das elisa kit  (DSMZ)

Journal: Nucleic Acids Research

Article Title: Potyviruses recruit host eIF4A3 to block m 6 A-mediated RNA decay by steric hindrance of viral RNA methylation in plants

doi: 10.1093/nar/gkaf1432

Figure Lengend Snippet: The m 6 A modification at A 6556 of the SCMV genomic RNA reduces viral pathogenesis. ( A ) Dot blot analysis of SCMV particles showed the presence of m 6 A modification on SCMV genomic RNA. Unmodified in vitro -transcribed RNAs (IVT RNAs) were used as the negative control. ( B ) RIP followed by dot blotting showed that SCMV genomic RNA undergoes m 6 A modification during viral infection. RIP assays were performed using an m 6 A-specific antibody to immunoprecipitate m 6 A-modified RNAs from SCMV-infected maize leaves. The immunoprecipitated RNAs were subsequently spotted onto a Hybond-N+ membrane and then detected using a biotin-labeled SCMV RNA-specific probe. ( C ) Methylated RNA immunoprecipitation with next-generation sequencing (MeRIP-seq) (representative of two independent biological replicates) of SCMV genomic RNA illustrated the distribution of m 6 A peaks (red bars) mapped to the SCMV genomic RNA. The signal from input samples was represented by blue bars. A schematic diagram of the SCMV genomic RNA was shown below, with green lines indicating the location of the m 6 A-enriched sequences. The strongest enrichment was observed within the NIa-Pro region. ( D, E ) Fluorescence amplification curves and bar plot of the threshold cycle (CT) from qPCR indicated that the presence of m 6 A modification at A 6556 in the SCMV genomic RNA. The m 6 A levels at position 6556 were assessed following treatment with the fat mass and obesity-associated protein (FTO), as shown in panel (D). In panel (E), the control probe targets a non-m 6 A site; the m 6 A6556 probe targets A 6556 . The significantly higher CT value detected by the m 6 A6556 probe demonstrated the presence of m 6 A modification at A 6556 . Rn represents the raw fluorescence of the associated well normalized to the fluorescence of the passive reference dye (ROX). Values are means ± SE (two-tailed Student’s t test, n = 3 independent experiments, * P < .05). ( F ) Schematic diagram of the SCMV-G 6556 -GFP mutation. The mutated nucleotide is marked in red. The black peak corresponds to the m 6 A peak. ( G ) SCMV-G 6556 -GFP caused stronger GFP fluorescence compared with SCMV-GFP. Photographs were taken at 5 days post-infection (dpi). Scale bar, 5 cm. ( H ) Percentages of maize seedlings infected with SCMV-GFP or SCMV-G 6556 -GFP were assessed based on different disease symptom grades. Seedlings infected with SCMV-G 6556 -GFP showed more severe mosaic symptoms, with an increased proportion of severe disease symptoms (grade III) and a decreased proportion of asymptomatic cases (grade N), relative to SCMV-GFP-infected seedlings. ( I ) Maize seedlings infected with SCMV-G 6556 -GFP displayed mosaic symptoms earlier, from 4 to 5 dpi, compared to those infected with SCMV-GFP. Data are presented as means ± SE ( n = 3 independent experiments). Statistical significance at same time point was determined by a two-tailed Student’s t -test (** P < .01; *** P < .001). ( J ) MeRIP-qPCR assays indicated reduced m 6 A levels at peak5 in SCMV-G 6556 -GFP-infected maize seedlings compared to SCMV-GFP-infected seedlings. Values are means ± SE (two-tailed Student’s t test, n = 3 independent experiments, ** P < .01). ( K, L ) SCMV-G 6556 -GFP exhibited higher levels of SCMV genomic RNA and coat protein (CP) compared to the SCMV-GFP. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to assess SCMV genomic RNA levels, with ZmUbi transcripts serving as internal controls. Immunoblotting assay showed viral CP levels at 5 dpi. Band intensity was quantified using ImageJ software, and values below the top panel represent the relative quantification of band intensity. The CP band intensity of SCMV-GFP was normalized to 1.00. In panel (K) and (L), data are presented as means ± SE (Student’s t test, n = 9 plants from three independent experiments, * P < .05; ** P < .01).

Article Snippet: Briefly, biotin-labeled SCMV RNA fragments were synthesized by Huada Biological Technology (Beijing, China).

Techniques: Modification, Dot Blot, In Vitro, Negative Control, Infection, Immunoprecipitation, Membrane, Labeling, Methylation, RNA Immunoprecipitation, Next-Generation Sequencing, Fluorescence, Amplification, Control, Two Tailed Test, Mutagenesis, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Software, Quantitative Proteomics

Journal: Nucleic Acids Research

Article Title: Potyviruses recruit host eIF4A3 to block m 6 A-mediated RNA decay by steric hindrance of viral RNA methylation in plants

doi: 10.1093/nar/gkaf1432

Figure Lengend Snippet: ZmMTA inhibits SCMV infection by catalyzing m 6 A modifications at A 6556 site on SCMV genomic RNA. ( A ) ZmMTA expression levels were significantly reduced in the 1 SL of CMV: ZmMTA -infected plants compared to CMV: GUS -infected (control) plants using qRT-PCR analysis at 18 days post vascular puncture inoculation (VPI). ( B ) Immunoblotting analysis revealed the decreased the ZmMTA protein levels using anti-ZmMTA antibody. ( C ) Dot blot analysis indicated a significant reduction in m 6 A modification levels of total RNA in CMV: ZmMTA -infected plants, compared to control plants. MB staining used as a loading control. ImageJ software was used to quantify the m 6 A dot blot intensity. Data are presented as means ± SE ( n = 3 independent experiments). ( D ) Knockdown of ZmMTA expression in maize plants inhibited m 6 A level at position A 6556 of SCMV genomic RNA compare to control plants. Data are shown as means ± SE (two-tailed Student’s t test, n = 5 plants, ** P < .01). ( E ) Stronger GFP fluorescence was observed in the 1 SL of SCMV-GFP-infected plants compared to control plants. Scale bar, 2 cm. ( F ) Plants infected with CMV: ZmMTA showed more severe mosaic symptoms relative to CMV: GUS -infected plants at 7 dpi. ( G ) SCMV genomic RNA accumulation levels in the 1 SL of ZmMTA -silenced or control plants were determined through qRT-PCR at 7 dpi. Data are expressed as the means ± SE ( n = 12 plants from three independent experiments). ( H ) Immunoblotting analysis showed SCMV CP accumulation in ZmMTA -silenced and control plant leaves ( n = 3 plants). ( I ) Co-localization of ZmMTA with SCMV RNA in cytoplasmic aggregates revealed by MS2-tethering assay. ZmMTA-DsRed (red) and MS2-tagged SCMV genomic RNA, labeled by MS2-CP-GFP (green), show clear co-localization in cytoplasmic aggregates (indicated by white arrows). Scale bar, 20 μm. ( J ) His-TF-ZmMTA directly bound to the biotinylated A 6556 probe through EMSA. A biotin-labeled 25 bp fragment of the peak5 containing GGA 6556 CA is highlighted in bold, red, and underlined. BSA served as a negative control. Bound and shifted probes were detected with Streptavidin-HRP (upper). ( K ) Upper leaves of plants infected with SCMV-G 6556 -GFP displayed similar GFP fluorescence in ZmMTA- silenced plants compared to control plants. Scale bar, 2 cm. ( L, M ) No significant differences were observed in the accumulation levels of SCMV-G 6556 -GFP genomic RNA and CP between CMV: ZmMTA and CMV: GUS -infected maize plants ( n = 4 plants). Statistical significance in panels (A–D) and (G, H) was assessed with two-tailed Student’s t -tests; panels (L, M) were analyzed by one-way analysis of variance (* P < .05; ** P < .01; *** P < .001; **** P < .0001; ns, no significant difference).

Article Snippet: Briefly, biotin-labeled SCMV RNA fragments were synthesized by Huada Biological Technology (Beijing, China).

Techniques: Infection, Expressing, Control, Quantitative RT-PCR, Western Blot, Dot Blot, Modification, Staining, Software, Knockdown, Two Tailed Test, Fluorescence, Labeling, Negative Control

Journal: Nucleic Acids Research

Article Title: Potyviruses recruit host eIF4A3 to block m 6 A-mediated RNA decay by steric hindrance of viral RNA methylation in plants

doi: 10.1093/nar/gkaf1432

Figure Lengend Snippet: ZmECT23 recognizes the m 6 A modification at position A 6556 of SCMV genomic RNA, leading to the destabilization of viral RNA. ( A ) AlphaFold3 predicted that ZmECT23 binds to the A 6556 site of the SCMV genomic RNA. Within the peak5 region, the central adenine in the GGm 6 ACA sequence was methylated. The amino acid residues Tyr-457, Asp-461, and Ala-472 of ZmECT23 may directly interact with m 6 A through hydrogen bonds. The peak5 RNA sequence was displayed in magenta, with the m 6 A modification at position A 6556 highlighted in bold black, and hydrogen bonds represented by yellow dashed lines. ( B ) RIP-qPCR analysis indicated that ZmECT23 binds to the peak5 region of SCMV genomic RNA containing the A 6556 site in N. benthamiana cells, but not to the corresponding region in the SCMV-G 6556 -GFP mutant. Protein-RNA complexes were isolated from N. benthamiana leaves ( n = 3 independent experiments) co-infiltrated with SCMV-GFP and ZmECT23-3Flag, or with SCMV-G 6556 -GFP and ZmECT23-3Flag, followed by immunoprecipitation using anti-Flag antibodies. (C, D) Lifetimes of SCMV-GFP/ΔGDD and SCMV-G 6556 -GFP/ΔGDD RNA. ( C ) RNA stability assays following transcriptional inhibition with actinomycin D showed that ZmECT23 markedly accelerated the degradation of SCMV-GFP/ΔGDD genomic RNA compared with the GUS control. ( D ) In N. benthamiana cells heterologously expressing ZmECT23, the degradation rate of SCMV-G 6556 -GFP/ΔGDD genomic RNA was significantly delayed compared to that of SCMV-GFP/ΔGDD. Data are presented as means ± SD ( n = 9 infiltrated leaf patches from three independent experiments). TI, transcription inhibition. ( E ) A 6556 -methylated reads carry significantly shorter poly(A) tails than unmethylated reads (probability threshold >0.5). Statistical significance was determined by Welch’s t -test (* P < .05). ( F ) ZmECT23 expression level was significantly reduced in the 1 SL of CMV: ZmECT23 -infected seedlings compared to the CMV: GUS (control) seedlings at 18 days VPI ( n = 12 plants from three independent experiments). ( G ) Stronger GFP fluorescence was observed in the 1 SL of SCMV-GFP-infected maize seedlings compared to control seedlings at 7 dpi. Scale bar, 2 cm. ( H ) Percentages of maize seedlings infected by SCMV-GFP with different disease symptom grades. Disease degrees (N, I, II, III) were visually assessed at 7 dpi. ZmECT23 -silenced seedlings infected with SCMV-GFP showed more severe mosaic symptoms, with an increased proportion of severe disease symptoms (grade III) and a decreased proportion of asymptomatic cases (grade N), relative to control plants. Data are presented as means ± SE from three independent experiments. ( I ) SCMV genomic RNA accumulation levels in the 1 SL of ZmECT23 -silenced or control seedlings were determined through qRT-PCR. Data are expressed as the means ± SE ( n = 11 plants from three independent experiments). ( J ) Immunoblotting analysis of SCMV CP accumulation in ZmECT23 -silenced and control seedlings ( n = 3 plants). The relative intensities of the CP bands were quantified using ImageJ. Statistical differences in panels (B, F) and (I, J) were determined using the two-tailed Student’s t -test (* P < .05; ** P < .01; *** P < .001; **** P < .0001).

Article Snippet: Briefly, biotin-labeled SCMV RNA fragments were synthesized by Huada Biological Technology (Beijing, China).

Techniques: Modification, Sequencing, Methylation, Mutagenesis, Isolation, Immunoprecipitation, Inhibition, Control, Expressing, Infection, Fluorescence, Quantitative RT-PCR, Western Blot, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Potyviruses recruit host eIF4A3 to block m 6 A-mediated RNA decay by steric hindrance of viral RNA methylation in plants

doi: 10.1093/nar/gkaf1432

Figure Lengend Snippet: ZmeIF4A3 enhances SCMV infection. ( A ) Schematic diagrams of pSCMV-GUS-3Flag and pSCMV-ZmeIF4A3-3Flag. ( B ) Stronger mosaic symptoms were observed in the 1 SL of SCMV-ZmeIF4A3-3Flag-infected plants compared to SCMV-GUS-3Flag-infected (control) plants. Photographs were taken at 5 dpi. Scale bar, 2 cm. ( C ) Percentages of maize plants infected by SCMV-GUS-3Flag or SCMV-ZmeIF4A3-3Flag with different disease symptom grades at 5 dpi. ( D, E ) Relative viral genomic RNA and CP accumulation levels were higher in SCMV-ZmeIF4A3-3Flag-infected plants compared to control plants ( n = 9 plants from three independent experiments), as determined by qRT-PCR and immunoblotting analysis. ( F ) Relative expression levels of ZmeIF4A3/ZmeIF4A3-like were reduced in ZmeIF4A3/ZmeIF4A3-like -silenced plants ( n = 9 plants from three independent experiments) using qRT-PCR. ( G ) Reduced GFP fluorescence was observed in the 1 SL of SCMV-GFP-infected plants compared to control (CMV: GUS ) plants at 7 dpi. Scale bars, 2 cm. ( H ) Percentages of maize plants infected by SCMV-GFP with different disease symptom grades. Data are presented means ± SE, derived from three independent experiments. ( I ) Symptom progression on individual plant was recorded from 6 to 10 dpi. In this study, 43 CMV: ZmeIF4A3 -infected plants (three experiments) and 57 CMV: GUS -infected plants (three experiments) were inoculated by SCMV-GFP. The values represent the means ± SE. ( J, K ) SCMV genomic RNA and CP accumulation in the 1 SL of ZmeIF4A3/ZmeIF4A3-like -silenced or control plants ( n = 9 plants from three independent experiments) were determined through qRT-PCR and immunoblotting analysis. Relative intensities of the CP bands were quantified using ImageJ software. CBB-stained gels were used to show sample loadings. In panels (D–F) and (I–K), data are presented as means ± SE (two-tailed Student’s t test, * P < .05; ** P < .01; *** P < .001; **** P < .0001).

Article Snippet: Briefly, biotin-labeled SCMV RNA fragments were synthesized by Huada Biological Technology (Beijing, China).

Techniques: Infection, Control, Quantitative RT-PCR, Western Blot, Expressing, Fluorescence, Derivative Assay, Software, Staining, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Potyviruses recruit host eIF4A3 to block m 6 A-mediated RNA decay by steric hindrance of viral RNA methylation in plants

doi: 10.1093/nar/gkaf1432

Figure Lengend Snippet: ZmeIF4A3 is recruited into SCMV VRCs via direct binding with NIa-Pro. ( A ) Subcellular localization of ZmeIF4A3-GFP in the leaf cells of H2B-RFP transgenic N. benthamiana by confocal microscopy at 3 days post-infiltration. Co-localization analysis of ZmeIF4A3-GFP with SCMV-DsRed or SCMV-6K2-mCherry, and ZmPPDK-GFP (negative control) with SCMV-6K2-mCherry in the leaf cells of N. benthamiana . The 6K2-mCherry served as a marker for SCMV VRCs. Confocal images of SCMV-ZmeIF4A3-mRFP in B2-GFP (a marker that binds double-stranded RNA (dsRNA) and labels active viral replication sites) transgenic N. benthamiana leaf cells at 5 days post-infiltration. Scale bars, 10 μm. The yellow line segment delineates the region for fluorescence intensity measurement. The line graph illustrates relative fluorescence intensity, with the y -axis depicting the relative fluorescence intensities of GFP or RFP and the x -axis representing transect length in micrometers. The blue line corresponds to fluorescence induced by ZmeIF4A3-GFP or B2-GFP, while the red line indicates fluorescence induced by H2B-RFP, SCMV-DsRed, or SCMV-ZmeIF4A3-mRFP. The graph shows Pearson’s correlation coefficients derived from three images, indicating significant co-localization between ZmeIF4A3-GFP and SCMV-6K2-mCherry. Data are presented as means ± SE (Student’s t -test; *** P < .001). ( B ) Y2H assays showed the interaction between ZmeIF4A3 and NIa-Pro. ( C ) LCI assay confirmed the ZmeIF4A3–NIa-Pro interaction. Agrobacterium strain GV3101 containing the LUC constructs was co-infiltrated into N. benthamiana leaves. Images of infiltrated leaves were captured after three days of incubation. ( D ) Co-IP analysis showed the interactions between SCMV-encoded NIa-Pro with ZmeIF4A3. N. benthamiana leaves were co-infiltrated with A. tumefaciens cells harboring expression vectors. Leaf protein extracts were incubated with anti-GFP nanobody agarose beads. Samples before (Input) and after (IP) were analyzed by immunoblotting using anti-GFP or Flag antibodies. ( E ) GST pull-down assays confirmed the direct interaction between SCMV NIa-Pro and ZmeIF4A3. Black arrowheads indicate GST-NIa-Pro or GST. ( F ) Deletion of 287–316 region (ZmeIF4A3 Δ287-316 ) abolished co-localization with SCMV-6K2-mCherry in N. benthamiana leaf cells. Photographs were taken at 5 days post-infiltration. Scale bar, 10 μm.

Article Snippet: Briefly, biotin-labeled SCMV RNA fragments were synthesized by Huada Biological Technology (Beijing, China).

Techniques: Binding Assay, Transgenic Assay, Confocal Microscopy, Negative Control, Marker, Fluorescence, Derivative Assay, Construct, Incubation, Co-Immunoprecipitation Assay, Expressing, Western Blot

Journal: Nucleic Acids Research

Article Title: Potyviruses recruit host eIF4A3 to block m 6 A-mediated RNA decay by steric hindrance of viral RNA methylation in plants

doi: 10.1093/nar/gkaf1432

Figure Lengend Snippet: ZmeIF4A3 binds to SCMV genomic RNA and reduces its m 6 A methylation levels by hindering the attachment of ZmMTA. ( A ) RIP-qPCR assays revealed the binding of ZmeIF4A3 to the peak5 region. Protein-RNA complex was extracted from N. benthamiana leaves ( n = 3 independent experiments) co-infiltrated with SCMV-GFP and ZmeIF4A3-3Flag, followed by immunoprecipitation with anti-Flag or mouse IgG (negative control). ( B ) EMSA results indicated that ZmeIF4A3 bound to the peak5 region of SCMV genomic RNA. The results showed that ZmeIF4A3 was capable of binding to the biotin-labeled probe, and the unlabeled probe could competitively interfere with the biotin-labeled probe binding to ZmeIF4A3. ( C ) SELECT-qPCR analysis revealed significantly elevated m 6 A levels of A 6556 in ZmeIF4A3/ZmeIF4A3-like -silenced (CMV: ZmeIF4A3/ZmeIF4A3-like ) plants compared to CMV: GUS -silenced (control) plants. Values are presented as means ± SD ( n = 9 plants from three independent experiments, two-tailed Student’s t test). ( D ) SELECT-qPCR analysis showed that overexpression of ZmeIF4A3-3Flag significantly reduced the m 6 A modification levels at the A 6556 of the SCMV genomic RNA ( n = 3 independent experiments). ( E ) ZmeIF4A3 inhibits ZmMTA binding to SCMV peak5 region, as determined by Flag-ZmMTA-RIP-qPCR assays. pSCMV-GFP and 3Flag-ZmMTA were agroinfiltrated into N. benthamiana leaves ( n = 4 independent experiments) along with GUS-3Myc or ZmeIF4A3-3Myc. Immunoblotting confirmed the expression of ZmMTA-3Flag, GUS-3Myc, and ZmeIF4A3-3Myc. Molecular weight markers are shown on the left. ( F ) ZmMTA-RIP-qPCR analysis revealed significantly enhanced ZmMTA occupancy on SCMV genomic RNA in CMV: ZmeIF4A3/ZmeIF4A3-like- infected plants compared to CMV: GUS -infected (control) plants. Data are means ± SE from three independent experiments. ( G ) ZmeIF4A3 inhibits ZmMTA binding to peak5 of SCMV genomic RNA validated by the biotinylated RNA pull-down assays. Bio-A 6556 probe was incubated with His-TF-3Flag-ZmMTA, or with His-TF-3Flag-ZmMTA and His-TF-ZmeIF4A3-3Myc together. Components pulled down using streptavidin beads were detected by immunoblotting with anti-Flag antibody. In panels (A), (D), (E), and (F), values are presented as means ± SE (two-tailed Student’s t test, * P < .05; ** P < .01; *** P < .001; **** P < .0001).

Article Snippet: Briefly, biotin-labeled SCMV RNA fragments were synthesized by Huada Biological Technology (Beijing, China).

Techniques: Methylation, Binding Assay, Immunoprecipitation, Negative Control, Labeling, Control, Two Tailed Test, Over Expression, Modification, Western Blot, Expressing, Molecular Weight, Infection, Incubation

Journal: Molecular Plant Pathology

Article Title: A single phosphorylatable amino acid residue is essential for the recognition of multiple potyviral HCPro effectors by potato Ny tbr

doi: 10.1111/mpp.70027

Figure Lengend Snippet: HCPro site 247 in diverse potyviruses determines Ny tbr recognition. Representative images of callose deposition responses and callose counts/mm 2 following transient expression of the indicated proteins following water or flg22 treatment. Callose counts/mm 2 are shown from PVY, TuMV and SCMV HCPro expressions in (a) Premier Russet (PR) plants ( n ≥ 20); (b) PR plants with flg22 ( n ≥ 15). Callose counts/mm 2 are also shown for the mutant SCMV HCPro S247A in (c) PR plants ( n ≥ 45); (d) PR plants with flg22 ( n ≥ 50). Kruskal–Wallis test ( α < 0.05) was used to test for statistical significance. Means marked with the same letter are not statistically different according to Dunn's test ( p < 0.05).

Article Snippet: Despite a generally low degree of amino acid sequence similarity to PVY O HCPro, a similar response was observed with the unrelated SCMV HCPro, which bears a phosphorylatable residue at position 247.

Techniques: Expressing, Mutagenesis

Journal: Molecular Plant Pathology

Article Title: A single phosphorylatable amino acid residue is essential for the recognition of multiple potyviral HCPro effectors by potato Ny tbr

doi: 10.1111/mpp.70027

Figure Lengend Snippet: Sequence identity of potyviral HCPro proteins compared to the PVY O HCPro and conservation among potyviruses at sites within the HCPro signature motif distinguishing the PVY O from PVY N strain groups.

Article Snippet: Despite a generally low degree of amino acid sequence similarity to PVY O HCPro, a similar response was observed with the unrelated SCMV HCPro, which bears a phosphorylatable residue at position 247.

Techniques: Sequencing

Journal: Molecular Plant Pathology

Article Title: A single phosphorylatable amino acid residue is essential for the recognition of multiple potyviral HCPro effectors by potato Ny tbr

doi: 10.1111/mpp.70027

Figure Lengend Snippet: Structural conservation of HCPro proteins that induced Ny tbr + defence. (a) Structural alignment of the HCPro proteins from PVY O , BsMoV, SCMV, and SCMoV. Each model was given a unique colour, and the regions of the BsMoV, SCMV, and SCMoV HCPro models that match the reference structure (PVY O HCPro) are in full colour, while unaligned regions are lighter. The regions highlighted correspond to residues 236, 238, 247, 252, 262, 269, 270, and 301 that define the signature region distinguishing PVY O from PVY N and that are within the central domain of HCPro, which encompasses residue 100–311. (b) The structure of the region encompassing amino acids 227–327 that includes the PVY signature residues from each virus are depicted individually. In both (a) and (b), the serine from each potyviral HCPro protein that aligns with the PVY O HCPro S247 is highlighted and indicated with an arrow.

Article Snippet: Despite a generally low degree of amino acid sequence similarity to PVY O HCPro, a similar response was observed with the unrelated SCMV HCPro, which bears a phosphorylatable residue at position 247.

Techniques: Residue, Virus