Modulation of host plant immunity by Tobamovirus proteins.

BACKGROUND: To establish successful infection, plant viruses produce profound alterations of host physiology, disturbing unrelated endogenous processes and contributing to the development of disease. In tobamoviruses, emerging evidence suggests that viral-encoded proteins display a great variety of functions beyond the canonical roles required for virus structure and replication. Among these, their modulation of host immunity appears to be relevant in infection progression. SCOPE: In this review, some recently described effects on host plant physiology of Tobacco mosaic virus (TMV)-encoded proteins, namely replicase, movement protein (MP) and coat protein (CP), are summarized. The discussion is focused on the effects of each viral component on the modulation of host defense responses, through mechanisms involving hormonal imbalance, innate immunity modulation and antiviral RNA silencing. These effects are described taking into consideration the differential spatial distribution and temporality of viral proteins during the dynamic process of replication and spread of the virus. CONCLUSION: In discussion of these mechanisms, it is shown that both individual and combined effects of viral-encoded proteins contribute to the development of the pathogenesis process, with the host plant's ability to control infection to some extent potentially advantageous to the invading virus.

Transgenic expression of Tobacco mosaic virus capsid and movement proteins modulate plant basal defense and biotic stress responses in Nicotiana tabacum.

Plant viruses cause metabolic and physiological changes associated with symptomatic disease phenotypes. Symptoms involve direct and indirect effects, which result in disruption of host physiology. We used transgenic tobacco expressing a variant of Tobacco mosaic virus (TMV) coat protein (CP(T42W)) or movement protein (MP), and a hybrid line (MP×CP(T42W)) that coexpresses both proteins, to study the plant response to individual viral proteins. Findings employing microarray analysis of MP×CP(T42W) plants and silenced mp×cp(T42W)* controls revealed that altered transcripts were mostly downregulated, suggesting a persistent shut-off due to MP×CP(T42W) expression. Next, we showed that MP triggered reactive oxygen species (ROS) accumulation, reduction of total ascorbate, and expression of ROS scavenging genes. These effects were enhanced when both proteins were coexpressed. MP and MP×CP(T42W) plants showed increased levels of salicylic acid (SA) and SA-responsive gene expression. Furthermore, these effects were partially reproduced in Nicotiana benthamiana when GMP1 transcript was silenced. CP(T42W) seems to be playing a negative role in the defense response by reducing the expression of PR-1 and RDR-1. MP and MP×CP(T42W) transgenic expression promoted a recovery-like phenotype in TMV RNA infections and enhanced susceptibility to Pseudomonas syringae and Sclerotinia sclerotiorum. The individual effects of viral proteins may reflect the ability of a virus to balance its own virulence.

Posttranscriptional Gene Silencing Does Not Play a Significant Role in Potato virus X Coat Protein-Mediated Resistance.

ABSTRACT The expression of a gene that encodes coat protein (CP) of Potato virus X (PVX) in transgenic tobacco plants confers a high level of CP-mediated rresistance (CP-MR) against PVX infection. To determine if posttranscriptional gene silencing (PTGS) plays a role in resistance, transgenic plants expressing PVX CP were challenged against PVX under conditions in which PTGS was suppressed by low temperatures or using viruses carrying PTGS suppressors. The data demonstrate that PTGS does not play a significant role in PVX CP-MR.

Identification of Echinococcus granulosus microRNAs and their expression in different life cycle stages and parasite genotypes.

The aetiological agent of cystic hydatid disease, the platyhelminth parasite Echinococcus granulosus, undergoes a series of metamorphic events during its complex life cycle. One of its developmental stages, the protoscolex, shows a remarkable degree of heterogeneous morphogenesis, being able to develop either into the vesicular or strobilar direction. Another level of complexity is added by the existence of genotypes or strains that differ in the range of intermediate hosts where they can develop and form fertile cysts. These features make E. granulosus an interesting model for developmental studies. Hence, we focused on the study of the regulation of gene expression by microRNAs (miRNAs), one of the key mechanisms that control development in metazoans and plants and which has not been analysed in E. granulosus yet. In this study, we cloned 38 distinct miRNAs, including four candidate new miRNAs that seem to be specific to Echinococcus spp. Thirty-four cloned sequences were orthologous to miRNAs already described in other organisms and were grouped in 16 metazoan miRNA families, some of them known for their role in the development of other organisms. The expression of some of the cloned miRNAs differs according to the parasite life cycle stage analysed, showing differential developmental expression. We did not detect differences in the expression of the analysed miRNAs between protoscoleces of two parasite genotypes. This work sets the scene for the study of gene regulation mediated by miRNAs in E. granulosus and provides a new approach to study the molecules involved in its developmental plasticity and intermediate host specificity. Understanding the developmental processes of E. granulosus may help to find new strategies for the control of cystic hydatid disease, caused by the metacestode stage of the parasite.

Infection and coaccumulation of tobacco mosaic virus proteins alter microRNA levels, correlating with symptom and plant development.

Infections by plant virus generally cause disease symptoms by interfering with cellular processes. Here we demonstrated that infection of Nicotiana tabacum (N.t) by plant viruses representative of the Tobamoviridae, Potyviridae, and Potexviridae families altered accumulation of certain microRNAs (miRNAs). A correlation was observed between symptom severity and alteration in levels of miRNAs 156, 160, 164,166, 169, and 171 that is independent of viral posttranscriptional gene silencing suppressor activity. Hybrid transgenic plants that produced tobacco mosaic virus (TMV) movement protein (MP) plus coat protein (CP)(T42W) (a variant of CP) exhibited disease-like phenotypes, including abnormal plant development. Grafting studies with a plant line in which both transgenes are silenced confirmed that the disease-like phenotypes are due to the coexpression of CP and MP. In hybrid MPxCP(T42W) plants and TMV-infected plants, miRNAs 156, 164, 165, and 167 accumulated to higher levels compared with nontransgenic and noninfected tissues. Bimolecular fluorescence complementation assays revealed that MP interacts with CP(T42W) in vivo and leads to the hypothesis that complexes formed between MP and CP caused increases in miRNAs that result in disease symptoms. This work presents evidence that virus infection and viral proteins influence miRNA balance without affecting posttranscriptional gene silencing and contributes to the hypothesis that viruses exploit miRNA pathways during pathogenesis.

Aggregation of TMV CP plays a role in CP functions and in coat-protein-mediated resistance.

Tobacco mosaic virus (TMV) coat protein (CP) in absence of RNA self-assembles into several different structures depending on pH and ionic strength. Transgenic plants that produce self-assembling CP are resistant to TMV infection, a phenomenon referred to as coat-protein-mediated resistance (CP-MR). The mutant CP Thr42Trp (CP(T42W)) produces enhanced CP-MR compared to wild-type CP. To establish the relationship between the formation of 20S CP aggregates and CP-MR, virus-like particles (VLPs) produced by TMV variants that yield high levels of CP-MR were characterized. We demonstrate that non-helical structures are found in VLPs formed in vivo by CP(T42W) but not by wild-type CP and suggest that the mutation shifts the intracellular equilibrium of aggregates from low to higher proportions of non-helical 20S aggregates. A similar shift in equilibrium of aggregates was observed with CP(D77R), another mutant that confers high level of CP-MR. The mutant CP(D50R) confers a level of CP-MR similar to wild-type CP and aggregates in a manner similar to wild-type CP. We conclude that increased CP-MR is correlated with a shift in intracellular equilibrium of CP aggregates, including aggregates that interfere with virus replication.

Tobacco mosaic virus (TMV) and potato virus X (PVX) coat proteins confer heterologous interference to PVX and TMV infection, respectively.

Replication of Potato virus X (PVX) was reduced in transgenic protoplasts that accumulated wild-type coat protein (CPWT) of Tobacco mosaic virus (TMV) or a mutant CP, CP(T42W), that produced highly ordered states of aggregation, including pseudovirions. This reaction is referred to as heterologous CP-mediated resistance. However, protoplasts expressing a CP mutant that abolished aggregation and did not produce pseudovirions, CPT28W, did not reduce PVX replication. Similarly, in transgenic tobacco plants producing TMV CPWT or CP(T42W), there was a delay in local cell-to-cell spread of PVX infection that was not observed in CP(T28W) plants or in non-transgenic plants. The results suggest that the quaternary structure of the TMV CP regulates the mechanism(s) of heterologous CP-mediated resistance. Similarly, transgenic protoplasts that produced PVX CP conferred transient protection against infection by TMV RNA. Transgenic plants that accumulated PVX CP reduced the cell-to-cell spread of infection and resulted in a delay in systemic infection following inoculation with TMV or TMV RNA. Heterologous CP-mediated resistance was characterized by a brief delay in systemic infection, whilst homologous CP-mediated resistance conferred reduced or no systemic infection.

Transgenic resistance in potato plants expressing potato leaf roll virus (PLRV) replicase gene sequences is RNA-mediated and suggests the involvement of post-transcriptional gene silencing.

Genetically engineered expression of replicase encoding sequences has been proposed as an efficient system to confer protection against virus diseases by eliciting protection mechanisms in the plant. Potato leaf-roll was one of the first diseases for which this kind of protection was engineered in potato plants. However, details of the protecting mechanism were not reported, so far. The ORF2b of an Argentinean strain of PLRV was cloned and sequenced finding 94% and 97% of homology with Australian and Dutch strains, respectively. To elucidate the mechanism of protection against PLRV infection, three versions of ORF2b (non-translatable sense, translatable sense with an engineered ATG and antisense) were constructed under the control of the 35S CaMV promoter and the nos terminator and introduced in potato plants (cv. Kennebec) by Agrobacterium tumefaciens-mediated transformation. Grafting infection experiments showed that resistant transgenic plants could be obtained with any of the constructs, suggesting that the mechanism of protection is independent of the expression of protein and is RNA mediated. Field trial infection confirmed that resistant transgenic events were obtained. Biolistic transient transformation experiments of leaves derived from transgenic plants using a gene coding for the fusion protein GUS-ORF2b, followed by scoring of the number of GUS expressing leaf spots, supported that the protection is mediated by a post-transcriptional gene silencing mechanism.

Coat protein regulates formation of replication complexes during tobacco mosaic virus infection.

The genome of tobacco mosaic virus (TMV) encodes replicase protein(s), movement protein (MP), and capsid protein (CP). On infection, one or more viral proteins direct the assembly of virus replication complexes (VRCs), in association with host-derived membranes. The impact of CP-mediated resistance on the structures of the replication complexes was examined in nontransgenic and transgenic BY-2 cell lines that produce wild-type CP, mutant CP(T42W), and Ds-Red, which was targeted to endoplasmic reticulum by using immunofluorescence and 3D microscopy. We developed a model of VRCs that shows a clear association of MP with and surrounding the endoplasmic reticulum. Replicase is located within the MP bodies, as well as isolated sites throughout the cell. CP surrounds the VRCs. CP enhances the production of MP and increases the size of the VRC; however, the mutant CP(T42W) reduces the amount of MP and interferes with the formation of VRCs. We propose a regulatory role of the CP in the establishment of the VRC. We suggest that the lack of formation of VRCs restricts the efficiency of virus replication and the formation of virus movement complexes, resulting in restriction of cell-cell spread of infection. This results in higher levels of plant CP-mediated protection provided by CP(T42W).

Direct labeling of monoclonal antibodies with 99mTc and radioimmunodetection of a murine mammary carcinoma with 99mTc-B2C114.

Two anti-CEA antibodies, B2C114 and IORCEA1, were radiolabeled with 99mTc by two direct methods (mercaptoethanol and ascorbic acid reduction), and the radio-immunoimaging properties of B2C114 were assessed in mice bearing an M3-reactive tumor. The labeling efficiency was greater than 90% as measured by ITLC in saline, methylethylketone and with serum albumin impregnated sheets using ethanol: water: NH4OH (2:5:1). The label was stable to challenge with excess DTPA, and in the case of ascorbic acid reduction, serum analysis showed that 10-15% of the radioactivity was lost during incubation. In vitro studies demonstrated that the radiolabeled antibodies retained their immunoreactivity. Biodistribution studies in normal Balb/c mice showed that the pattern of uptake was quite similar for both antibodies. Biodistribution of the 99mTc-B2C114 and image studies in the animal model showed that the tumor was clearly visualized and that B2C114 labeled with 99mTc is a possible candidate for human radioimmunodetection of CEA-expressing tumors.