Browsing by Subject "PVA"
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(2021)Potyviruses are positive-sense single-stranded RNA viruses that can alter several functions of their host plants and consequently, cause significant economic losses in the infected crop plants. During the viral infection, the host transcriptome changes. Stress related genes are triggered, and genes allowing for susceptibility are target for viral-induced modifications. Therefore, in this study, we investigated whether the expression of potential proviral genes SUO1, AGO1, and the major antiviral player AGO2 change in Nicotiana benthamiana (N. benthamiana) in response to potato virus A (PVA; genus Potyvirus) infection. Moreover, we aimed to determine whether helper component protease (HCPro) and active replication have a role in the transcriptional regulation of these genes. Leaves infected with PVA tagged with Renilla luciferase were collected at 3, 6, and 9 days postinoculation, and the viral gene expression was quantified with a dual-luciferase assay. Total RNA was isolated, cDNA was synthesized, and samples were analyzed through qPCR. BLAST hit results revealed that N. benthamiana has three homologs of the SUO1 gene. qPCR data showed no significant change in neither the expression of SUO1 homologs nor the expression of AGO1 during wild-type PVA infection. Moreover, the lack of HCPro or viral replication did not affect the expression of these genes. On the other hand, the expression of AGO2 was approximately 6 and 5 fold up-regulated at 6 and 9 days post-inoculation, respectively. In contrast with the wild-type PVA infection, the mutated viruses had a pronounced effect on AGO2 transcripts at 3 days post-inoculation. Replication-deficient viral RNA increased AGO2 expression circa four-fold, followed by the HCPro-deficient viral RNA increasing expression circa two-fold. AGO2, the major player involved in antiviral defense, was up-regulated during the wild-type infection. Active viral replication and functional HCPro played a role in AGO2 regulation. However, Agrobacterium infiltration can be accounted for interfering with the interpretation of the AGO2 results. Although SUO1 and AGO1 may be potential genes allowing for susceptibility, this study revealed that the PVA infection does not affect the mRNA expression of these genes. Furthermore, it is concluded that active HCPro and viral replication do not have a role in the expression of these genes on mRNA level. To have a clearer view, integrating small RNA, mRNA, and protein quantification analysis of SUO1 homologs will be necessary. Keywords
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(2017)The leaves of healthy potato (Solanum tuberosum L.) plants and leaves systemically infected with Potato virus A (PVA; a monopartite positive sense single stranded RNA genome, genus: Potyvirus, family: Potyviridae) have been previously analysed and compared for the nuclear proteome to better understand the role of nucleus in plant virus infection. The analysis indicated that a small GTP-binding protein (ras-related protein; rabE1) is found in the nuclei of PVA-infected leaves but is absent from nuclei of healthy leaves. Therefore, to confirm the differential localization, this study was conducted. Subcellular localization of proteins was studied by tagging the protein with a marker protein, green fluorescent protein (GFP). The mRNA of ras-like protein, rabE1, of potato cv. Pentland Crown was cloned and expressed with GFP fused to the N- or C-terminus of rabE1 (GFP-ras and ras-GFP respectively). Plants of Nicotiana benthamiana were inoculated with PVA by two methods. Firstly, the Agrobacterium strains expressing GFP-ras or ras-GFP were mixed with the Agrobacterium strain expressing red fluorescent protein (RFP) or PVA-RFP and introduced to the healthy leaves of N. benthamiana plants and observed under epifluorescence microscope three days post inoculation. Secondly, PVA-RFP was agroinoculated to two or three basal leaves of young N. benthamiana plants and plants were grown for 12 days to allow systemic infection. Thereafter, PVA infected upper leaves and healthy leaves were infiltrated with GFP-ras or ras-GFP and the leaves were observed under epifluorescence microscope three days after infiltration. In healthy plants, GFP-ras localized to the cytoplasm and ras-GFP localized mostly to the cytoplasm and in few cells it was found both in the nucleus and cytoplasm. Whereas, in PVA infected cells, GFP-ras localized to the cytoplasm and in some cells to both nucleus and cytoplasm suggesting that PVA infection increases GFP-ras localization to the nucleus. However, no difference was observed in localization of ras-GFP in PVA infected and healthy leaves. The hypothesis of the research is thus partially true. Our study shows that rabE1 can be differentially localized in the healthy and PVA-infected plant cells.
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