Development of Tobacco Mosaic Virus Infection Sites in Nicotiana benthamiana

To monitor infection of Nicotiana benthamiana by tobacco mosaic virus (TMV), leaves were inoculated with viral constructs expressing the green fluorescent protein (GFP) from jellyfish (Aequorea victoria) fused to the movement protein (MP) of TMV (MP:GFP) or as a free GFP in place of the coat protein (CP). Infection sites produced by TMV expressing the MP:GFP appeared as fluorescent rings larger in diameter and less fluorescent than fluorescent disks induced by constructs encoding free GFP. These results suggest that protein expression driven by the MP subgenomic promoter (sgp) initiates and ends earlier and is at lower level than that observed for proteins driven by the CP sgp. Similarly, analyses of cross sections through the infection sites revealed that in different cell types the accumulation of MP:GFP was regulated differently than the accumulation of free GFP. Immunocytochemistry and electron microscopy showed that near the leading edge of the fluorescent ring the MP:GFP and the viral 126 kDa and 183 kDa replicase proteins accumulated in paired cytoplasmic bodies that formed often on opposite sides of adjacent cell walls containing plasmodesmata. In the dimly fluorescent center of the rings the 126 kDa and 183 kDa proteins, but not the MP:GFP, were localized in unpaired cytoplasmic bodies containing ropelike, fibrillar structures. The paired bodies were similar to previously described viroplasms, while the unpaired bodies were similar to X-bodies. These data indicate that the accumulation of proteins expressed from different sgps of TMV has a specific spatial and temporal pattern in planta. In addition, the cytoplasmic bodies containing the 126 kDa and 183 kDa proteins are dynamic entities whose protein content and subcellular location change during infection.

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Heterologous Expression of CLIBASIA_03915/CLIBASIA_04250 by Tobacco Mosaic Virus Resulted in Phloem Necrosis in the Senescent Leaves of Nicotiana benthamiana

International Journal of Molecular Sciences ◽

10.3390/ijms21041414 ◽

2020 ◽

Vol 21 (4) ◽

pp. 1414 ◽

Cited By ~ 5

Author(s):

Hui Li ◽

Xiaobao Ying ◽

Lina Shang ◽

Bryce Redfern ◽

Nicholas Kypraios ◽

...

Keyword(s):

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Nicotiana Benthamiana ◽

Fluorescent Protein ◽

Nuclear Localization Sequence ◽

Localization Sequence ◽

Candidatus Liberibacter ◽

Phloem Necrosis ◽

Liberibacter Asiaticus ◽

Green Fluorescent

Huanglongbing (HLB), also known as citrus greening, is the most notorious citrus disease worldwide. Candidatus Liberibacter asiaticus (CaLas) is a phloem-restricted bacterium associated with HLB. Because there is no mutant library available, the pathogenesis of CaLas is obscure. In this study, we employed tobacco mosaic virus (TMV) to express two mature secretion proteins CLIBASIA_03915 (m03915) and CLIBASIA_04250 (m04250) in Nicotiana benthamiana (N. benthamiana). Phloem necrosis was observed in the senescent leaves of N. benthamiana that expressed the two low molecular weight proteins, while no phloem necrosis was observed in the plants that expressed the control, green fluorescent protein (GFP). Additionally, no phloem necrosis was observed in the senescent leaves of N. benthamiana that expressed the null mutation of m03915 and frameshifting m04250. The subcellular localizations of m03915 and m04250 were determined by fusion with GFP using confocal microscopy. The subcellular localization of m03915 was found to be as free GFP without a nuclear localization sequence (NLS). However, m04250 did have an NLS. Yeast two-hybrid (Y2H) was carried out to probe the citrus proteins interacting with m03915 and m04250. Six citrus proteins were found to interact with m03915. The identified proteins were involved in the metabolism of compounds, transcription, response to abiotic stress, ubiquitin-mediated protein degradation, etc. The prey of m04250 was involved in the processing of specific pre-mRNAs. Identification of new virulence factors of CaLas will give insight into the pathogenesis of CaLas, and therefore, it will eventually help develop the HLB-resistant citrus.

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ORF6 of Tobacco mosaic virus is a determinant of viral pathogenicity in Nicotiana benthamiana

Journal of General Virology ◽

10.1099/vir.0.80270-0 ◽

2004 ◽

Vol 85 (10) ◽

pp. 3123-3133 ◽

Cited By ~ 19

Author(s):

Tomas Canto ◽

Stuart A. MacFarlane ◽

Peter Palukaitis

Keyword(s):

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Nicotiana Benthamiana ◽

Fluorescent Protein ◽

Tobacco Rattle Virus ◽

Potato Virus X ◽

Host Responses ◽

Reading Frame ◽

Virus Accumulation ◽

Green Fluorescent

Tobacco mosaic virus (TMV) contains a sixth open reading frame (ORF6) that potentially encodes a 4·8 kDa protein. Elimination of ORF6 from TMV attenuated host responses in Nicotiana benthamiana without alteration in virus accumulation. Furthermore, heterologous expression of TMV ORF6 from either potato virus X (PVX) or tobacco rattle virus (TRV) vectors enhanced the virulence of both viruses in N. benthamiana, also without effects on their accumulation. By contrast, the presence or absence of TMV ORF6 had no effect on host response or virus accumulation in N. tabacum plants infected with TMV or PVX. TMV ORF6 also had no effect on the synergism between TMV and PVX in N. tabacum. However, the presence of the TMV ORF6 did have an effect on the pathogenicity of a TRV vector in N. tabacum. In three different types of assay carried out in N. benthamiana plants, expression of TMV ORF6 failed to suppress gene silencing. Expression in N. benthamiana epidermal cells of the encoded 4·8 kDa protein fused to the green fluorescent protein at either end showed, in addition to widespread cytosolic fluorescence, plasmodesmatal targeting specific to both fusion constructs. The role of the ORF6 in host responses is discussed.

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In Planta Synthesis of Designer-Length Tobacco Mosaic Virus-Based Nano-Rods That Can Be Used to Fabricate Nano-Wires

Frontiers in Plant Science ◽

10.3389/fpls.2017.01335 ◽

2017 ◽

Vol 8 ◽

Cited By ~ 9

Author(s):

Keith Saunders ◽

George P. Lomonossoff

Keyword(s):

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

In Planta ◽

Nano Rods ◽

Nano Wires

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Cellular Targets of Functional and Dysfunctional Mutants of Tobacco Mosaic Virus Movement Protein Fused to Green Fluorescent Protein

Journal of Virology ◽

10.1128/jvi.74.23.11339-11346.2000 ◽

2000 ◽

Vol 74 (23) ◽

pp. 11339-11346 ◽

Cited By ~ 58

Author(s):

Vitaly Boyko ◽

Jessica van der Laak ◽

Jacqueline Ferralli ◽

Elena Suslova ◽

Myoung-Ok Kwon ◽

...

Keyword(s):

Amino Acids ◽

Green Fluorescent Protein ◽

Tobacco Mosaic Virus ◽

Inclusion Bodies ◽

Fluorescent Protein ◽

Movement Protein ◽

Leading Edge ◽

Intercellular Transport ◽

C Terminus ◽

Green Fluorescent

ABSTRACT Intercellular transport of tobacco mosaic virus (TMV) RNA involves the accumulation of virus-encoded movement protein (MP) in plasmodesmata (Pd), in endoplasmic reticulum (ER)-derived inclusion bodies, and on microtubules. The functional significance of these interactions in viral RNA (vRNA) movement was tested in planta and in protoplasts with TMV derivatives expressing N- and C-terminal deletion mutants of MP fused to the green fluorescent protein. Deletion of 55 amino acids from the C terminus of MP did not interfere with the vRNA transport function of MP:GFP but abolished its accumulation in inclusion bodies, indicating that accumulation of MP at these ER-derived sites is not a requirement for function in vRNA intercellular movement. Deletion of 66 amino acids from the C terminus of MP inactivated the protein, and viral infection occurred only upon complementation in plants transgenic for MP. The functional deficiency of the mutant protein correlated with its inability to associate with microtubules and, independently, with its absence from Pd at the leading edge of infection. Inactivation of MP by N-terminal deletions was correlated with the inability of the protein to target Pd throughout the infection site, whereas its associations with microtubules and inclusion bodies were unaffected. The observations support a role of MP-interacting microtubules in TMV RNA movement and indicate that MP targets microtubules and Pd by independent mechanisms. Moreover, accumulation of MP in Pd late in infection is insufficient to support viral movement, confirming that intercellular transport of vRNA relies on the presence of MP in Pd at the leading edge of infection.

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Autophagy is involved in assisting the replication of Bamboo mosaic virus in Nicotiana benthamiana

Journal of Experimental Botany ◽

10.1093/jxb/erz244 ◽

2019 ◽

Vol 70 (18) ◽

pp. 4657-4670 ◽

Cited By ~ 5

Author(s):

Ying-Ping Huang ◽

Ying-Wen Huang ◽

Yung-Jen Hsiao ◽

Siou-Cen Li ◽

Yau-Huei Hsu ◽

...

Keyword(s):

Mosaic Virus ◽

Nicotiana Benthamiana ◽

Fluorescent Protein ◽

Rna Virus ◽

Critical Role ◽

Viral Rna ◽

Autophagosome Formation ◽

Orange Fluorescent Protein ◽

Positive Sense ◽

Trigger Cell Death

Abstract Autophagy plays a critical role in plants under biotic stress, including the response to pathogen infection. We investigated whether autophagy-related genes (ATGs) are involved in infection with Bamboo mosaic virus (BaMV), a single-stranded positive-sense RNA virus. Initially, we observed that BaMV infection in Nicotiana benthamiana leaves upregulated the expression of ATGs but did not trigger cell death. The induction of ATGs, which possibly triggers autophagy, increased rather than diminished BaMV accumulation in the leaves, as revealed by gene knockdown and transient expression experiments. Furthermore, the inhibitor 3-methyladenine blocked autophagosome formation and the autophagy inducer rapamycin, which negatively and positively affected BaMV accumulation, respectively. Pull-down experiments with an antibody against orange fluorescent protein (OFP)-NbATG8f, an autophagosome marker protein, showed that both plus- and minus-sense BaMV RNAs could associate with NbATG8f. Confocal microscopy revealed that ATG8f-enriched vesicles possibly derived from chloroplasts contained both the BaMV viral RNA and its replicase. Thus, BaMV infection may induce the expression of ATGs possibly via autophagy to selectively engulf a portion of viral RNA-containing chloroplast. Virus-induced vesicles enriched with ATG8f could provide an alternative site for viral RNA replication or a shelter from the host silencing mechanism.

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Phenotypes and Functional Effects Caused by Various Viral RNA Silencing Suppressors in Transgenic Nicotiana benthamiana and N. tabacum

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-21-2-0178 ◽

2008 ◽

Vol 21 (2) ◽

pp. 178-187 ◽

Cited By ~ 50

Author(s):

Shahid Aslam Siddiqui ◽

Cecilia Sarmiento ◽

Erkki Truve ◽

Harry Lehto ◽

Kirsi Lehto

Keyword(s):

Mosaic Virus ◽

Potato Virus ◽

Nicotiana Benthamiana ◽

Rna Silencing ◽

Fluorescent Protein ◽

Potato Virus X ◽

Mottle Virus ◽

African Cassava Mosaic Virus ◽

Rice Yellow Mottle Virus ◽

Green Fluorescent

RNA silencing suppressor genes derived from six virus genera were transformed into Nicotiana benthamiana and N. tabacum plants. These suppressors were P1 of Rice yellow mottle virus (RYMV), P1 of Cocksfoot mottle virus, P19 of Tomato bushy stunt virus, P25 of Potato virus X, HcPro of Potato virus Y (strain N), 2b of Cucumber mosaic virus (strain Kin), and AC2 of African cassava mosaic virus (ACMV). HcPro caused the most severe phenotypes in both Nicotiana spp. AC2 also produced severe effects in N. tabacum but a much milder phenotype in N. benthamiana, although both HcPro and AC2 affected the leaf tissues of the two Nicotiana spp. in similar ways, causing hyperplasia and hypoplasia, respectively. P1-RYMV caused high lethality in the N. benthamiana plants but only mild effects in the N. tabacum plants. Phenotypic alterations produced by the other transgenes were minor in both species. Interestingly, the suppressors had very different effects on crucifer-infecting Tobamovirus (crTMV) infections. AC2 enhanced both spread and brightness of the crTMV-green fluorescent protein (GFP) lesions, whereas 2b and both P1 suppressors enhanced spread but not brightness of these lesions. P19 promoted spread of the infection into new foci within the infiltrated leaf, whereas HcPro and P25 suppressed the spread of crTMV-GFP lesions.

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Tobacco Mosaic Virus Movement Protein Interacts with Green Fluorescent Protein-Tagged Microtubule End-Binding Protein 1

PLANT PHYSIOLOGY ◽

10.1104/pp.108.117481 ◽

2008 ◽

Vol 147 (2) ◽

pp. 611-623 ◽

Cited By ~ 56

Author(s):

Katrin Brandner ◽

Adrian Sambade ◽

Emmanuel Boutant ◽

Pascal Didier ◽

Yves Mély ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Fluorescent Protein ◽

Movement Protein ◽

Binding Protein ◽

Virus Movement ◽

Green Fluorescent

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The Tobacco mosaic virus Replicase Protein Disrupts the Localization and Function of Interacting Aux/IAA Proteins

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-19-0864 ◽

2006 ◽

Vol 19 (8) ◽

pp. 864-873 ◽

Cited By ~ 53

Author(s):

Meenu S. Padmanabhan ◽

Haiymanot Shiferaw ◽

James N. Culver

Keyword(s):

Transgenic Plants ◽

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Cellular Localization ◽

Fluorescent Protein ◽

Response Regulator ◽

Disease Symptoms ◽

Severe Stunting ◽

Replicase Protein ◽

Viral Replicase

Previously, we identified a correlation between the interaction of the Tobacco mosaic virus (TMV) 126/183-kDa replicase with the auxin response regulator indole acetic acid (IAA)26/PAP1 and the development of disease symptoms. In this study, the TMV replicase protein is shown to colocalize with IAA26 in the cytoplasm and prevent its accumulation within the nucleus. Furthermore, two additional auxin (Aux)/IAA family members, IAA27 and IAA18, were found to interact with the TMV replicase and displayed alterations in their cellular localization or accumulation that corresponded with their ability to interact with the TMV replicase. In contrast, the localization and accumulation of noninteracting Aux/IAA proteins were unaffected by the presence of the viral replicase. To investigate the effects of the replicase interaction on Aux/IAA function, transgenic plants expressing a proteolysis-resistant IAA26-P108H-green fluorescent protein (GFP) protein were created. Transgenic plants accumulating IAA26-P108H-GFP displayed an abnormal developmental phenotype that included severe stunting and leaf epinasty. However, TMV infection blocked the nuclear localization of IAA26-P108H-GFP and attenuated the developmental phenotype displayed by the transgenic plants. Combined, these findings suggest that TMV-induced disease symptoms can be attributed, in part, to the ability of the viral replicase protein to disrupt the localization and subsequent function of interacting Aux/IAA proteins.

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