FATTY ACID DESATURASE4 enhances plant RNA virus replication and undergoes host vacuolar ATPase-mediated degradation.

Emerging evidence indicates that fatty acid (FA) metabolic pathways regulate host immunity to vertebrate viruses. However, information on FA signaling in plant virus infection remains elusive. In this study, we demonstrate the importance of fatty acid desaturase (FAD), an enzyme that catalyzes the rate-limiting step in the conversion of saturated FAs into unsaturated FAs, during infection by a plant RNA virus. We previously found that the rare Kua-ubiquitin conjugating enzyme (Kua-UEV1) fusion protein FAD4 from Nicotiana benthamiana (NbFAD4) was down-regulated upon turnip mosaic virus (TuMV) infection. We now demonstrate that NbFAD4 is unstable and is degraded as TuMV infection progresses. NbFAD4 is required for TuMV replication, as it interacts with TuMV replication protein 6K2 and colocalizes with viral replication complexes. Moreover, NbFAD4 overexpression dampened the accumulation of immunity-related phytohormones and FA metabolites, and its catalytic activity appears to be crucial for TuMV infection. Finally, a yeast two-hybrid library screen identified the vacuolar H+-ATPase component ATP6V0C as involved in NbFAD4 degradation and further suppression of TuMV infection. This study reveals the intricate role of FAD4 in plant virus infection, and shed lights on a new mechanism by which a V-ATPase is involved in plant antiviral defense.

Analysis of Plant Virus-Induced Immunity by Using Viral-Derived Double-Stranded RNA in Arabidopsis thaliana.

Pattern-triggered immunity is the first line of defense against infection by pathogens such as bacteria and fungi in plants, and this mechanism remains poorly defined in plant viruses. Double-stranded RNA (dsRNA) is an intermediate in the replication of plant RNA viruses, and is considered to be a conserved structure of plant viruses similar to pathogen-associated molecular pattern. Whether dsRNA is the elicitor that activates plant immunity in response to virus infection remains obscure. In this method, we use the cDNA of turnip mosaic virus genome as the template to in vitro synthesis of viral dsRNA and examine whether viral dsRNA could activate plant immunity in Arabidopsis thaliana, including MAPK kinase cascade and reactive oxygen burst. In order to provide some references for researchers studying dsRNA in terms of research methodology and experimental methods, we use western blot to measure MAPK kinase cascade and luminol-based assay to measure ROS burst in Arabidopsis thaliana treated by viral dsRNA.

Proteomics Identified UDP-Glycosyltransferase Family Members as Pro-Viral Factors for Turnip Mosaic Virus Infection in Nicotiana benthamiana.

Viruses encounter numerous host factors that facilitate or suppress viral infection. Although some host factors manipulated by viruses were uncovered, we have limited knowledge of the pathways hijacked to promote viral replication and activate host defense responses. Turnip mosaic virus (TuMV) is one of the most prevalent viral pathogens in many regions of the world. Here, we employed an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomics approach to characterize cellular protein changes in the early stages of infection of Nicotiana benthamiana by wild type and replication-defective TuMV. A total of 225 differentially accumulated proteins (DAPs) were identified (182 increased and 43 decreased). Bioinformatics analysis showed that a few biological pathways were associated with TuMV infection. Four upregulated DAPs belonging to uridine diphosphate-glycosyltransferase (UGT) family members were validated by their mRNA expression profiles and their effects on TuMV infection. NbUGT91C1 or NbUGT74F1 knockdown impaired TuMV replication and increased reactive oxygen species production, whereas overexpression of either promoted TuMV replication. Overall, this comparative proteomics analysis delineates the cellular protein changes during early TuMV infection and provides new insights into the role of UGTs in the context of plant viral infection.

Rice stripe virus p2 protein interacts with ATG5 and is targeted for degradation by autophagy.

Autophagy can be induced by viral infection and plays antiviral roles in plants, but the underlying mechanism is not well understood. In our previous reports, we have demonstrated that the plant ATG5 plays an essential role in activating autophagy in rice stripe virus (RSV)-infected plants. We also showed that eIF4A, a negative factor of autophagy, interacts with and inhibits ATG5. We here found that RSV p2 protein interacts with ATG5 and can be targeted by autophagy for degradation. Expression of p2 protein induced autophagy and p2 protein was shown to interfere with the interaction between ATG5 and eIF4A, while eIF4A had no effect on the interaction between ATG5 and p2. These results indicate an additional information on the induction of autophagy in RSV-infected plants.

Acidic dileucine motifs in the cylindrical inclusion protein of turnip mosaic virus are crucial for endosomal targeting and viral replication.

Previously we reported that the multifunctional cylindrical inclusion (CI) protein of turnip mosaic virus (TuMV) is targeted to endosomes through the interaction with the medium subunit of adaptor protein complex 2 (AP2ß), which is essential for viral infection. Although several functionally important regions in the CI have been identified, little is known about the determinant(s) for endosomal trafficking. The CI protein contains seven conserved acidic dileucine motifs [(D/E)XXXL(L/I)] typical of endocytic sorting signals recognized by AP2ß. Here, we selected five motifs for further study and identified that they all were located in the regions of CI interacting with AP2ß. Coimmunoprecipitation assays revealed that alanine substitutions in the each of these acidic dileucine motifs decreased binding with AP2ß. Moreover, these CI mutants also showed decreased accumulation of punctate bodies, which enter endocytic-tracking styryl-stained endosomes. The mutations were then introduced into a full-length infectious clone of TuMV, and each mutant had reduced viral replication and systemic infection. The data suggest that the acidic dileucine motifs in CI are indispensable for interacting with AP2ß for efficient viral replication. This study provides new insights into the role of endocytic sorting motifs in the intracellular movement of viral proteins for replication.

FvZFP1 confers transgenic Nicotiana benthamiana resistance against plant pathogens and improves tolerance to abiotic stresses.

Zinc finger proteins can induce plant resistance and activate the expression of molecules involved in the resistance pathway in response to harsh environmental conditions. Previously, we found that a novel Fragaria vesca zinc finger protein interacts with the P6 protein encoded by a strawberry vein banding virus. However, the molecular mechanism of the zinc finger protein in plant stress resistance is still unknown. In this study, we reported the identification and functional characterization of the RING finger and CHY zinc finger domain-containing protein 1 (FvZFP1). The overexpression of FvZFP1 in Nicotiana benthamiana enhanced resistance to tobacco mosaic virus (TMV) and Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) infection by increasing ROS content. Additionally, FvZFP1 overexpression upregulated salicylic acid (SA) response-related gene expression as well as SA accumulation following TMV and Pst DC3000 infection. Furthermore, FvZFP1 overexpression resulted in increased salinity and drought stress tolerance by increasing SOD activity and decreasing MDA content. Overexpression of FvZFP1 also activated the ABA pathway under salinity or drought conditions. To our knowledge, this is the first study on the involvement of F. vesca zinc finger protein in crosstalk between biotic and abiotic stress signaling pathways, suggesting that FvZFP1 is a candidate gene for the improvement of resistance in response to multiple stresses.

Strawberry Vein Banding Virus P6 Protein Is a Translation Trans-Activator and Its Activity Can be Suppressed by FveIF3g.

The strawberry vein banding virus (SVBV) open reading frame (ORF) VI encodes a P6 protein known as the RNA silencing suppressor. This protein is known to form inclusion like granules of various sizes and accumulate in both the nuclei and the cytoplasm of SVBV-infected plant cells. In this study, we have determined that the P6 protein is the only trans-activator (TAV) encoded by SVBV, and can efficiently trans-activate the translation of downstream gfp mRNA in a bicistron derived from the SVBV. Furthermore, the P6 protein can trans-activate the expression of different bicistrons expressed by different caulimovirus promoters. The P6 protein encoded by SVBV from an infectious clone can also trans-activate the expression of bicistron. Through protein-protein interaction assays, we determined that the P6 protein could interact with the cell translation initiation factor FveIF3g of Fragaria vesca and co-localize with it in the nuclei of Nicotiana benthamiana cells. This interaction reduced the formation of P6 granules in cells and its trans-activation activity on translation.