWV disease control, but only two genes have been found to confer resistance against a wide spectrum of TSWV isolates: Sw-5 in tomato and Tsw in pepper. However, TSWV resistance-breaking isolates have emerged in different countries a few years after using resistant cultivars. In this paper, we report the first complete nucleotide sequences of three Spanish TSWV isolates with different biotypes according to their abilities to overcome resistance: LL-N.05 (wild type, WT), Pujol1TL3 (Sw-5 resistance breaking, SBR) and PVR (Tsw resistance-breaking, TBR). The genome of these TSWV isolates consisted of three segments: L (8913-8914 nt), M (4752-4825 nt) and (S 2924-2961 nt). Variations in nucleotide sequences and genomic RNA lengths among the different virus biotypes are reported here. Phylogenetic analysis of the five TSWV open reading frames showed evidence of reassortment between genomic segments of LL-N.05 and Pujol1TL3, which was supported by analysis with different recombination-detecting algorithms.

Genetic variation and possible mechanisms driving the evolution of worldwide fig mosaic virus isolates.

Fig mosaic virus (FMV) is a multipartite negative-sense RNA virus infecting fig trees worldwide. FMV is transmitted by vegetative propagation and grafting of plant materials, and by the eriophyid mite Aceria ficus. In this work, the genetic variation and evolutionary mechanisms shaping FMV populations were characterized. Nucleotide sequences from four genomic regions (each within the genomic RNAs 1, 2, 3, and 4) from FMV isolates from different countries were determined and analyzed. FMV genetic variation was low, as is seen for many other plant viruses. Phylogenetic analysis showed some geographically distant FMV isolates which clustered together, suggesting long-distance migration. The extent of migration was limited, although varied, between countries, such that FMV populations of different countries were genetically differentiated. Analysis using several recombination algorithms suggests that genomes of some FMV isolates originated by reassortment of genomic RNAs from different genetically similar isolates. Comparison between nonsynonymous and synonymous substitutions showed selection acting on some amino acids; however, most evolved neutrally. This and neutrality tests together with the limited gene flow suggest that genetic drift plays an important role in shaping FMV populations.

Characterization of Hovi-mEH1, a microsomal epoxide hydrolase from the glassy-winged sharpshooter Homalodisca vitripennis.

Epoxide hydrolase (EH) is an enzyme in the α/ß-hydrolase fold superfamily that uses a water molecule to transform an epoxide to its corresponding diol. In insects, EHs metabolize among other things critical developmental hormones called juvenile hormones (JHs). EHs also play roles in the detoxification of toxic compounds that are found in the insect's diet or environment. In this study, a full-length cDNA encoding an epoxide hydrolase, Hovi-mEH1, was obtained from the xylem-feeding insect Homalodisca vitripennis. H. vitripennis, commonly known as the glassy-winged sharpshooter, is an economically important vector of plant pathogenic bacteria such as Xylella fastidiosa. Hovi-mEH1 hydrolyzed the general EH substrates cis-stilbene oxide and trans-diphenylpropene oxide with specific activities of 47.5 ± 6.2 and 1.3 ± 0.5 nmol of diol formed min⁻¹ mg⁻¹, respectively. Hovi-mEH1 metabolized JH III with a Vmax of 29.3 ± 1.6 nmol min⁻¹ mg⁻¹, kcat of 0.03 s⁻¹, and KM of 13.8 ± 2.0 µM. These Vmax and kcat values are similar to those of known JH metabolizing EHs from lepidopteran and coleopteran insects. Hovi-mEH1 showed 99.1% identity to one of three predicted EH-encoding sequences that were identified in the transcriptome of H. vitripennis. Of these three sequences only Hovi-mEH1 clustered with known JH metabolizing EHs. On the basis of biochemical, phylogenetic, and transcriptome analyses, we hypothesize that Hovi-mEH1 is a biologically relevant JH-metabolizing enzyme in H. vitripennis.

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.

Bipartite and tripartite Cucumber mosaic virus-based vectors for producing the Acidothermus cellulolyticus endo-1,4-ß-glucanase and other proteins in non-transgenic plants.

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-ß-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-ß-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.

A new satellite RNA is associated with natural infections of cucumber mosaic virus in succulent snap bean.

Cucumber mosaic virus (CMV) was consistently recovered from symptomatic snap bean plants during surveys conducted in 2007 and 2008 in central Wisconsin. A large proportion of these CMV-infected plants contained a single-stranded linear RNA molecule consisting of 339 nucleotides and sharing 90-94% sequence identity with other satellite (sat) RNAs of CMV. Comparison of this satRNA sequence with currently available CMV satRNA sequences suggests this to be a novel satRNA.

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.

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.

A mutation in the Lettuce infectious yellows virus minor coat protein disrupts whitefly transmission but not in planta systemic movement.

The Lettuce infectious yellows virus (LIYV) RNA 2 mutant p1-5b was previously isolated from Bemisia tabaci-transmitted virus maintained in Chenopodium murale plants. p1-5b RNA 2 contains a single-nucleotide deletion in the minor coat protein (CPm) open reading frame (ORF) that is predicted to result in a frameshift and premature termination of the protein. Using the recently developed agroinoculation system for LIYV, we tested RNA 2 containing the p1-5b CPm mutant genotype (agro-pR6-5b) in Nicotiana benthamiana plants. We showed that plant infection triggered by agro-pR6-5b spread systemically and resulted in the formation of virions similar to those produced in p1-5b-inoculated protoplasts. However, virions derived from these mutant CPm genotypes were not transmitted by whiteflies, even though virion concentrations were above the typical transmission thresholds. In contrast, and as demonstrated for the first time, an engineered restoration mutant (agro-pR6-5bM1) was capable of both systemic movement in plants and whitefly transmission. These results provide strong molecular evidence that the full-length LIYV-encoded CPm is dispensable for systemic plant movement but is required for whitefly transmission.