For biofuel applications, synthetic endoglucanase E1 and xylanase (Xyn10A) derived from Acidothermus cellulolyticus were transiently expressed in detached whole sunflower (Helianthus annuus L.) leaves using vacuum infiltration. Three different expression systems were tested, including the constitutive CaMV 35S-driven, CMVar (Cucumber mosaic virus advanced replicating), and TRBO (Tobacco mosaic virus RNA-Based Overexpression Vector) systems. For 6-day leaf incubations, codon-optimized E1 and xylanase driven by the CaMV 35S promoter were successfully expressed in sunflower leaves. The two viral expression vectors, CMVar and TRBO, were not successful although we found high expression in Nicotiana benthamiana leaves previously for other recombinant proteins. To further enhance transient expression, we demonstrated two novel methods: using the plant hormone methyl jasmonic acid in the agroinfiltration buffer and two-phase optimization of the leaf incubation temperature. When methyl jasmonic acid was added to Agrobacterium tumefaciens cell suspensions and infiltrated into plant leaves, the functional enzyme production increased 4.6-fold. Production also increased up to 4.2-fold when the leaf incubation temperature was elevated above the typical temperature, 20C, to 30C in the late incubation phase, presumably due to enhanced rate of protein synthesis in plant cells. Finally, we demonstrated co-expression of E1 and xylanase in detached sunflower leaves. To our knowledge, this is the first report of (co)expression of heterologous plant cell wall-degrading enzymes in sunflower.
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.
Cucumber mosaic virus (CMV) has a worldwide distribution and the widest host range of any known plant virus. From 2000 to 2012, epidemics of CMV severely affected the production of snap bean (Phaseulos vulgaris L.) in the Midwest and Northeastern United States. Virus diversity leading to emergence of new strains is often considered a significant factor in virus epidemics. In addition to epidemics, new disease phenotypes arising from genetic exchanges or mutation can compromise effectiveness of plant disease management strategies. Here, we captured a snapshot of genetic variation of 32 CMV isolates collected from different regions of the U.S including new field as well as historic isolates. Nucleotide diversity (?) was low for U.S. CMV isolates. Sequence and phylogenetic analyses revealed that CMV subgroup I is predominant in the US and further showed that the CMV population is a mixture of subgroups IA and IB. Furthermore, phylogenetic analysis suggests likely reassortment between subgroups IA and IB within five CMV isolates. Based on phylogenetic and computational analysis, recombination between subgroups I and II as well as IA and IB in RNA 3 was detected. This is the first report of recombination between CMV subgroups I and II. Neutrality tests illustrated that negative selection was the major force operating upon the CMV genome, although some positively selected sites were detected for all encoded proteins. Together, these data suggest that different regions of the CMV genome are under different evolutionary constraints. These results also delineate composition of the CMV population in the US, and further suggest that recombination and reassortment among strain subgroups does occur but at a low frequency, and point towards CMV genomic regions that differ in types of selection pressure.
Epoxide hydrolase (EH) is an enzyme in the ?/?-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 insects 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 ?M. 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.
Next generation sequence analyses were used to assess virus-derived small RNA (vsRNA) profiles for Homalodisca coagulata virus-1 (HoCV-1), family Dicistroviridae, and Homalodisca vitripennis reovirus (HoVRV), family Reoviridae, from virus-infected H. vitripennis, the glassy-winged sharpshooter. The vsRNA reads were mapped against the monopartite genome of HoCV-1 and all 12 genome segments of HoVRV, and 21nt vsRNAs were most common. However, strikingly contrasting patterns for the HoCV-1 and HoVRV genomic RNAs were observed. The majority of HoCV-1 vsRNAs mapped to the genomic positive-strand RNA and, although minor hotspots were observed, vsRNAs mapped across the entire genomic RNA. In contrast, HoVRV vsRNAs mapped to both positive and negative-sense strands for all genome segments, but different genomic segments showed distinct hotspots. The HoVRV vsRNAs were more common for 5 and 3 regions of HoVRV regions of all segments. These data suggest that taxonomically different viruses in the same host offer different targets for RNA-antiviral defense.
The glassy-winged sharpshooter Homalodisca vitripennis (Hemiptera: Cicadellidae), is a xylem-feeding leafhopper and important vector of the bacterium Xylella fastidiosa; the causal agent of Pierces disease of grapevines. The functional complexity of the transcriptome of H. vitripennis has not been elucidated thus far. It is a necessary blueprint for an understanding of the development of H. vitripennis and for designing efficient biorational control strategies including those based on RNA interference.
The citrus mealybug, Planococcus citri, is an important plant pest with a very broad plant host range. P. citri is a phloem feeder and loss of plant vigor and stunting are characteristic symptoms induced on a range of host plants, but P. citri also reduces fruit quality and causes fruit drop leading to significant yield reductions. Better strategies for managing this pest are greatly needed. RNA interference (RNAi) is an emerging tool for functional genomics studies and is being investigated as a practical tool for highly targeted insect control. Here we investigated whether RNAi effects can be induced in P. citri and whether candidate mRNAs could be identified as possible targets for RNAi-based P. citri control. RNAi effects were induced in P. citri, as demonstrated by specific target reductions of P. citri actin, chitin synthase 1 and V-ATPase mRNAs after injection of the corresponding specific double-stranded RNA inducers. We also used recombinant Tobacco mosaic virus (TMV) to express these RNAi effectors in Nicotiana benthamiana plants. We found that P. citri showed lower fecundity and pronounced death of crawlers after feeding on recombinant TMV-infected plants. Taken together, our data show that actin, chitin synthase 1 and V-ATPase mRNAs are potential targets for RNAi against P. citri, and that recombinant TMV is an effective tool for evaluating candidate RNAi effectors in plants.
The potato/tomato psyllid, Bactericera cockerelli (B. cockerelli), is an important plant pest and the vector of the phloem-limited bacterium Candidatus Liberibacter psyllaurous (solanacearum), which is associated with the zebra chip disease of potatoes. Previously, we reported induction of RNA interference effects in B. cockerelli via in vitro-prepared dsRNA/siRNAs after intrathoracic injection, and after feeding of artificial diets containing these effector RNAs. In order to deliver RNAi effectors via plant hosts and to rapidly identify effective target sequences in plant-feeding B. cockerelli, here we developed a plant virus vector-based in planta system for evaluating candidate sequences. We show that recombinant Tobacco mosaic virus (TMV) containing B. cockerelli sequences can efficiently infect and generate small interfering RNAs in tomato (Solanum lycopersicum), tomatillo (Physalis philadelphica) and tobacco (Nicotiana tabacum) plants, and more importantly delivery of interfering sequences via TMV induces RNAi effects, as measured by actin and V-ATPase mRNA reductions, in B. cockerelli feeding on these plants. RNAi effects were primarily detected in the B. cockerelli guts. In contrast to our results with TMV, recombinant Potato virus X (PVX) and Tobacco rattle virus (TRV) did not give robust infections in all plants and did not induce detectable RNAi effects in B. cockerelli. The greatest RNA interference effects were observed when B. cockerelli nymphs were allowed to feed on leaf discs collected from inoculated or lower expanded leaves from corresponding TMV-infected plants. Tomatillo plants infected with recombinant TMV containing B. cockerelli actin or V-ATPase sequences also showed phenotypic effects resulting in decreased B. cockerelli progeny production as compared to plants infected by recombinant TMV containing GFP. These results showed that RNAi effects can be achieved in plants against the phloem feeder, B. cockerelli, and the TMV-plant system will provide a faster and more convenient method for screening of suitable RNAi target sequences in planta.
Criniviruses comprise one of the genera within the family Closteroviridae. Members in this family are restricted to the phloem and rely on whitefly vectors of the genera Bemisia and/or Trialeurodes for plant-to-plant transmission. All criniviruses have bipartite, positive-sense single-stranded RNA genomes, although there is an unconfirmed report of one having a tripartite genome. Lettuce infectious yellows virus (LIYV) is the type species of the genus, the best studied so far of the criniviruses and the first for which a reverse genetics system was developed. LIYV RNA 1 encodes for proteins predicted to be involved in replication, and alone is competent for replication in protoplasts. Replication results in accumulation of cytoplasmic vesiculated membranous structures which are characteristic of most studied members of the Closteroviridae. These membranous structures, often referred to as Beet yellows virus (BYV)-type vesicles, are likely sites of RNA replication. LIYV RNA 2 is replicated in trans when co-infecting cells with RNA 1, but is temporally delayed relative to RNA 1. Efficient RNA 2 replication also is dependent on the RNA 1-encoded RNA-binding protein, P34. No LIYV RNA 2-encoded proteins have been shown to affect RNA replication, but at least four, CP (major coat protein), CPm (minor coat protein), Hsp70h, and P59 are virion structural components and CPm is a determinant of whitefly transmissibility. Roles of other LIYV RNA 2-encoded proteins are largely as yet unknown, but P26 is a non-virion protein that accumulates in cells as characteristic plasmalemma deposits which in plants are localized within phloem parenchyma and companion cells over plasmodesmata connections to sieve elements. The two remaining crinivirus-conserved RNA 2-encoded proteins are P5 and P9. P5 is 39 amino acid protein and is encoded at the 5 end of RNA 2 as ORF 1 and is part of the hallmark closterovirus gene array. The orthologous gene in BYV has been shown to play a role in cell-to-cell movement and indicated to be localized to the endoplasmic reticulum as a Type III integral membrane protein. The other small protein, P9, is encoded by ORF 4 overlaps with ORF 3 that encodes the structural protein, P59. P9 seems to be unique to viruses in the genus Crinivirus, as no similar protein has been detected in viruses of the other two genera of the Closteroviridae.
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.
The potato/tomato psyllid, Bactericerca cockerelli (B. cockerelli), and the Asian citrus psyllid, Diaphorina citri (D. citri), are very important plant pests, but they are also vectors of phloem-limited bacteria that are associated with two devastating plant diseases. B. cockerelli is the vector of Candidatus Liberibacter psyllaurous (solanacearum), which is associated with zebra chip disease of potatoes, and D. citri is the vector of Ca. Liberibacter asiaticus, which is associated with the Huanglongbing (citrus greening) disease that currently threatens the entire Florida citrus industry. Here we used EST sequence information from D. citri to identify potential targets for RNA interference in B. cockerelli. We targeted ubiquitously expressed and gut-abundant mRNAs via injection and oral acquisition of double-stranded RNAs and siRNAs and were able to induce mortality in recipient psyllids. We also showed knockdown of target mRNAs, and that oral acquisition resulted primarily in mRNA knockdown in the psyllid gut. Concurrent with gene knockdown was the accumulation of target specific ? 21 nucleotide siRNAs for an abundant mRNA for BC-Actin. These results showed that RNAi can be a powerful tool for gene function studies in psyllids, and give support for continued efforts for investigating RNAi approaches as possible tools for psyllid and plant disease control.
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.
The Crinivirus, Lettuce infectious yellows virus (LIYV) has a bipartite, positive-sense ssRNA genome. LIYV RNA 1 encodes replication-associated proteins while RNA 2 encodes proteins needed for other aspects of the LIYV life cycle. LIYV RNA 1 ORF 2 encodes P34, a trans enhancer for RNA 2 accumulation. Here we show that P34 is a sequence non-specific ssRNA-binding protein in vitro. P34 binds ssRNA in a cooperative manner, and the C-terminal region contains the RNA-binding domain. Topology predictions suggest that P34 is a membrane-associated protein and the C-terminal region is exposed outside of the membrane. Furthermore, fusions of P34 to GFP localized to the perinuclear region of transfected protoplasts, and colocalized with an ER-specific dye. This localization was of interest since LIYV RNA 1 replication (with or without P34 protein) induced strong ER rearrangement to the perinuclear region. Together, these data provide insight into LIYV replication and possible functions of P34.
The xylem feeding leafhopper Homalodisaca vitripennis (H. vitripennis) is an unusually robust and efficient vector of Xylella fastidiosa, a Gram-negative bacterium which causes several very important plant diseases. Here we investigated RNA interference (RNAi) to target actin, a key component of insect cells and whole bodies, in H. vitripennis cells. RNAi effectors were delivered via lipid based transfection and real-time RT-PCR, RNA hybridization, and microscopic analyses were employed to verify RNAi effects. When actin dsRNAs were used, a 10-fold decrease in the target H. vitripennis actin mRNA level was seen in cells. Altered phenotypic effects also were evident in transfected cells, as were small interfering RNAs, hallmarks of RNAi. The use of H. vitripennis cells and RNAi offers new opportunities to research hemipterans, the most important insect vectors of plant pathogens.
Previously we described Tomato bushy stunt virus (TBSV) vectors, which retained their capsid protein gene and were engineered with magnesium chelatase (ChlH) and phytoene desaturase (PDS) gene sequences from Nicotiana benthamiana. Upon plant infection, these vectors eventually lost the inserted sequences, presumably as a result of recombination. Here, we modified the same vectors to also contain the plant miR171 or miR159 target sequences immediately 3 of the silencing inserts. We inoculated N. benthamiana plants and sequenced recombinant RNAs recovered from noninoculated upper leaves. We found that while some of the recombinant RNAs retained the microRNA (miRNA) target sites, most retained only the 3 10 and 13 nucleotides of the two original plant miRNA target sequences, indicating in planta miRNA-guided RNA-induced silencing complex cleavage of the recombinant TBSV RNAs. In addition, recovered RNAs also contained various fragments of the original sequence (ChlH and PDS) upstream of the miRNA cleavage site, suggesting that the 3 portion of the miRNA-cleaved TBSV RNAs served as a template for negative-strand RNA synthesis by the TBSV RNA-dependent RNA polymerase (RdRp), followed by template switching by the RdRp and continued RNA synthesis resulting in loss of nonessential nucleotides.
Use of transient expression for the rapid, large-scale production of recombinant proteins in plants requires optimization of existing methods to facilitate scale-up of the process. We have demonstrated that the techniques used for agroinfiltration and induction greatly impact transient production levels of heterologous protein. A Cucumber mosaic virus inducible viral amplicon (CMViva) expression system was used to transiently produce recombinant alpha-1-antitrypsin (rAAT) by co-infiltrating harvested Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains, one containing the CMViva expression cassette carrying the AAT gene and the other containing a binary vector carrying the gene silencing suppressor p19. Harvested leaves were both infiltrated and induced by either pressure or vacuum infiltration. Using the vacuum technique for both processes, maximum levels of functional and total rAAT were elevated by (190 +/- 8.7)% and (290 +/- 7.5)%, respectively, over levels achieved when using the pressure technique for both processes. The bioprocessing conditions for vacuum infiltration and induction were optimized and resulted in maximum rAAT production when using an A. tumefaciens concentration at OD(600) of 0.5 and a 0.25-min vacuum infiltration, and multiple 1-min vacuum inductions further increased production 25% and resulted in maximum levels of functional and total rAAT at (2.6 +/- 0.09)% and (4.1 +/- 0.29)% of the total soluble protein, respectively, or (90 +/- 1.7) and (140 +/- 10) mg per kg fresh weight leaf tissue at 6 days post-induction. Use of harvested plant tissue with vacuum infiltration and induction demonstrates a bioprocessing route that is fully amenable to scale-up.
Lettuce infectious yellows virus (LIYV) is phloem-limited, non-mechanically transmissible, and is transmitted to plants only by Bemisia tabaci. Here, we developed agroinoculation to deliver LIYV to plants thereby obviating the need for B. tabaci. Agroinfiltration of RNA 1 containing a green fluorescent protein gene into Nicotiana benthamiana leaves resulted in subliminal infections, as judged by green fluorescence. Agroinfiltration of LIYV wild-type RNA 1 and 2 constructs resulted in systemic infections in N. benthamiana plants and typical LIYV symptoms. In addition, partially purified LIYV virions from agroinoculated N. benthamiana plants were successfully acquired via membrane-feeding and transmitted to lettuce plants by B. tabaci. Agroinoculation coupled with targeted mutagenesis technologies will greatly enhance LIYV reverse genetics studies to characterize LIYV gene functions in planta for processes such as virus replication, recombination, trafficking, symptom elicitation and virus-vector interactions.
Interactions of Lettuce infectious yellows virus (LIYV)-encoded proteins were tested by yeast-two-hybrid (Y2H) assays. LIYV-encoded P34, Hsp70h, P59, CP, CPm, and P26 were tested in all possible pairwise combinations. Interaction was detected only for the P26-P26 combination. P26 self-interaction domains were mapped using a series of N- and C-terminal truncations. Orthologous P26 proteins from the criniviruses Beet pseudoyellows virus (BPYV), Cucurbit yellow stunting disorder virus (CYSDV), and Lettuce chlorosis virus (LCV) were also tested, and each exhibited strong self-interaction but no interaction with orthologous proteins. Two small putative proteins encoded by LIYV RNA2, P5 and P9, were also tested for interactions with the six aforementioned LIYV proteins and each other. No interactions were detected for P5, but P9-P9 self-interaction was detected. P26- and P9-encoding genes are present in all described members of the genus Crinivirus, but are not present in other members of the family Closteroviridae. LIYV P26 has previously been demonstrated to induce a unique LIYV cytopathology, plasmalemma deposits (PLDs), but no role is yet known for P9.
Lettuce infectious yellows virus (LIYV) encodes a 26 kDa protein (P26) previously shown to associate with plasmalemma deposits (PLDs), unique LIYV-induced cytopathologies located at the plasmalemma over plasmodesmata pit fields in companion cells and phloem parenchyma. To further characterize the relationship of P26 and PLDs, we assessed localization and cytopathology induction of P26 expressed from either LIYV or a heterologous Tobacco mosaic virus (TMV) vector using green fluorescent protein (GFP) fusions, immunofluorescence microscopy, biochemical fractionation, and transmission electron microscopy (TEM). TEM analyses demonstrated that P26 not only associated with, but induced formation of PLDs in the absence of other LIYV proteins. Interestingly, PLDs induced by P26-expressing TMV were no longer confined to phloem cells. Putative P26 orthologs from two other members of the genus Crinivirus which do not induce conspicuous PLDs exhibited fractionation properties similar to LIYV P26 but were not associated with any PLD-like cytopathology.
Lettuce infectious yellows virus (LIYV), the type member of the genus Crinivirus in the family Closteroviridae, is specifically transmitted by the sweet potato whitefly (Bemisia tabaci) in a semipersistent manner. LIYV infections result in a low virus titer in plants and protoplasts, impeding reverse genetic efforts to analyze LIYV gene/protein functions. We found that synergistic interactions occurred in mixed infections of LIYV and Turnip mosaic virus (TuMV) in Nicotiana benthamiana plants, and these resulted in enhanced accumulation of LIYV. Furthermore, we examined the ability of transgenic plants and protoplasts expressing only the TuMV P1/HC-Pro sequence to enhance the accumulation of LIYV. LIYV RNA and protein titers increased by as much as 8-fold in these plants and protoplasts relative to control plants. LIYV infections remained phloem-limited in P1/HC-Pro transgenic plants, suggesting that enhanced accumulation of LIYV in these plants was due primarily to increased replication efficiency, not to greater spread.
Plant cell culture production of recombinant products offers a number of advantages over traditional eukaryotic expression systems, particularly if the product can be targeted to and purified from the cell culture broth. However, one of the main obstacles is product degradation by proteases that are produced during cell culture, and/or the loss of biological activity of secreted (extracellular) products as a result of alteration in the protein conformation. Because proteolysis activity and target protein stability can be significantly influenced by culture conditions, it is important to evaluate bioprocess conditions that minimize these effects. In this study, a bioreactor strategy using a protocol involving pH adjustment and medium exchange during plant cell culture is proposed for improving the production of functional recombinant alpha(1)-antitrypsin (rAAT), a human blood protein, produced using several alternative expression systems, including a Cauliflower mosaic virus (CaMV) 35S constitutive promoter expression system, a chemically inducible, estrogen receptor-based promoter (XVE) expression system, and a novel Cucumber mosaic virus (CMV) inducible viral amplicon (CMViva) expression system developed by our group. We have demonstrated that higher medium pH help reduce protease activity derived from cell cultures and improve the inherent stability of human AAT protein as well. This strategy resulted in a fourfold increase in the productivity of extracellular functional rAAT (100 microg/L) and a twofold increase in the ratio of functional rAAT to total rAAT (48%) in transgenic N. benthamiana cell cultures using a chemically inducible viral amplicon expression system.
A series of Lettuce infectious yellows virus (LIYV) RNA 1 mutants was created to evaluate their ability to replicate in tobacco protoplasts. Mutants DeltaEcoRI, DeltaE-LINK, and Delta1B, having deletions in open reading frames (ORFs) 1A and 1B, did not replicate when individually inoculated to protoplasts or when co-inoculated with wild-type RNA1 as a helper virus. A fragment of the green fluorescent protein (GFP) gene was inserted into the RNA 1 ORF 2 (P34) in order to provide a unique sequence tag. This mutant, P34-GFP TAG, was capable of independent replication in protoplasts. Mutants derived from P34-GFP TAG having frameshift mutations in the ORF 1A or 1B were unable to replicate in protoplasts alone or in trans when co-inoculated with wild-type RNA1 as a helper virus. Taken together, these data strongly suggest that LIYV RNA 1 replication is cis-preferential.
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-?-glucanase (E1) in non-transgenic plants.
Fig mosaic virus (FMV), a member of the newly described genus Emaravirus, has four negative-sense single-stranded genomic RNAs, and each codes for a single protein in the viral complementary RNA (vcRNA). In this study we show that FMV mRNAs for genome segments 2 and 3 contain short (12-18 nucleotides) heterogeneous nucleotide leader sequences at their 5 termini. Furthermore, by using the high affinity cap binding protein eIF4E(K119A), we also determined that a 5 cap is present on a population of the FMV positive-sense RNAs, presumably as a result of cap-snatching. Northern hybridization results showed that the 5 capped RNA3 segments are slightly smaller than the homologous vcRNA3 and are not polyadenylated. These data suggest that FMV generates 5 capped mRNAs via cap-snatching, similar to strategies used by other negative-sense multipartite ssRNA viruses.
The glassy winged sharpshooter, Homalodisca vitripennis, is an unusually robust and efficient leafhopper vector of Xylella fastidiosa. X. fastidiosa is the causative agent of Pierces disease, almond scorch, citrus variegated chlorosis and other serious plant diseases. The present study was conducted to establish whether RNA interference (RNAi) was induced in nymphal H. vitripennis that were injected with actin dsRNAs and other dsRNAs.
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