NSvc4 Encoded by Rice Stripe Virus Targets Host Chloroplasts to Suppress Chloroplast-Mediated Defense

Our previous research found that NSvc4, the movement protein of rice stripe virus (RSV), could localize to the actin filaments, endoplasmic reticulum, plasmodesmata, and chloroplast, but the roles of NSvc4 played in the chloroplast were opaque. Here, we confirm the accumulation of NSvc4 in the chloroplasts and the N-terminal 1–73 amino acids of NSvc4 are sufficient to localize to chloroplasts. We provide evidence to show that chloroplast-localized NSvc4 can impair the chloroplast-mediated immunity. Expressing NSvc4 in Nicotiana benthamiana leaves results in the decreased expression of defense-related genes NbPR1, NbPR2, and NbWRKY12 and the inhibition of chloroplast-derived ROS production. In addition, generation of an infectious clone of potato virus X (PVX) carrying NSvc4 facilitates PVX infection in N. benthamiana plants. Moreover, we identify two chloroplast-related host factors, named NbGAPDH-A and NbPsbQ1, both of which can interact with NSvc4. Knockdown of NbGAPDH-A or NbPsbQ1 can both promote RSV infection. Our results decipher a detailed function of NSvc4 in the chloroplast.

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Identification of a Movement Protein of the Tenuivirus Rice Stripe Virus

Journal of Virology ◽

10.1128/jvi.01696-08 ◽

2008 ◽

Vol 82 (24) ◽

pp. 12304-12311 ◽

Cited By ~ 93

Author(s):

Ruyi Xiong ◽

Jianxiang Wu ◽

Yijun Zhou ◽

Xueping Zhou

Keyword(s):

Nicotiana Benthamiana ◽

Fluorescent Protein ◽

Severe Disease ◽

Rice Stripe Virus ◽

Potato Virus X ◽

Infected Leaf ◽

Biolistic Bombardment ◽

Movement Proteins ◽

Green Fluorescent

ABSTRACT Rice stripe virus (RSV) is the type member of the genus Tenuivirus. RSV has four single-stranded RNAs and causes severe disease in rice fields in different parts of China. To date, no reports have described how RSV spreads within host plants or the viral and/or host factor(s) required for tenuivirus movement. We investigated functions of six RSV-encoded proteins using trans-complementation experiments and biolistic bombardment. We demonstrate that NSvc4, encoded by RSV RNA4, supports the intercellular trafficking of a movement-deficient Potato virus X in Nicotiana benthamiana leaves. We also determined that upon biolistic bombardment or agroinfiltration, NSvc4:enhanced green fluorescent protein (eGFP) fusion proteins localize predominantly near or within the walls of onion and tobacco epidermal cells. In addition, the NSvc4:eGFP fusion protein can move from initially bombarded cells to neighboring cells in Nicotiana benthamiana leaves. Immunocytochemistry using tissue sections from RSV-infected rice leaves and an RSV NSvc4-specific antibody showed that the NSvc4 protein accumulated in walls of RSV-infected leaf cells. Gel retardation assays revealed that the NSvc4 protein interacts with single-stranded RNA in vitro, a common feature of many reported plant viral movement proteins (MPs). RSV NSvc4 failed to interact with the RSV nucleocapsid protein using yeast two-hybrid assays. Taken together, our data indicate that RSV NSvc4 is likely an MP of the virus. This is the first report describing a tenuivirus MP.

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Development of a mini-replicon-based reverse-genetics system for rice stripe tenuivirus

10.1101/2021.04.04.438373 ◽

2021 ◽

Author(s):

Mingfeng Feng ◽

Luyao Li ◽

Ruixiang Cheng ◽

Yulong Yuan ◽

Yongxin Dong ◽

...

Keyword(s):

Nicotiana Benthamiana ◽

Reverse Genetics ◽

Molecular Mechanisms ◽

Rice Stripe Virus ◽

Egfp Expression ◽

Asian Countries ◽

Genomic Rnas ◽

Gene Functions ◽

Rsv Infection ◽

Rice Pathogens

ABSTRACTNegative-stranded RNA (NSR) viruses include both animal- and plant-infecting viruses that often cause serious diseases in human and livestock, and in agronomic crops. Rice stripe tenuivirus (RSV), a plant NSR virus with four negative-stranded/ambisense RNA segments, is one of the most destructive rice pathogens in many Asian countries. Due to the lack of a reliable reverse-genetics technology, molecular studies of RSV gene functions and its interaction with host plants are severely hampered. To overcome this obstacle, we developed a mini-replicon-based reverse-genetics system for RSV gene functional analysis in Nicotiana benthamiana. We first developed a mini-replicon system expressing RSV genomic RNA3 eGFP reporter (MR3(-)eGFP), a nucleocapsid (NP), and a codon usage optimized RNA-dependent RNA polymerase (RdRpopt), respectively. Using this mini-replicon system we determined that RSV NP and RdRpopt are indispensable for the eGFP expression from MR3(-)eGFP. The expression of eGFP from MR3(-)eGFP can be significantly enhanced in the presence of NSs and P19-HcPro-γb. In addition, NSvc4, the movement protein of RSV, facilitated eGFP trafficking between cells. We also developed an antigenomic RNA3-based replicon in N. benthamiana. However, we found that the RSV NS3 coding sequence acts as a cis-element to regulate viral RNA expression. Finally, we made mini-replicons representing all four RSV genomic RNAs. This is the first mini-replicon-based reverse-genetics system for monocot-infecting tenuivirus. We believe that this mini-replicon system described here will allow the studies of RSV replication, transcription, cell-to-cell movement and host machinery underpinning RSV infection in plants.IMPORTANCEPlant-infecting segmented negative-stranded RNA (NSR) viruses are grouped into 3 genera: Orthotospovirus, Tenuivirus and Emaravirus. The reverse-genetics systems have been established for members in the genera Orthotospovirus and Emaravirus, respectively. However, there is still no reverse-genetics system available for Tenuivirus. Rice stripe virus (RSV) is a monocot-infecting tenuivirus with four negative-stranded/ambisense RNA segments. It is one of the most destructive rice pathogens and causes significant damages to rice industry in Asian countries. Due to the lack of a reliable reverse-genetics system, molecular characterizations of RSV gene functions and the host machinery underpinning RSV infection in plants are extremely difficult. To overcome this obstacle, we developed a mini-replicon-based reverse-genetics system for RSV in Nicotiana benthamiana. This is the first mini-replicon-based reverse-genetics system for tenuivirus. We consider that this system will provide researchers a new working platform to elucidate the molecular mechanisms dictating segmented tenuivirus infections in plant.

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Identification of a movement protein of Mirafiori lettuce big-vein ophiovirus

Journal of General Virology ◽

10.1099/vir.0.050005-0 ◽

2013 ◽

Vol 94 (5) ◽

pp. 1145-1150 ◽

Cited By ~ 5

Author(s):

Akihiro Hiraguri ◽

Shoko Ueki ◽

Hideki Kondo ◽

Koji Nomiyama ◽

Takumi Shimizu ◽

...

Keyword(s):

Mosaic Virus ◽

Nicotiana Benthamiana ◽

Protein Gene ◽

Movement Protein ◽

Rna Virus ◽

Infectious Clone ◽

Microprojectile Bombardment ◽

Tomato Mosaic Virus ◽

Intercellular Movement ◽

Heterologous Virus

Mirafiori lettuce big-vein virus (MiLBVV) is a member of the genus Ophiovirus, which is a segmented negative-stranded RNA virus. In microprojectile bombardment experiments to identify a movement protein (MP) gene of ophioviruses that can trans-complement intercellular movement of an MP-deficient heterologous virus, a plasmid containing an infectious clone of a tomato mosaic virus (ToMV) derivative expressing the GFP was co-bombarded with plasmids containing one of three genes from MiLBVV RNAs 1, 2 and 4 onto Nicotiana benthamiana. Intercellular movement of the movement-defective ToMV was restored by co-expression of the 55 kDa protein gene, but not with the two other genes. Transient expression in epidermal cells of N. benthamiana and onion showed that the 55 kDa protein with GFP was localized on the plasmodesmata. The 55 kDa protein encoded in the MiLBVV RNA2 can function as an MP of the virus. This report is the first to describe an ophiovirus MP.

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The unfolded protein response plays dual roles in rice stripe virus infection through fine-tuning the movement protein accumulation

PLoS Pathogens ◽

10.1371/journal.ppat.1009370 ◽

2021 ◽

Vol 17 (3) ◽

pp. e1009370

Author(s):

Chenyang Li ◽

Yi Xu ◽

Shuai Fu ◽

Yu Liu ◽

Zongdi Li ◽

...

Keyword(s):

Virus Infection ◽

Movement Protein ◽

Rice Stripe Virus ◽

Fine Tuning ◽

Dual Roles ◽

Unfolded Protein ◽

Rsv Infection ◽

Autophagic Degradation ◽

The Unfolded Protein Response ◽

Protein Response

The movement of plant viruses is a complex process that requires support by the virus-encoded movement protein and multiple host factors. The unfolded protein response (UPR) plays important roles in plant virus infection, while how UPR regulates viral infection remains to be elucidated. Here, we show that rice stripe virus (RSV) elicits the UPR in Nicotiana benthamiana. The RSV-induced UPR activates the host autophagy pathway by which the RSV-encoded movement protein, NSvc4, is targeted for autophagic degradation. As a counteract, we revealed that NSvc4 hijacks UPR-activated type-I J-domain proteins, NbMIP1s, to protect itself from autophagic degradation. Unexpectedly, we found NbMIP1 stabilizes NSvc4 in a non-canonical HSP70-independent manner. Silencing NbMIP1 family genes in N. benthamiana, delays RSV infection, while over-expressing NbMIP1.4b promotes viral cell-to-cell movement. Moreover, OsDjA5, the homologue of NbMIP1 family in rice, behaves in a similar manner toward facilitating RSV infection. This study exemplifies an arms race between RSV and the host plant, and reveals the dual roles of the UPR in RSV infection though fine-tuning the accumulation of viral movement protein.

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Phosphorylation of the TGBp1 movement protein of Potato virus X by a Nicotiana tabacum CK2-like activity

Virus Research ◽

10.1016/j.virusres.2008.04.007 ◽

2008 ◽

Vol 137 (1) ◽

pp. 16-23 ◽

Cited By ~ 27

Author(s):

Natalia Andrea Módena ◽

Alicia Mercedes Zelada ◽

Florencia Conte ◽

Alejandro Mentaberry

Keyword(s):

Nicotiana Tabacum ◽

Potato Virus ◽

Movement Protein ◽

Potato Virus X

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Corrigendum to “Nicotiana benthamiana protein, NPCIP1, interacting with Potato virus X coat protein plays a role as susceptible factor for viral infection” [Virology 386 (2009) 257–269]

Virology ◽

10.1016/j.virol.2009.06.019 ◽

2009 ◽

Vol 391 (1) ◽

pp. 149-150

Author(s):

Mi-Ri Park ◽

Sang-Ho Park ◽

Sang-Yun Cho ◽

Kook-Hyung Kim

Keyword(s):

Coat Protein ◽

Viral Infection ◽

Potato Virus ◽

Nicotiana Benthamiana ◽

Potato Virus X

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Host Factors Modulating RSV Infection: Use of Small Interfering RNAs to Probe Functional Importance

Human Respiratory Syncytial Virus - Methods in Molecular Biology ◽

10.1007/978-1-4939-3687-8_8 ◽

2016 ◽

pp. 93-117 ◽

Cited By ~ 5

Author(s):

Leon Caly ◽

Hong-mei Li ◽

David Jans

Keyword(s):

Host Factors ◽

Small Interfering Rnas ◽

Functional Importance ◽

Rsv Infection

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Interplay between potato virus X and RNA granules in Nicotiana benthamiana

Virus Research ◽

10.1016/j.virusres.2019.197823 ◽

2020 ◽

Vol 276 ◽

pp. 197823

Author(s):

Gabriel Robles-Luna ◽

Nicolás Furman ◽

María Florencia Barbarich ◽

Nicolás Carlotto ◽

Alejandra Attorresi ◽

...

Keyword(s):

Potato Virus ◽

Nicotiana Benthamiana ◽

Potato Virus X ◽

Rna Granules

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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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