Double-Stranded-RNA-Binding Protein 2 Participates in Antiviral Defense.

Double-stranded RNA (dsRNA) is a common pattern formed during the replication of both RNA and DNA viruses. Perception of virus-derived dsRNAs by specialized receptor molecules leads to the activation of various antiviral measures. In plants, these defensive processes include the adaptive RNA interference (RNAi) pathway and innate pattern-triggered immune (PTI) responses. While details of the former process have been well established in recent years, the latter are still only partially understood at the molecular level. Nonetheless, emerging data suggest extensive cross talk between the different antiviral mechanisms. Here, we demonstrate that dsRNA-binding protein 2 (DRB2) of Nicotiana benthamiana plays a direct role in potato virus X (PVX)-elicited systemic necrosis. These results establish that DRB2, a known component of RNAi, is also involved in a virus-induced PTI response. In addition, our findings suggest that RNA-dependent polymerase 6 (RDR6)-dependent dsRNAs play an important role in the triggering of PVX-induced systemic necrosis. Based on our data, a model is formulated whereby competition between different DRB proteins for virus-derived dsRNAs helps establish the dominant antiviral pathways that are activated in response to virus infection.IMPORTANCE Plants employ multiple defense mechanisms to restrict viral infections, among which RNA interference is the best understood. The activation of innate immunity often leads to both local and systemic necrotic responses, which confine the virus to the infected cells and can also provide resistance to distal, noninfected parts of the organism. Systemic necrosis, which is regarded as a special form of the local hypersensitive response, results in necrosis of the apical stem region, usually causing the death of the plant. Here, we provide evidence that the dsRNA-binding protein 2 of Nicotiana benthamiana plays an important role in virus-induced systemic necrosis. Our findings are not only compatible with the recent hypothesis that DRB proteins act as viral invasion sensors but also extends it by proposing that DRBs play a critical role in establishing the dominant antiviral measures that are triggered during virus infection.

Ultrahigh-activity immune inducer from Endophytic Fungi induces tobacco resistance to virus by SA pathway and RNA silencing.

BACKGROUND: Plant viruses cause severe economic losses in agricultural production. An ultrahigh activity plant immune inducer (i.e., ZhiNengCong, ZNC) was extracted from endophytic fungi, and it could promote plant growth and enhance resistance to bacteria. However, the antiviral function has not been studied. Our study aims to evaluate the antiviral molecular mechanisms of ZNC in tobacco. RESULTS: Here, we used Potato X virus (PVX), wild-type tobacco and NahG transgenic tobacco as materials to study the resistance of ZNC to virus. ZNC exhibited a high activity in enhancing resistance to viruses and showed optimal use concentration at 100-150 ng/mL. ZNC also induced reactive oxygen species accumulation, increased salicylic acid (SA) content by upregulating the expression of phenylalanine ammonia lyase (PAL) gene and activated SA signaling pathway. We generated transcriptome profiles from ZNC-treated seedlings using RNA sequencing. The first GO term in biological process was positive regulation of post-transcriptional gene silencing, and the subsequent results showed that ZNC promoted RNA silencing. ZNC-sprayed wild-type leaves showed decreased infection areas, whereas ZNC failed to induce a protective effect against PVX in NahG leaves. CONCLUSION: All results indicate that ZNC is an ultrahigh-activity immune inducer, and it could enhance tobacco resistance to PVX at low concentration by positively regulating the RNA silencing via SA pathway. The antiviral mechanism of ZNC was first revealed in this study, and this study provides a new antiviral bioagent.

[A Recombinant Rotavirus Antigen Based on the Coat Protein of Alternanthera Mosaic Virus].

Thanks to their strong immunostimulating properties and safety for humans, plant viruses represent an appropriate basis for the design of novel vaccines. The coat protein of Alternanthera mosaic virus can form virus-like particles that are stable under physiological conditions and have adjuvant properties. This work presents a recombinant human rotavirus A antigen based on the epitope of rotavirus structural protein VP6, using Alternanthera mosaic virus coat protein as a carrier. An expression vector containing the gene of Alternanthera mosaic virus (MU strain) coat protein fused to the epitope of rotavirus protein VP6 was designed. Immunoblot analysis showed that the chimeric protein was effectively recognized by commercial polyclonal antibodies to rotavirus and therefore is a suitable candidate for development of a vaccine prototype. Interaction of the chimeric recombinant protein with the native coat protein of Alternanthera mosaic virus and its RNA resulted in the formation of ribonucleoprotein complexes that were recognized by anti-rotavirus antibodies.

Distinct Roles of Non-Overlapping Surface Regions of the Coiled-Coil Domain in the Potato Immune Receptor Rx1.

The intracellular immune receptor Rx1 of potato (Solanum tuberosum), which confers effector-triggered immunity to Potato virus X, consists of a central nucleotide-binding domain (NB-ARC) flanked by a carboxyl-terminal leucine-rich repeat (LRR) domain and an amino-terminal coiled-coil (CC) domain. Rx1 activity is strictly regulated by interdomain interactions between the NB-ARC and LRR, but the contribution of the CC domain in regulating Rx1 activity or immune signaling is not fully understood. Therefore, we used a structure-informed approach to investigate the role of the CC domain in Rx1 functionality. Targeted mutagenesis of CC surface residues revealed separate regions required for the intramolecular and intermolecular interaction of the CC with the NB-ARC-LRR and the cofactor Ran GTPase-activating protein2 (RanGAP2), respectively. None of the mutant Rx1 proteins was constitutively active, indicating that the CC does not contribute to the autoinhibition of Rx1 activity. Instead, the CC domain acted as a modulator of downstream responses involved in effector-triggered immunity. Systematic disruption of the hydrophobic interface between the four helices of the CC enabled the uncoupling of cell death and disease resistance responses. Moreover, a strong dominant negative effect on Rx1-mediated resistance and cell death was observed upon coexpression of the CC alone with full-length Rx1 protein, which depended on the RanGAP2-binding surface of the CC. Surprisingly, coexpression of the N-terminal half of the CC enhanced Rx1-mediated resistance, which further indicated that the CC functions as a scaffold for downstream components involved in the modulation of disease resistance or cell death signaling.

The quest for a nanoparticle-based vaccine inducing broad protection to influenza viruses.

Influenza virus infections are a significant public threat and the best approach to prevent them is through vaccination. Because of the perpetual changes of circulating influenza strains, the efficacy of influenza vaccines rarely exceeds 50%. To improve the protection efficacy, we have designed a novel vaccine formulation that shows a broad range of protection. The formulation is made of the matrix protein 2 (M2e) and the nucleoprotein (NP) antigens. The multimerization of NP into nanoparticles improved significantly the immune response to NP. The combination of the NP nanoparticles with the PapMV-M2e nanoparticles enhances significantly the immune response directed to NP revealing the adjuvant property of the PapMV platform. The vaccine formulation combining these two types of nanoparticles protects mice from infectious challenges by two different influenza strains (H1N1 and H3N2) and is a promising influenza A vaccine capable to elicit a broad protection.

Activation of innate immunity in primary human cells using a plant virus derived nanoparticle TLR7/8 agonist.

Rod-shaped virus-like nanoparticles (VLNP) made of papaya mosaic virus (PapMV) coat proteins (CP) self-assembled around a single stranded RNA (ssRNA) were showed to be a TLR7 agonist. Their utilization as an immune modulator in cancer immunotherapy was shown to be promising. To establish a clinical relevance in human for PapMV VLNP, we showed that stimulation of human peripheral blood mononuclear cells (PBMC) with VLNP induces the secretion of interferon alpha (IFNα) and other pro-inflammatory cytokines and chemokines. Plasmacytoid dendritic cells (pDCs) were activated and secreted IFN-α upon VLNP exposure. Monocyte-derived dendritic cells upregulate maturation markers and produce IL-6 in response to PapMV VLNP stimulation, which suggests the activation of TLR8. Finally, when co-cultured with NK cells, PapMV induced pDCs promoted the NK cytolytic activity against cancer cells. These data obtained with primary human immune cells further strengthen the clinical relevance of PapMV VLNPs as a cancer immunotherapy agent.

Combination of Plant Virus Nanoparticle-Based in Situ Vaccination with Chemotherapy Potentiates Antitumor Response.

Immunotherapeutics are gaining more traction in the armamentarium used to combat cancer. Specifically, in situ vaccination strategies have gained interest because of their ability to alter the tumor microenvironment to an antitumor state. Herein, we investigate whether flexuous plant virus-based nanoparticles formed by the potato virus X (PVX) can be used as an immunotherapeutic for in situ vaccine monotherapy. We further developed dual chemo-immunotherapeutics by incorporating doxorubicin (DOX) into PVX yielding a dual-functional nanoparticle (PVX-DOX) or by coadministration of the two therapeutic regimes, PVX immunotherapy and DOX chemotherapy (PVX+DOX). In the context of B16F10 melanoma, PVX was able to elicit delayed tumor progression when administered as an intratumoral in situ vaccine. Furthermore, the coadministration of DOX via PVX+DOX enhanced the response of the PVX monotherapy through increased survival, which was also represented in the enhanced antitumor cytokine/chemokine profile stimulated by PVX+DOX when compared to PVX or DOX alone. Importantly, coadministered PVX+DOX was better for in situ vaccination than PVX loaded with DOX (PVX-DOX). Whereas the nanomedicine field strives to design multifunctional nanoparticles that integrate several functions and therapeutic regimens into a single nanoparticle, our data suggest a paradigm shift; some therapeutics may need to be administered separately to synergize and achieve the most potent therapeutic outcome. Altogether, our studies show that development of plant viral nanoparticles for in situ vaccines for treatment is a possibility, and dual mechanistic therapeutics can increase efficacy. Nonetheless, combining immunotherapeutics with cytolytic chemotherapy requires detailed investigation to inform optimal integration of cytolytic and immunotherapies and maximize synergy and efficacy.

A versatile papaya mosaic virus (PapMV) vaccine platform based on sortase-mediated antigen coupling.

BACKGROUND: Flexuous rod-shaped nanoparticles made of the coat protein (CP) of papaya mosaic virus (PapMV) have been shown to trigger innate immunity through engagement of toll-like receptor 7 (TLR7). PapMV nanoparticles can also serve as a vaccine platform as they can increase the immune response to fused peptide antigens. Although this approach shows great potential, fusion of antigens directly to the CP open reading frame (ORF) is challenging because the fused peptides can alter the structure of the CP and its capacity to self assemble into nanoparticles-a property essential for triggering an efficient immune response to the peptide. This represents a serious limitation to the utility of this approach as fusion of small peptides only is tolerated. RESULTS: We have developed a novel approach in which peptides are fused directly to pre-formed PapMV nanoparticles. This approach is based on the use of a bacterial transpeptidase (sortase A; SrtA) that can attach the peptide directly to the nanoparticle. An engineered PapMV CP harbouring the SrtA recognition motif allows efficient coupling. To refine our engineering, and to predict the efficacy of coupling with SrtA, we modeled the PapMV structure based on the known structure of PapMV CP and on recent reports revealing the structure of two closely related potexviruses: pepino mosaic virus (PepMV) and bamboo mosaic virus (BaMV). We show that SrtA can allow the attachment of long peptides [Influenza M2e peptide (26 amino acids) and the HIV-1 T20 peptide (39 amino acids)] to PapMV nanoparticles. Consistent with our PapMV structural model, we show that around 30% of PapMV CP subunits in each nanoparticle can be fused to the peptide antigen. As predicted, engineered nanoparticles were capable of inducing a strong antibody response to the fused antigen. Finally, in a challenge study with influenza virus, we show that mice vaccinated with PapMV-M2e are protected from infection. CONCLUSIONS: This technology will allow the development of vaccines harbouring long peptides containing several B and/or T cell epitopes that can contribute to a broad and robust protection from infection. The design can be fast, versatile and can be adapted to the development of vaccines for many infectious diseases as well as cancer vaccines.

The effector SPRYSEC-19 of Globodera rostochiensis suppresses CC-NB-LRR-mediated disease resistance in plants.

The potato cyst nematode Globodera rostochiensis invades roots of host plants where it transforms cells near the vascular cylinder into a permanent feeding site. The host cell modifications are most likely induced by a complex mixture of proteins in the stylet secretions of the nematodes. Resistance to nematodes conferred by nucleotide-binding-leucine-rich repeat (NB-LRR) proteins usually results in a programmed cell death in and around the feeding site, and is most likely triggered by the recognition of effectors in stylet secretions. However, the actual role of these secretions in the activation and suppression of effector-triggered immunity is largely unknown. Here we demonstrate that the effector SPRYSEC-19 of G. rostochiensis physically associates in planta with the LRR domain of a member of the SW5 resistance gene cluster in tomato (Lycopersicon esculentum). Unexpectedly, this interaction did not trigger defense-related programmed cell death and resistance to G. rostochiensis. By contrast, agroinfiltration assays showed that the coexpression of SPRYSEC-19 in leaves of Nicotiana benthamiana suppresses programmed cell death mediated by several coiled-coil (CC)-NB-LRR immune receptors. Furthermore, SPRYSEC-19 abrogated resistance to Potato virus X mediated by the CC-NB-LRR resistance protein Rx1, and resistance to Verticillium dahliae mediated by an unidentified resistance in potato (Solanum tuberosum). The suppression of cell death and disease resistance did not require a physical association of SPRYSEC-19 and the LRR domains of the CC-NB-LRR resistance proteins. Altogether, our data demonstrated that potato cyst nematodes secrete effectors that enable the suppression of programmed cell death and disease resistance mediated by several CC-NB-LRR proteins in plants.

Virus adaptation to quantitative plant resistance: erosion or breakdown?

Adaptation of populations to new environments is frequently costly due to trade-offs between life history traits, and consequently, parasites are expected to be locally adapted to sympatric hosts. Also, during adaptation to the host, an increase in parasite fitness could have direct consequences on its aggressiveness (i.e. the quantity of damages caused to the host by the virus). These two phenomena have been observed in the context of pathogen adaptation to host's qualitative and monogenic resistances. However, the ability of pathogens to adapt to quantitative polygenic plant resistances and the consequences of these potential adaptations on other pathogen life history traits remain to be evaluated. Potato virus Y and two pepper genotypes (one susceptible and one with quantitative resistance) were used, and experimental evolutions showed that adaptation to a quantitative resistance was possible and resulted in resistance breakdown. This adaptation was associated to a fitness cost on the susceptible cultivar, but had no consequence either in terms of aggressiveness, which could be explained by a high tolerance level, or in terms of aphid transmission efficiency. We concluded that quantitative resistances are not necessarily durable but management strategies mixing susceptible and resistant cultivars in space and/or in time should be useful to preserve their efficiency.

Engineering broad-spectrum resistance against RNA viruses in potato.

RNA silencing technology has become the tool of choice for inducing resistance against viruses in plants. A significant discovery of this technology is that double-stranded RNA (dsRNA), which is diced into small interfering RNAs (siRNAs), is a potent trigger for RNA silencing. By exploiting this phenomenon in transgenic plants, it is possible to confer high level of virus resistance by specific targeting of cognate viral RNA. In order to maximize the efficiency and versatility of the vector-based siRNA approach, we have constructed a chimeric expression vector containing three partial gene sequences derived from the ORF2 gene of Potato virus X, Helper Component Protease gene of Potato virus Y and Coat protein gene of Potato leaf roll virus. Solanum tuberosum cv. Desiree and Kuroda were transformed with this chimeric gene cassette via Agrobacterium tumefaciens-mediated transformation and transgenic status was confirmed by PCR, Southern and double antibody sandwich ELISA detection. Due to simultaneous RNA silencing, as demonstrated by accumulation of specific siRNAs, the expression of partial triple-gene sequence cassette depicted 20% of the transgenic plants are immune against all three viruses. Thus, expression of a single transgene construct can effectively confer resistance to multiple viruses in transgenic plants.

Factors influencing the detection limit of the lateral-flow sandwich immunoassay: a case study with potato virus X.

Key factors influencing the analyte detection limit of the sandwich immunochromatographic assay (ICA), namely, the size of gold nanoparticles, the antibody concentration, the conjugation pH, and characteristics of membranes, are discussed. The impacts of these factors were quantitatively characterized and compared for the first time using the same antigen (potato virus X). The antibody-colloidal gold conjugates synthesized at pH 9.0-9.5 (the pH was examined in the range from 7.5 to 10.0) and at an antibody concentration of 15 µg/mL (the concentration was tested from 10 to 100 µg/mL) demonstrated maximum binding with the analyte. The relationship between the size of gold nanoparticles and the ICA detection limit was determined. The detection limit decreases from 80 to 3 ng/mL (for antibodies with K (D) = 1.0 × 10(-9) M, data were obtained using a BIAcore X instrument) for a series of particles with a diameter from 6.4 to 33.4 nm (electron microscopy and dynamic light scattering data). In the case of larger particles (52 nm in diameter), the detection limit increases and reaches 9 ng/mL. A 10 mM phosphate buffer, pH 8, and a 50 mM phosphate buffer, pH 7, were the conditions of choice for the deposition of reactants. Taking into account these facts, we developed a lateral-flow test system for the rapid (10 min) detection of potato virus X in plant leaves. The ICA provided a visual detection limit of 3 ng/mL. In the case of the instrumental processing, potato virus X can be determined in the concentration range from 3 to 300 ng/mL with a detection limit 2 ng/mL.

Highly sensitive field test lateral flow immunodiagnostics of PVX infection.

A test system is described and expanded upon for mass field immunochromatography assay on porous membrane carriers for rapid diagnostics of potato virus X (PVX) in potato leaf tissue and sprout extracts using colloidal gold nanoparticles as a marker. Sensitivity of the assay developed for PVX identification is found to be comparable to the sensitivity of solid-phase sandwich-ELISA. Complete assay time does not exceed 15 min, and the lower limit of the PVX detection in non-clarified leaf extract is 2 ng/ml. A single measurement requires 0.1-0.2 ml (3-5 drops) of tested solution only (extracted from 10-20 mg of potato leaf tissue or sprouts). The simplicity and reliability of the method makes it especially efficient in direct rapid monitoring of many infected potato specimens in the field, as verified by field trials of 360 clones of 28 domestic and foreign cultivars of potato. A diagnostic kit for routine analyses of potato viral infections both in the laboratory and in the field is described and expanded upon.

Expression of Human papillomavirus 16 E7ggg oncoprotein on N- and C-terminus of Potato virus X coat protein in bacterial and plant cells.

The E7 oncoprotein from Human papillomavirus type 16 (HPV16) is an attractive candidate for anti-cancer therapeutical vaccine development. In this study, we engineered different fusions of mutagenized coding sequence of E7 oncoprotein (E7ggg) with coat protein of Potato virus X (PVX CP) both on 5'- and 3'-terminus of PVX CP and evaluated the influence of the length of linker (no linker, 4, 15aa) connecting PVX CP and E7ggg on their production. At first the expression in Escherichia coli was conducted to assess the characteristics of the recombinant protein prior to be further produced in plants, that is, resultant proteins were used for screening of their immunological reactivity with antibodies against PVX CP and E7. Fusion proteins successfully expressed in bacteria and plants were partially purified and their reactivity and ability to form virus-like particles were evaluated with anti-E7 antibodies.

Stable Display of Artificially Long Foreign Antigens on Chimeric Bamboo mosaic virus Particles.

Plant viruses can be genetically modified to generate chimeric virus particles (CVPs) carrying heterologous peptides fused on the surface of coat protein (CP) subunits as vaccine candidates. However, some factors may be especially significant in determining the properties of chimeras. In this study, peptides from various sources and of various lengths were inserted into the Bamboo mosaic virus-based (BaMV) vector CP N-terminus to examine the chimeras infecting and accumulating in plants. Interestingly, it was found that the two different strains Foot-and-mouth disease virus (FMDV) VP1 antigens with flexible linker peptides (77 or 82 amino acids) were directly expressed on the BaMV CP, and the chimeric particles self-assembled and continued to express FMDV antigens. The chimeric CP, when directly fused with a large foreign protein (117 amino acids), can self-fold into incomplete virus particles or disks. The physicochemical properties of heterologus peptides N-terminus, complex strand structures of heterologus peptides C-terminus and different flexible linker peptides, can affect the chimera accumulation. Based on these findings, using plant virus-based chimeras to express foreign proteins can increase their length limitations, and engineered plant-made CVP-based vaccines have increasing potential for further development as novel vaccines.

Convenient Auto-Processing Vector Based on Bamboo Mosaic Virus for Presentation of Antigens Through Enzymatic Coupling.

We have developed a new binary epitope-presenting CVP platform based on bamboo mosaic virus (BaMV) by using the sortase A (SrtA)-mediated ligation technology. The reconstructed BaMV genome harbors two modifications: 1) a coat protein (CP) with N-terminal extension of the tobacco etch virus (TEV) protease recognition site plus 4 extra glycine (G) residues as the SrtA acceptor; and 2) a TEV protease coding region replacing that of the triple-gene-block proteins. Inoculation of such construct, pKB5G, on Nicotiana benthamiana resulted in the efficient production of filamentous CVPs ready for SrtA-mediated ligation with desired proteins. The second part of the binary platform includes an expression vector for the bacterial production of donor proteins. We demonstrated the applicability of the platform by using the recombinant envelope protein domain III (rEDIII) of Japanese encephalitis virus (JEV) as the antigen. Up to 40% of the BaMV CP subunits in each CVP were loaded with rEDIII proteins in 1 min. The rEDIII-presenting BaMV CVPs (BJLPET5G) could be purified using affinity chromatography. Immunization assays confirmed that BJLPET5G could induce the production of neutralizing antibodies against JEV infections. The binary platform could be adapted as a useful alternative for the development and mass production of vaccine candidates.

Multiple coat protein mutations abolish recognition of Pepino mosaic potexvirus (PepMV) by the potato rx resistance gene in transgenic tomatoes.

Despite the fact that Pepino mosaic virus (PepMV) and Potato virus X (PVX) share less than 40% identity in their coat proteins (CP), the known PVX elicitor of Rx, transgenic tomato (cv. Microtom) plants expressing a functional potato Rx resistance gene showed resistance toward PepMV. However, in a low percentage of plants, PepMV accumulation was observed and back inoculation experiments demonstrated that these plants contained resistance-breaking PepMV variants. Sequencing of the CP gene of these variants showed the accumulation of mutations in the amino acid 41 to 125 region the CP, whereas no mutations were observed in the nonevolved isolates. Agroinfiltration-mediated transient expression of the mutant CP demonstrated that they had a greatly attenuated or abolished ability to induce a hypersensitive reaction in Rx-expressing Nicotiana benthamiana leaves. The transient expression of truncated forms of the PepMV CP allowed the identification of a minimal elicitor domain (amino acids 30 to 136). These results demonstrate that the Rx-based sensing system is able to recognize the PepMV CP but, contrary to the situation with PVX, for which only two closely spaced resistance-breaking mutations are known, many mutations over a significant stretch of the PepMV CP allow escape from recognition by Rx.

Salicylic acid is involved in the Nb-mediated defense responses to Potato virus X in Solanum tuberosum.

To evaluate the role of salicylic acid (SA) in Nb-mediated hypersensitive resistance to Potato virus X (PVX) avirulent strain ROTH1 in Solanum tuberosum, we have constructed SA-deficient transgenic potato plant lines by overexpressing the bacterial enzyme salicylate hydroxylase (NahG), which degrades SA. Evaluation of these transgenic lines revealed hydrogen peroxide accumulation and spontaneous lesion formation in an age- and light-dependent manner. In concordance, NahG potato plants were more sensitive to treatment with methyl viologen, a reactive oxygen species-generating compound. In addition, when challenged with PVX ROTH1, NahG transgenic lines showed a decreased disease-resistance response to infection and were unable to induce systemic acquired resistance. However, the avirulent viral effector, the PVX 25-kDa protein, does induce expression of the pathogenesis-related gene PR-1a in NahG potato plants. Taken together, our data indicate that SA is involved in local and systemic defense responses mediated by the Nb gene in Solanum tuberosum. This is the first report to show that basal levels of SA correlate with hypersensitive resistance to PVX.

Identification of Pns6, a putative movement protein of RRSV, as a silencing suppressor.

RNA silencing is a potent antiviral response in plants. As a counterdefense, most plant and some animal viruses encode RNA silencing suppressors. In this study, we showed that Pns6, a putative movement protein of Rice ragged stunt virus (RRSV), exhibited silencing suppressor activity in coinfiltration assays with the reporter green fluorescent protein (GFP) in transgenic Nicotiana benthamiana line 16c. Pns6 of RRSV suppressed local silencing induced by sense RNA but had no effect on that induced by dsRNA. Deletion of a region involved in RNA binding abolished the silencing suppressor activity of Pns6. Further, expression of Pns6 enhanced Potato virus × pathogenicity in N. benthamiana. Collectively, these results suggested that RRSV Pns6 functions as a virus suppressor of RNA silencing that targets an upstream step of the dsRNA formation in the RNA silencing pathway. This is the first silencing suppressor to be identified from the genus Oryzavirus.

Differential Accumulation of Innate- and Adaptive-Immune-Response-Derived Transcripts during Antagonism between Papaya Ringspot Virus and Papaya Mosaic Virus.

Papaya ringspot virus (PRSV), a common potyvirus infecting papaya plants worldwide, can lead to either antagonism or synergism in mixed infections with Papaya mosaic virus (PapMV), a potexvirus. These two unrelated viruses produce antagonism or synergism depending on their order of infection in the plant. When PRSV is inoculated first or at the same time as PapMV, the viral interaction is synergistic. However, an antagonistic response is observed when PapMV is inoculated before PRSV. In the antagonistic condition, PRSV is deterred from the plant and its drastic effects are overcome. Here, we examine differences in gene expression by high-throughput RNA sequencing, focused on immune system pathways. We present the transcriptomic expression of single and mixed inoculations of PRSV and PapMV leading to synergism and antagonism. Upregulation of dominant and hormone-mediated resistance transcripts suggests that the innate immune system participates in synergism. In antagonism, in addition to innate immunity, upregulation of RNA interference-mediated resistance transcripts suggests that adaptive immunity is involved.