Development of an assay system for the analysis of host RISC activity in the presence of a potyvirus RNA silencing suppressor, HC-Pro.

BACKGROUND: To investigate the mechanism of RNA silencing suppression, the genetic transformation of viral suppressors of RNA silencing (VSRs) in Arabidopsis integrates ectopic VSR expression at steady state, which overcomes the VSR variations caused by different virus infections or limitations of host range. Moreover, identifying the insertion of the transgenic VSR gene is necessary to establish a model transgenic plant for the functional study of VSR. METHODS: Developing an endogenous AGO1-based in vitro RNA-inducing silencing complex (RISC) assay prompts further investigation into VSR-mediated suppression. Three P1/HC-Pro plants from turnip mosaic virus (TuMV) (P1/HC-ProTu), zucchini yellow mosaic virus (ZYMV) (P1/HC-ProZy), and tobacco etch virus (TEV) (P1/HC-ProTe) were identified by T-DNA Finder and used as materials for investigations of the RISC cleavage efficiency. RESULTS: Our results indicated that the P1/HC-ProTu plant has slightly lower RISC activity than P1/HC-ProZy plants. In addition, the phenomena are consistent with those observed in TuMV-infected Arabidopsis plants, which implies that HC-ProTu could directly interfere with RISC activity. CONCLUSIONS: In this study, we demonstrated the application of various plant materials in an in vitro RISC assay of VSR-mediated RNA silencing suppression.

Complete genome sequence of Amazon lily mosaic virus isolated from amaryllis (Hippeastrum hybridum Hort.).

In April 2011, a virus was isolated by single-lesion isolation on Chenopodium quinoa leaves from an amaryllis plant with chlorotic ringspots in a private garden in Changhua County, Taiwan. An Illumina MiSeq sequencing system was used to determine the genomic nucleotide (nt) sequence of the virus. A de novo-assembled contig with 9377 nt, containing an open reading frame encoding a putative potyviral polyprotein, was annotated as the potyvirus Amazon lily mosaic virus (ALiMV), sharing 95.5% nt sequence identity with a partial genomic sequence of ALiMV available in the GenBank database. Therefore, the amaryllis virus was designated as ALiMV-TW. Through 5´ and 3´ rapid amplification of cDNA ends (RACE), the complete 9618-nt genome sequence of ALiMV-TW was determined. Sequence comparisons indicated that the genome and polyprotein of ALiMV-TW share 52.3-65.1% nt and 30.1-64.2% aa sequence identity, respectively, with those of other potyviruses. This is the first report of a complete genome sequence of ALiMV.

Investigating the Viral Suppressor HC-Pro Inhibiting Small RNA Methylation through Functional Comparison of HEN1 in Angiosperm and Bryophyte.

In plants, HEN1-facilitated methylation at 3' end ribose is a critical step of small-RNA (sRNA) biogenesis. A mutant of well-studied Arabidopsis HEN1 (AtHEN1), hen1-1, showed a defective developmental phenotype, indicating the importance of sRNA methylation. Moreover, Marchantia polymorpha has been identified to have a HEN1 ortholog gene (MpHEN1); however, its function remained unfathomed. Our in vivo and in vitro data have shown MpHEN1 activity being comparable with AtHEN1, and their substrate specificity towards duplex microRNA (miRNA) remained consistent. Furthermore, the phylogenetic tree and multiple alignment highlighted the conserved molecular evolution of the HEN1 family in plants. The P1/HC-Pro of the turnip mosaic virus (TuMV) is a known RNA silencing suppressor and inhibits HEN1 methylation of sRNAs. Here, we report that the HC-Pro physically binds with AtHEN1 through FRNK motif, inhibiting HEN1's methylation activity. Moreover, the in vitro EMSA data indicates GST-HC-Pro of TuMV lacks sRNA duplex-binding ability. Surprisingly, the HC-Pro also inhibits MpHEN1 activity in a dosage-dependent manner, suggesting the possibility of interaction between HC-Pro and MpHEN1 as well. Further investigations on understanding interaction mechanisms of HEN1 and various HC-Pros can advance the knowledge of viral suppressors.

A Conserved Helix in the C-Terminal Region of Watermelon Silver Mottle Virus Nonstructural Protein S Is Imperative For Protein Stability Affecting Self-Interaction, RNA Silencing Suppression, and Pathogenicity.

In orthotospovirus, the nonstructural protein S (NSs) is the RNA-silencing suppressor (RSS) and pathogenicity determinant. Here, we demonstrate that a putative α-helix, designated H8, spanning amino acids 338 to 369 of the C-terminal region of the NSs protein, is crucial for self-interaction of watermelon silver mottle virus NSs protein and that the H8 affects RSS function. Co-immunoprecipitation, yeast two-hybrid, and bimolecular fluorescence complementation analyses revealed that the triple point mutation (TPM) of H8 amino acids Y338A, H350A, and F353A resulted in NSs protein self-interaction dysfunction. Transient expression of H8-deleted (ΔH8) and TPM NSs proteins in Nicotiana benthamiana plants by agroinfitration indicated that these proteins have weaker RSS activity and are far less stable than wild-type (WT) NSs. However, an electrophoretic mobility assay revealed that small interfering RNA (siRNA) binding ability of TPM NSs protein is not compromised. The pathogenicity assay of WT NSs protein expressed by the attenuated turnip mosaic virus vector restored severe symptoms in recombinant-infected N. benthamiana plants but not for ΔH8 or TPM proteins. Taken together, we conclude that the H8 helix in the C-terminal region of NSs protein is crucial for stabilizing NSs protein through self-interaction to maintain normal functions of RSS and pathogenicity, but not for NSs-siRNA binding activity.

Lfo-miR164b and LfNAC1 as autumn leaf senescence regulators in Formosan sweet gum (Liquidambar formosana Hance).

In this study, a microRNA microarray was used to investigate the microRNA profiles from young green leaves, and senescent red leaves and yellow leaves of Formosan sweet gum (Liquidambar formosana Hance). The conserved microRNA miR164 was highly expressed in green leaves compared to senescent leaves. The pri-microRNA of miR164 was identified and named lfo-miR164b based on its secondary structure. In Agrobacterium-mediated transient expression experiment, lfo-miR164b was confirmed to regulate the leaf senescence-associated gene LfNAC1 and LfNAC100. Transient overexpression of LfNAC1 induced the expression of leaf senescence genes in Nicotiana benthamiana. In addition, LfNAC1 activated the expression of proLfSGR::YFP, suggesting the regulatory role of LfNAC1 in leaf senescence. In summary, miR164 inhibits the expression of LfNAC1 in spring and summer, later on LfNAC1 actives leaf senescence-associated genes to cause leaf senescence following a gradual decline of miR164 as the seasons change. The "miR164-NAC" regulatory mechanism was confirmed in Formosan sweet gum autumn leaf senescence.

MicroRNAs in Marchantia polymorpha.

Contents Summary 409 I. Introduction 409 II. RNA silencing machinery in Marchantia polymorpha 410 III. miRNA prediction by integrating omics approach 410 IV. miRNAs and their targets in Marchantia polymorpha 410 V. Mpo-miR390-mediated MpTAS3 tasiRNA biogenesis and potential tasiARF target MpARF2 414 VI. Artificial miRNA and CRISPR-CAS9 edited MIR gene in Marchantia polymorpha 414 VII. Conclusions 415 Acknowledgements 415 References 415 SUMMARY: The liverwort Marchantia polymorpha occupies an important phylogenetic position for comparative studies of land plant gene regulation. Multiple gene regulatory pathways mediated by small RNAs, including microRNAs (miRNAs), trans-acting short-interfering RNAs, and heterochromatic siRNAs often associated with RNA-dependent DNA methylation, have been characterized in flowering plants. Genes for essential components for all of these small RNA-mediated gene silencing pathways are found in M. polymorpha as well as the moss Phsycomitrella patens, indicating that these pathways existed in the ancestral land plant. However, only seven miRNAs are conserved across land plants, with both ancestral and novel targets identified in M. polymorpha. There is little or no evidence that any of these conserved miRNAs are present in algae. As with other plants investigated, most miRNAs in M. polypmorpha exhibit lineage-specific evolution. Application of artificial miRNA and CRISPR-Cas9 technologies in genetic studies of M. polymorpha provide avenues to further investigate miRNA biology.

Gene-to-Gene Network Analysis of the Mediation of Plant Innate Immunity by the Eliciting Plant Response-Like 1 (Epl1) Elicitor of Trichoderma formosa.

A new clade, Trichoderma formosa, secretes eliciting plant response-like 1 (Epl1), a small peptide elicitor that stimulates plant immunity. Nicotiana benthamiana pretreated with Epl1 for 3 days developed immunity against Tomato mosaic virus (ToMV) infection. The transcriptome profiles of T. formosa and N. benthamiana were obtained by deep sequencing; the transcript of Epl1 is 736 nt in length and encodes a 12-kDa peptide. Identifying critical genes in Epl1-mediated immunity was challenging due to high similarity between the transcriptome expression profiles of Epl1-treated and ToMV-infected N. benthamiana samples. Therefore, an efficient bioinformatics data mining approach was used for high-throughput transcriptomic assays in this study. We integrated gene-to-gene network analysis into the ContigViews transcriptome database, and genes related to jasmonic acid and ethylene signaling, salicylic acid signaling, leucine-rich repeats, transcription factors, and histone variants were hubs in the gene-to-gene networks. In this study, the Epl1 of T. formosa triggers plant immunity against various pathogen infections. Moreover, we demonstrated that high-throughput data mining and gene-to-gene network analysis can be used to identify critical candidate genes for further studies on the mechanisms of plant immunity.

Shrimp miR-10a Is Co-opted by White Spot Syndrome Virus to Increase Viral Gene Expression and Viral Replication.

Members of the microRNA miR-10 family are highly conserved and play many important roles in diverse biological mechanisms, including immune-related responses and cancer-related processes in certain types of cancer. In this study, we found the most highly upregulated shrimp microRNA from Penaeus vannamei during white spot syndrome virus (WSSV) infection was miR-10a. After confirming the expression level of miR-10a by northern blot and quantitative RT-PCR, an in vivo experiment showed that the viral copy number was decreased in miR-10a-inhibited shrimp. We found that miR-10a targeted the 5' untranslated region (UTR) of at least three viral genes (vp26, vp28, and wssv102), and plasmids that were controlled by the 5' UTR of these genes produced enhanced luciferase signals in transfected SF9 cells. These results suggest a previously unreported role for shrimp miR-10a and even a new type of host-virus interaction, whereby a co-opts the key cellular regulator miR-10a to globally enhance the translation of viral proteins.

Isolation and Identification of Post-Transcriptional Gene Silencing-Related Micro-RNAs by Functionalized Silicon Nanowire Field-effect Transistor.

Many transcribed RNAs are non-coding RNAs, including microRNAs (miRNAs), which bind to complementary sequences on messenger RNAs to regulate the translation efficacy. Therefore, identifying the miRNAs expressed in cells/organisms aids in understanding genetic control in cells/organisms. In this report, we determined the binding of oligonucleotides to a receptor-modified silicon nanowire field-effect transistor (SiNW-FET) by monitoring the changes in conductance of the SiNW-FET. We first modified a SiNW-FET with a DNA probe to directly and selectively detect the complementary miRNA in cell lysates. This SiNW-FET device has 7-fold higher sensitivity than reverse transcription-quantitative polymerase chain reaction in detecting the corresponding miRNA. Next, we anchored viral p19 proteins, which bind the double-strand small RNAs (ds-sRNAs), on the SiNW-FET. By perfusing the device with synthesized ds-sRNAs of different pairing statuses, the dissociation constants revealed that the nucleotides at the 3'-overhangs and pairings at the terminus are important for the interactions. After perfusing the total RNA mixture extracted from Nicotiana benthamiana across the device, this device could enrich the ds-sRNAs for sequence analysis. Finally, this bionanoelectronic SiNW-FET, which is able to isolate and identify the interacting protein-RNA, adds an additional tool in genomic technology for the future study of direct biomolecular interactions.

Two Novel Motifs of Watermelon Silver Mottle Virus NSs Protein Are Responsible for RNA Silencing Suppression and Pathogenicity.

The NSs protein of Watermelon silver mottle virus (WSMoV) is the RNA silencing suppressor and pathogenicity determinant. In this study, serial deletion and point-mutation mutagenesis of conserved regions (CR) of NSs protein were performed, and the silencing suppression function was analyzed through agroinfiltration in Nicotiana benthamiana plants. We found two amino acid (aa) residues, H113 and Y398, are novel functional residues for RNA silencing suppression. Our further analyses demonstrated that H113 at the common epitope (CE) ((109)KFTMHNQ(117)), which is highly conserved in Asia type tospoviruses, and the benzene ring of Y398 at the C-terminal ß-sheet motif ((397)IYFL(400)) affect NSs mRNA stability and protein stability, respectively, and are thus critical for NSs RNA silencing suppression. Additionally, protein expression of other six deleted (ΔCR1-ΔCR6) and five point-mutated (Y15A, Y27A, G180A, R181A and R212A) mutants were hampered and their silencing suppression ability was abolished. The accumulation of the mutant mRNAs and proteins, except Y398A, could be rescued or enhanced by co-infiltration with potyviral suppressor HC-Pro. When assayed with the attenuated Zucchini yellow mosaic virus vector in squash plants, the recombinants carrying individual seven point-mutated NSs proteins displayed symptoms much milder than the recombinant carrying the wild type NSs protein, suggesting that these aa residues also affect viral pathogenicity by suppressing the host silencing mechanism.

Application of an Integrated Omics Approach for Identifying Host Proteins That Interact With Odontoglossum ringspot virus Capsid Protein.

The glutamic acid at position 100 (E(100)) in the capsid protein (CP) of Odontoglossum ringspot virus (ORSV) plays an important role in long-distance viral movement in Nicotiana benthamiana. The ORSV(E100A) mutant, which has a glutamic acid to alanine substitution, shows a loss of systemic infectivity in N. benthamiana. Transmission electron microscopy and size-exclusion chromatography assays showed that E(100) is essential for CP-CP interaction and viral particle assembly. To identify the ORSV triggering or response genes and CP-interacting proteins (CP-IP), an integrated omics approach based on next-generation sequencing and proteomics profiling was used in this study. The whole-transcriptomes of healthy and ORSV-infected leaves of N. benthamiana were analyzed, and the gene information was used to create a N. benthamiana protein database that was used for protein identification following mass spectrometry analysis. The integrated omics approach identified several putative host proteins that interact with ORSV CP(WT) and were categorized as photosystem subunits, defense-associated proteins, and cell division components. The expression pattern and CP interaction of these CP-IP were examined by semiquantitative reverse transcription polymerase chain reaction and an in vitro binding assay, respectively, to verify the in silico data. Among these proteins, a proteinase inhibitor of N. benthamiana (NbPI2) was highly associated with CP(E100A) as compared with CP(WT), and NbPI1 and NbPI2 were highly induced in ORSV-infected plants. NbPI1- and NbPI2-silenced plants (via a Tobacco rattle virus-induced gene-silencing system) did not exhibit a difference in ORSV infection. Thus, whether NbPI1 and NbPI2 play a role in plant immunity requires further investigation. In summary, the integrated omics approach provides massive and valuable information to identify the ORSV CP-IP and these CP-IP will help us to understand the movement of this virus and plant-virus interaction.

Genetic analyses of the FRNK motif function of Turnip mosaic virus uncover multiple and potentially interactive pathways of cross-protection.

Cross-protection triggered by a mild strain of virus acts as a prophylaxis to prevent subsequent infections by related viruses in plants; however, the underling mechanisms are not fully understood. Through mutagenesis, we isolated a mutant strain of Turnip mosaic virus (TuMV), named Tu-GK, that contains an Arg182Lys substitution in helper component-proteinase (HC-Pro(K)) that confers complete cross-protection against infection by a severe strain of TuMV in Nicotiana benthamiana, Arabidopsis thaliana Col-0, and the Arabidopsis dcl2-4/dcl4-1 double mutant defective in DICER-like ribonuclease (DCL)2/DCL4-mediated silencing. Our analyses showed that HC-Pro(K) loses the ability to interfere with microRNA pathways, although it retains a partial capability for RNA silencing suppression triggered by DCL. We further showed that Tu-GK infection triggers strong salicylic acid (SA)-dependent and SA-independent innate immunity responses. Our data suggest that DCL2/4-dependent and -independent RNA silencing pathways are involved, and may crosstalk with basal innate immunity pathways, in host defense and in cross-protection.

Improving initial infectivity of the Turnip mosaic virus (TuMV) infectious clone by an mini binary vector via agro-infiltration.

BACKGROUND: The in vivo infectious clone of Turnip mosaic virus (TuMV), p35S-TuMV, was used on plant pathology research for many years. To activate p35S-TuMV, the plasmid was mechanically introduced to the local lesion host Chenopodium quinoa. However, low infectivity occurred when the TuMV from C. quinoa was transferred to the systemic host Nicotiana benthamiana. RESULTS: To increase the efficiency of initial infectivity on N. benthamiana, the expression of the TuMV infectious clone by a binary vector that directly activates viral RNA through agro-infiltration is considered to be a good alternative. The size of the binary vector by agro-infiltration is usually large and its backbone has numerous restriction sites that increase difficulty for construction. In this study, we attempted to construct a mini binary vector (pBD003) with less restriction sites. The full-length cDNA of TuMV genome, with or without green fluorescence protein, was inserted in pBD003 to generate pBD-TuMV constructs, which were then individually introduced to N. benthamiana plants by agro-infiltration. Symptom development and ELISA positivity with TuMV antiserum indicated that the pBD-TuMV constructs are infectious. Moreover, the initial infectivity of a mild strain TuMV-GK, which contains an R182K mutation on HC-Pro, constructed in the pBD003 vector was significantly increased by agro-infiltration. CONCLUSION: Thus, we concluded that the newly constructed mini binary vector provides a more feasible tool for TuMV researches in areas, such as creating a mild strain for cross-protection, or a viral vector for foreign gene expression. In addition, the multiple cloning sites will be further cloned in pBD003 for convenience in constructing other viral infectious clones.

Multiple domains of the tobacco mosaic virus p126 protein can independently suppress local and systemic RNA silencing.

Small RNA-mediated RNA silencing is a widespread antiviral mechanism in plants and other organisms. Many viruses encode suppressors of RNA silencing for counter-defense. The p126 protein encoded by Tobacco mosaic virus (TMV) has been reported to be a suppressor of RNA silencing but the mechanism of its function remains unclear. This protein is unique among the known plant viral silencing suppressors because of its large size and multiple domains. Here, we report that the methyltransferase, helicase, and nonconserved region II (NONII) of p126 each has silencing-suppressor function. The silencing-suppression activities of methyltransferase and helicase can be uncoupled from their enzyme activities. Specific amino acids in NONII previously shown to be crucial for viral accumulation and symptom development are also crucial for silencing suppression. These results suggest that some viral proteins have evolved to possess modular structural domains that can independently interfere with host silencing, and that this may be an effective mechanism of increasing the robustness of a virus.

Multiple artificial microRNAs targeting conserved motifs of the replicase gene confer robust transgenic resistance to negative-sense single-stranded RNA plant virus.

MicroRNAs (miRNAs) regulate the abundance of target mRNAs by guiding cleavage at sequence complementary regions. In this study, artificial miRNAs (amiRNAs) targeting conserved motifs of the L (replicase) gene of Watermelon silver mottle virus (WSMoV) were constructed using Arabidopsis pre-miRNA159a as the backbone. The constructs included six single amiRNAs targeting motifs A, B1, B2, C, D of E, and two triple amiRNAs targeting motifs AB1E or B2DC. Processing of pre-amiRNAs was confirmed by agro-infiltration, and transgenic Nicotiana benthamiana plants expressing each amiRNA were generated. Single amiRNA transgenic lines expressing amiR-LB2 or amiR-LD showed resistance to WSMoV by delaying symptom development. Triple amiRNA lines expressing amiR-LB2, amiR-LD and amiR-LC provided complete resistance against WSMoV, with no indication of infection 28 days after inoculation. Resistance levels were positively correlated with amiRNA expression levels in these single and triple amiRNA lines. The triple amiR-LAB1E line did not provide resistance to WSMoV. Similarly, the poorly expressed amiR-LC and amiR-LE lines did not provide resistance to WSMoV. The amiR-LA- and amiR-LB1-expressing lines were susceptible to WSMoV, and their additional susceptibility to the heterologous Turnip mosaic virus harbouring individual target sequences indicated that these two amiRNAs have no effect in vivo. Transgenic lines expressing amiR-LB2 exhibited delayed symptoms after challenge with Peanut bud necrosis virus having a single mismatch in the target site. Overall, our results indicate that two amiRNAs, amiR-LB2 and amiR-LD, of the six designed amiRNAs confer moderate resistance against WSMoV, and the triple construct including the two amiRNAs provides complete resistance.

Discriminating mutations of HC-Pro of zucchini yellow mosaic virus with differential effects on small RNA pathways involved in viral pathogenicity and symptom development.

Helper component-proteinase (HC-Pro), the gene-silencing suppressor of Potyvirus spp., interferes with microRNA (miRNA) and short-interfering RNA (siRNA) pathways. Our previous studies showed that three mutations of highly conserved amino acids of HC-Pro, R(180)I (mutation A), F(205)L (B), and E(396)N (C), of Zucchini yellow mosaic virus (ZYMV) affect symptom severity and viral pathogenicity. The mutant ZYMV GAC (ZGAC) with double mutations, R(180)I/E(396)N, induces transient leaf mottling in host plants followed by recovery. This mutant confers complete cross protection against subsequent infection by the parental ZYMV (ZG) strain. Here, we sought to obtain molecular evidence on the roles of the three highly conserved amino acids of HC-Pro in miRNA and siRNA pathways using transgenic Arabidopsis plants expressing comparable levels of wild-type and mutant HC-Pro proteins. We demonstrated that amino acid residues 180, 205, and 396 of HC-Pro are critical for suppression of miRNA, trans-acting siRNA (ta-siRNA), and virus-induced gene silencing (VIGS) pathways but not for sense-post transcriptional gene silencing (s-PTGS). Because the HC-Pro double mutant (R(180)I/E(396)N) does not interfere with miRNA and ta-siRNA pathways, the ZGAC mutant virus elicits only attenuated symptoms. Furthermore, the recovery seen on ZGAC-infected plants likely results from the weak VIGS suppression by the HC-Pro double AC mutant. Thus, through manipulating these three conserved amino acids on HC-Pro, symptom severity of diseases caused by Potyvirus spp. can be modulated to generate useful cross protectants for field application. Although some of our mutated HC-Pro proteins do not interfere with miRNA and ta-siRNA pathways, they still retain the ability to suppress s-PTGS.

Molecular evolution of a viral non-coding sequence under the selective pressure of amiRNA-mediated silencing.

Plant microRNAs (miRNA) guide cleavage of target mRNAs by DICER-like proteins, thereby reducing mRNA abundance. Native precursor miRNAs can be redesigned to target RNAs of interest, and one application of such artificial microRNA (amiRNA) technology is to generate plants resistant to pathogenic viruses. Transgenic Arabidopsis plants expressing amiRNAs designed to target the genome of two unrelated viruses were resistant, in a highly specific manner, to the appropriate virus. Here, we pursued two different goals. First, we confirmed that the 21-nt target site of viral RNAs is both necessary and sufficient for resistance. Second, we studied the evolutionary stability of amiRNA-mediated resistance against a genetically plastic RNA virus, TuMV. To dissociate selective pressures acting upon protein function from those acting at the RNA level, we constructed a chimeric TuMV harboring a 21-nt, amiRNA target site in a non-essential region. In the first set of experiments designed to assess the likelihood of resistance breakdown, we explored the effect of single nucleotide mutation within the target 21-nt on the ability of mutant viruses to successfully infect amiRNA-expressing plants. We found non-equivalency of the target nucleotides, which can be divided into three categories depending on their impact in virus pathogenicity. In the second set of experiments, we investigated the evolution of the virus mutants in amiRNA-expressing plants. The most common outcome was the deletion of the target. However, when the 21-nt target was retained, viruses accumulated additional substitutions on it, further reducing the binding/cleavage ability of the amiRNA. The pattern of substitutions within the viral target was largely dominated by G to A and C to U transitions.

Effectiveness and stability of heterologous proteins expressed in plants by Turnip mosaic virus vector at five different insertion sites.

The N-terminal (NT) regions of particular protein-coding sequences are generally used for in-frame insertion of heterologous open reading frames (ORFs) in potyviral vectors for protein expression in plants. An infectious cDNA clone of Turnip mosaic virus (TuMV) isolate YC5 was engineered at the generally used NT regions of HC-Pro and CP, and other possibly permissive sites to investigate their effectiveness to express the GFP (jellyfish green fluorescent protein) and Der p 5 (allergen from the dust mite, Dermatophagoides pteronyssinus) ORFs. The results demonstrated the permissiveness of the NT regions of P3, CIP and NIb to carry the ORFs and express the translates as part of the viral polyprotein, the processing of which released free-form proteins in the host cell milieu. However, these sites varied in their permissiveness to retain the ORFs intact and hence affect the heterologous protein expression. Moreover, strong influence of the inserted ORF and host plants in determining the permissiveness of a viral genomic context to stably carry the alien ORFs and hence to support their prolonged expression was also noticed. In general, the engineered sites were relatively more permissive to the GFP ORF than to the Der p 5 ORF. Among the hosts, the local lesion host, Chenopodium quinoa Willd. showed the highest extent of support to TuMV to stably carry the heterologous ORFs at the engineered sites and the protein expression therefrom. Among the systemic hosts, Nicotiana benthamiana Domin proved more supportive to TuMV to carry and express the heterologous ORFs than the Brassica hosts, whereas the protein expression levels were significantly higher and more stable in the plants of Brassica campestris L. var. chinensis and B. campestris L. var. ching-geeng than those in the plants of B. juncea L. and B. campestris L. var. pekinensis.