Traffic jam on the cellular secretory pathway generated by a replication protein from a plant RNA virus

Although positive-strand RNA [(+)RNA] viruses have a limited coding capacity, they can replicate efficiently in host cells because of their ability to use host-derived proteins, membranes, lipids, and metabolites, and to rewire cellular trafficking pathways. Previously, we showed that a plant RNA virus, Red clover necrotic mosaic virus (RCNMV), hijacked Arf1 and Sar1, which are small GTPases that regulate the biogenesis of COPI and COPII vesicles, respectively, for viral RNA replication. These small GTPases are relocated from appropriate subcellular compartments to the viral RNA replication sites by p27 replication protein, which raises the possibility that RCNMV interferes with the cellular secretory pathway. Here, we examined this possibility by using green fluorescent protein-fused rice SCAMP1 and Arabidopsis LRR84A as secretory pathway marker proteins and showed that p27 inhibited the trafficking of these proteins. RCNMV-mediated inhibition of the host secretion pathway and its possible impact on plant–virus interaction are discussed

Validating Plant Genes Involved in Pepper Yellow Leaf Curl Indonesia Virus Infection Using VIGS in Model Plant Nicotiana benthamiana

Pepper yellow leaf curl disease caused by Pepper yellow leaf curl Indonesia virus (PepYLCIV) has become a challenge to chili pepper cultivation. Development of resistant variety by utilizing recessive resistance gene is expected to control the disease in the field. This study aimed to validate three plant genes, namely deltaCOP, hsc70, and BAM1, in PepYLCIV infection by applying Virus-induced Gene Silencing (VIGS) in a model plant, wild type Nicotiana benthamiana. PepYLCIV and construct of Tobacco rattle virus (TRV) which induced silencing of each gene were co-inoculated into N. benthamiana plants through agroinfiltration. Gene expression and the relative amount of viral DNA were determined by quantitative reverse transcription PCR (qRT-PCR) and quantitative PCR (qPCR), respectively, at 15 days post inoculation. The results showed a decreased level of deltaCOP, hsc70, and BAM1 expressions to 66.4%, 53.0%, and 47.0%, respectively, compared to that in the control (100%). Silencing of the three genes decreased the accumulation of PepYLCIV to 0.1%, 18.4%, and 63.0%, respectively, compared to that in the control. deltaCOP and hsc70 genes were indicated to be involved in the viral infection and could be good candidate genes for obtaining chili pepper varieties resistant to PepYLCIV. This result affirmed that the reverse genetics technique could be an alternative approach for identifying plant genes involved in viral infection, including PepYLCIV. The use of an infectious clone in this study allows the virus inoculations could be carried out without rearing and maintaining its natural vector, hence reduces the risk of virus transmission to healthy plants

Endoplasmic reticulum targeting of the Red clover necrotic mosaic virus movement protein is associated with the replication of viral RNA1 but not that of RNA2

Red clover necrotic mosaic virus (RCNMV) is a positive-strand RNA virus with a bipartite genome. The movement protein (MP) encoded by RNA2 is essential for viral movement. To obtain further insights into the viral movement mechanism, subcellular localizations of RCNMV MP fused with green fluorescent protein (MP:GFP) were examined in Nicotiana benthamiana epidermal cells and protoplasts. The MP:GFP expressed from the recombinant virus first appeared in the cell wall and subsequently was observed on the cortical endoplasmic reticulum (ER) as punctate spots. In contrast, the MP:GFP expressed transiently in the absence of other viral components was localized exclusively in the cell wall. Transient expression of the MP:GFP with a variety of RCNMV components revealed that the ER localization of the MP:GFP was associated with RNA1 replication, or its negative-strand RNA synthesis, but not those of RNA2 or replicase proteins per se. A model of RCNMV cell-to-cell movement is discussed

Subcellular dynamics of red clover necrotic mosaic virus double-stranded RNAs in infected plant cells

New evidences are emerging to support the importance of viral replication complexes (VRCs) in not only viral replication, but also viral cell-to-cell movement. Currently, how VRCs grow in size and colocalize with viral movement proteins (MPs) remains unclear. Herein, we performed live-cell imaging of red clover necrotic mosaic virus (RCNMV) dsRNA by using reporter B2-GFP plants. Tiny granules of dsRNA were formed along the endoplasmic reticulum (ER) at an early stage of infection. Importantly, the colocalization of the dsRNA granules with the virus-encoded p27 replication protein showed that these structures are components of VRCs. These granules moved throughout the cytoplasm, driven by the acto–myosin system, and coalesced with each other to form larger aggregates; the MPs were not associated with these processes. Notably, the MPs colocalized preferentially with large dsRNA aggregates, rather than with tiny dsRNA granules, suggesting that the increase in the size of VRCs promotes their colocalization with MPs

Interactions between p27 and p88 replicase proteins of Red clover necrotic mosaic virus play an essential role in viral RNA replication and suppression of RNA silencing via the 480-kDa viral replicase complex assembly

AbstractRed clover necrotic mosaic virus (RCNMV), a positive-sense RNA virus with a bipartite genome, encodes p27 and p88 replicase proteins that are required for viral RNA replication and suppression of RNA silencing. In this study, we indentified domains in p27 and p88 responsible for their protein–protein interactions using in vitro pull-down assays with the purified recombinant proteins. Coimmunoprecipitation analysis in combination with blue-native polyacrylamide gel electrophoresis using mutated p27 proteins showed that both p27–p27 and p27–p88 interactions are essential for the formation of the 480-kDa complex, which has RCNMV-specific RNA-dependent RNA polymerase activity. Furthermore, we found a good correlation between the accumulated levels of the 480-kDa complex and replication levels and the suppression of RNA silencing activity. Our results indicate that interactions between RCNMV replicase proteins play an essential role in viral RNA replication and in suppressing RNA silencing via the 480-kDa replicase complex assembly

Roles of Phosphatidic Acid in Virus RNA Replication

Eukaryotic positive-strand RNA [(+)RNA] viruses are intracellular obligate parasites replicate using the membrane-bound replicase complexes that contain multiple viral and host components. To replicate, (+)RNA viruses exploit host resources and modify host metabolism and membrane organization. Phospholipase D (PLD) is a phosphatidylcholine- and phosphatidylethanolamine-hydrolyzing enzyme that catalyzes the production of phosphatidic acid (PA), a lipid second messenger that modulates diverse intracellular signaling in various organisms. PA is normally present in small amounts (less than 1% of total phospholipids), but rapidly and transiently accumulates in lipid bilayers in response to different environmental cues such as biotic and abiotic stresses in plants. However, the precise functions of PLD and PA remain unknown. Here, we report the roles of PLD and PA in genomic RNA replication of a plant (+)RNA virus, Red clover necrotic mosaic virus (RCNMV). We found that RCNMV RNA replication complexes formed in Nicotiana benthamiana contained PLDα and PLDβ. Gene-silencing and pharmacological inhibition approaches showed that PLDs and PLDs-derived PA are required for viral RNA replication. Consistent with this, exogenous application of PA enhanced viral RNA replication in plant cells and plant-derived cell-free extracts. We also found that a viral auxiliary replication protein bound to PA in vitro, and that the amount of PA increased in RCNMV-infected plant leaves. Together, our findings suggest that RCNMV hijacks host PA-producing enzymes to replicate

GAPDH-A Facilitates Intercellular Movement of RCNMV

The formation of virus movement protein (MP)-containing punctate structures on the cortical endoplasmic reticulum is required for efficient intercellular movement of Red clover necrotic mosaic virus (RCNMV), a bipartite positive-strand RNA plant virus. We found that these cortical punctate structures constitute a viral replication complex (VRC) in addition to the previously reported aggregate structures that formed adjacent to the nucleus. We identified host proteins that interacted with RCNMV MP in virus-infected Nicotiana benthamiana leaves using a tandem affinity purification method followed by mass spectrometry. One of these host proteins was glyceraldehyde 3-phosphate dehydrogenase-A (NbGAPDH-A), which is a component of the Calvin-Benson cycle in chloroplasts. Virus-induced gene silencing of NbGAPDH-A reduced RCNMV multiplication in the inoculated leaves, but not in the single cells, thereby suggesting that GAPDH-A plays a positive role in cell-to-cell movement of RCNMV. The fusion protein of NbGAPDH-A and green fluorescent protein localized exclusively to the chloroplasts. In the presence of RCNMV RNA1, however, the protein localized to the cortical VRC as well as the chloroplasts. Bimolecular fluorescence complementation assay and GST pulldown assay confirmed in vivo and in vitro interactions, respectively, between the MP and NbGAPDH-A. Furthermore, gene silencing of NbGAPDH-A inhibited MP localization to the cortical VRC. We discuss the possible roles of NbGAPDH-A in the RCNMV movement process

Cellular analysis of SOD1 protein-aggregation propensity and toxicity: a case of ALS with slow progression harboring homozygous SOD1-D92G mutation

Mutations within Superoxide dismutase 1 (SOD1) cause amyotrophic lateral sclerosis (ALS), accounting for approximately 20% of familial cases. The pathological feature is a loss of motor neurons with enhanced formation of intracellular misfolded SOD1. Homozygous SOD1-D90A in familial ALS has been reported to show slow disease progression. Here, we reported a rare case of a slowly progressive ALS patient harboring a novel SOD1 homozygous mutation D92G (homD92G). The neuronal cell line overexpressing SOD1-D92G showed a lower ratio of the insoluble/soluble fraction of SOD1 with fine aggregates of the misfolded SOD1 and lower cellular toxicity than those overexpressing SOD1-G93A, a mutation that generally causes rapid disease progression. Next, we analyzed spinal motor neurons derived from induced pluripotent stem cells (iPSC) of a healthy control subject and ALS patients carrying SOD1-homD92G or heterozygous SOD1-L144FVX mutation. Lower levels of misfolded SOD1 and cell loss were observed in the motor neurons differentiated from patient-derived iPSCs carrying SOD1-homD92G than in those carrying SOD1-L144FVX. Taken together, SOD1-homD92G has a lower propensity to aggregate and induce cellular toxicity than SOD1-G93A or SOD1-L144FVX, and these cellular phenotypes could be associated with the clinical course of slowly progressive ALS

Inappropriate expression of an NLP effector in colletotrichum orbiculare impairs infection on cucurbitaceae cultivars via plant recognition of the C-terminal region

The hemibiotrophic pathogen Colletotrichum orbiculare preferentially expresses a necrosis and ethylene-inducing peptide 1 (Nep1)-like protein named NLP1 during the switch to necrotrophy. Here, we report that the constitutive expression of NLP1 in C. orbiculare blocks pathogen infection in multiple Cucurbitaceae cultivars via their enhanced defense responses. NLP1 has a cytotoxic activity that induces cell death in Nicotiana benthamiana. However, C. orbiculare transgenic lines constitutively expressing a mutant NLP1 lacking the cytotoxic activity still failed to infect cucumber, indicating no clear relationship between cytotoxic activity and the NLP1-dependent enhanced defense. NLP1 also possesses the microbe-associated molecular pattern (MAMP) sequence called nlp24, recognized by Arabidopsis thaliana at its central region, similar to NLPs of other pathogens. Surprisingly, inappropriate expression of a mutant NLP1 lacking the MAMP signature is also effective for blocking pathogen infection, uncoupling the infection block from the corresponding MAMP. Notably, the deletion analyses of NLP1 suggested that the C-terminal region of NLP1 is critical to enhance defense in cucumber. The expression of mCherry fused with the C-terminal 32 amino acids of NLP1 was enough to trigger the defense of cucurbits, revealing that the C-terminal region of the NLP1 protein is recognized by cucurbits and, then, terminates C. orbiculare infection

RNAレプリコンを発現する形質転換植物によるin vivo mRNA増幅システム

京都大学0048新制・課程博士博士(農学)甲第6548号農博第898号新制||農||728(附属図書館)学位論文||H8||N2959(農学部図書室)UT51-96-T494京都大学大学院農学研究科農林生物学専攻(主査)教授 古澤 巖, 教授 泉井 桂, 教授 津田 盛也学位規則第4条第1項該当Doctor of Agricultural ScienceKyoto UniversityDFA