Although the complete nucleotide sequence of strawberry vein banding virus(SVBV) has been determined and bioinformatic analysis has revealed that the SVBV genome could encode seven proteins, the precise function of ea...Although the complete nucleotide sequence of strawberry vein banding virus(SVBV) has been determined and bioinformatic analysis has revealed that the SVBV genome could encode seven proteins, the precise function of each protein is unclear. This study provided evidence that the P1 protein of SVBV(SVBV-P1) possesses the following features. Bioinformatic and subcellular localization analyses showed that SVBV-P1 is localized in the cytoplasm and cell walls of epidermal cells in Nicotiana benthamiana, and it forms inclusion bodies associated with microtubules and the endoplasmic reticulum. Dilution experiments demonstrated that SVBV-P1 could move from the original agro-infiltrated cells to adjacent cells in N. benthamiana leaves. Further trans-complementation experiments demonstrated that SVBV-P1 could facilitate the intercellular movement of a movement-deficient potato virus X mutant in N. benthamiana leaves. Finally, yeast twohybrid and bimolecular fluorescence complementation assays revealed that SVBV-P1 could interact with the SVBV coat protein, which is a major component of Caulimovirus virions. Results of the electrophoretic mobility shift assay indicated that SVBV-P1 lacks DNA-binding capability. In summary, the results suggest that SVBV-P1 is probably a movement protein of SVBV, providing new insights into the function of movement proteins of the Caulimovirus genus.展开更多
Strawberry vein banding virus （SVBV）-infected strawberry cells contain cytoplasmic inclusions with isometric particles. To identify the components of the inclusions, green fluorescent protein （GFP） was fused to th...Strawberry vein banding virus （SVBV）-infected strawberry cells contain cytoplasmic inclusions with isometric particles. To identify the components of the inclusions, green fluorescent protein （GFP） was fused to the carboxy-terminus （C-terminus） of SVBV open reading frames, these constructs were separately transformed into Agrobacterium tumefaciens and infiltrated into Nicotiana benthamiana leaves. Results showed that the SVBV P6 protein assembled into prominent and amorphous inclusion bodies （IBs）. To investigate P6 subcellular localization, P6-GFP was ectopically expressed in N. benthamiana leaves by agroinfiltration and then stained with 4＂,6-diamidino-2-phenylindole （DAPI）. We found the P6 protein accumulated in the nuclei and also formed cytoplasmic IBs with different sizes. To further determine the location of P6 IBs in the cytoplasm, and explore whether the P6 IBs move freely or depend on cytoskeleton and endoplasmic reticulum （ER）, the microfilament marker protein （GFP-ABD2-GFP）, microtubules marker protein （mCherry-MAP65-1） and ER marker protein （mCherry-HDEL） were separately coexpressed with P6-GFP and into N. benthamiana leaves by agroinfiltration, exhibiting that P6 IBs aligned with cytoskeleton and endoplasmic reticulum. Meanwhile, coinfiltration of P1 and P6 indicated the P6 colocalized with the P1 protein at periphery of cells. The P6 protein contains one C-terminal nuclear localization signal （NLS） region, a P6 protein mutant with a deleted NLS did not localize in the nucleus, did not form IBs, and was unable to facilitate exogenous GFP expression. These results demonstrate that the deleted NLS region is an important P6 domain required for biological functions. In summary, the mobile P6 IBs are associated with ER, microfilaments and microtubules and move along microfilaments to the SVBV P1 protein in the PD.展开更多
基金supported by the grants from the National Natural Science Foundation of China(32072386 and 31801700)the Key Research and Development Project of Anhui Province,China(202004a06020013)the Postdoctoral Science Fund of Anhui Province,China(2019B360)。
文摘Although the complete nucleotide sequence of strawberry vein banding virus(SVBV) has been determined and bioinformatic analysis has revealed that the SVBV genome could encode seven proteins, the precise function of each protein is unclear. This study provided evidence that the P1 protein of SVBV(SVBV-P1) possesses the following features. Bioinformatic and subcellular localization analyses showed that SVBV-P1 is localized in the cytoplasm and cell walls of epidermal cells in Nicotiana benthamiana, and it forms inclusion bodies associated with microtubules and the endoplasmic reticulum. Dilution experiments demonstrated that SVBV-P1 could move from the original agro-infiltrated cells to adjacent cells in N. benthamiana leaves. Further trans-complementation experiments demonstrated that SVBV-P1 could facilitate the intercellular movement of a movement-deficient potato virus X mutant in N. benthamiana leaves. Finally, yeast twohybrid and bimolecular fluorescence complementation assays revealed that SVBV-P1 could interact with the SVBV coat protein, which is a major component of Caulimovirus virions. Results of the electrophoretic mobility shift assay indicated that SVBV-P1 lacks DNA-binding capability. In summary, the results suggest that SVBV-P1 is probably a movement protein of SVBV, providing new insights into the function of movement proteins of the Caulimovirus genus.
基金supported by the Fund of State Key Laboratory for Biology of Plant Diseases and Insect Pests (SKLOF201710)the National Natural Science Foundation of China (31671999,31371915)the Zhejiang Natural Science Foundation of China (LY17C140001)
文摘Strawberry vein banding virus （SVBV）-infected strawberry cells contain cytoplasmic inclusions with isometric particles. To identify the components of the inclusions, green fluorescent protein （GFP） was fused to the carboxy-terminus （C-terminus） of SVBV open reading frames, these constructs were separately transformed into Agrobacterium tumefaciens and infiltrated into Nicotiana benthamiana leaves. Results showed that the SVBV P6 protein assembled into prominent and amorphous inclusion bodies （IBs）. To investigate P6 subcellular localization, P6-GFP was ectopically expressed in N. benthamiana leaves by agroinfiltration and then stained with 4＂,6-diamidino-2-phenylindole （DAPI）. We found the P6 protein accumulated in the nuclei and also formed cytoplasmic IBs with different sizes. To further determine the location of P6 IBs in the cytoplasm, and explore whether the P6 IBs move freely or depend on cytoskeleton and endoplasmic reticulum （ER）, the microfilament marker protein （GFP-ABD2-GFP）, microtubules marker protein （mCherry-MAP65-1） and ER marker protein （mCherry-HDEL） were separately coexpressed with P6-GFP and into N. benthamiana leaves by agroinfiltration, exhibiting that P6 IBs aligned with cytoskeleton and endoplasmic reticulum. Meanwhile, coinfiltration of P1 and P6 indicated the P6 colocalized with the P1 protein at periphery of cells. The P6 protein contains one C-terminal nuclear localization signal （NLS） region, a P6 protein mutant with a deleted NLS did not localize in the nucleus, did not form IBs, and was unable to facilitate exogenous GFP expression. These results demonstrate that the deleted NLS region is an important P6 domain required for biological functions. In summary, the mobile P6 IBs are associated with ER, microfilaments and microtubules and move along microfilaments to the SVBV P1 protein in the PD.
