Journal: Journal of experimental botany

Article Title: Evidence for a positive regulatory role of strawberry (Fragaria x ananassa) Fa WRKY1 and Arabidopsis At WRKY75 proteins in resistance.

doi: 10.1093/jxb/erp152

Figure Lengend Snippet: Fig. 3. Relative Fa WRKY1 expression in Camarosa strawberry fruit tissues (R stage) naturally infected with C. acutatum. Red fruits (R stage) showing increasing symptoms of natural infection with C. acutatum (ID1 to ID4) were collected from the field. Pools made of red fruits with similar symptoms were prepared. Total RNA was extracted from these pools to generate RNA-infected (ID1 to ID4) and control samples (C). Transcript level of Fa WRKY1 was determined by qRT-PCR with cDNA generated from these RNA samples. Mean expression and SD values from three independent determinations are shown. The expression values of the Fa WRKY1 gene were normalized as described in Fig. 2 and were relative to that in uninfected R stage fruits (C).

Article Snippet: DAB staining were carried out as previously described (Alvarez et al., 1998). qRT-PCR analysis For strawberry, total RNA extraction was performed as previously described (Medina-Escobar et al., 1997) and for Arabidopsis using the AurumTM Total RNA mini kit (BioRad).

Techniques: Expressing, Infection, Control, Quantitative RT-PCR, Generated

Journal: Journal of experimental botany

Article Title: Evidence for a positive regulatory role of strawberry (Fragaria x ananassa) Fa WRKY1 and Arabidopsis At WRKY75 proteins in resistance.

doi: 10.1093/jxb/erp152

Figure Lengend Snippet: Fig. 2. Fa WRKY1 expression in strawberry. (A) Relative Fa WRKY1 expression in several strawberry plant tissues. G1 (rec), fruit receptacle of green-1 stage; G1 (aq), achene of green-1 stage; R, root; L, leaf; S, stolon; C, crown; P, petiole. (B) Relative Fa WRKY1 expression during strawberry fruit developmental and ripening stages. G1 (green-1), G2 (green-2), G3 (green-3), W (white), I (intermediate), R (red), OR1 (overripening-1), OR2 (over- ripening-2). Values for OR1 and OR2 stages (grey bars) are for fruit tissue (receptacle+achenes). Black bars, receptacle. White bars, achenes. In both (A) and (B), quantitative RT-PCR analysis was performed on total RNA prepared from these samples to de- termine transcript level of Fa WRKY1. Mean expression and SD values from three independent determinations are shown. The expression values of the Fa WRKY1 gene were normalized using the expression level of Fa RIB413 gene as an internal standard and were relative to that in G1 (rec) that was set as one as described in Casado-Dı´az et al. (2006).

Article Snippet: DAB staining were carried out as previously described (Alvarez et al., 1998). qRT-PCR analysis For strawberry, total RNA extraction was performed as previously described (Medina-Escobar et al., 1997) and for Arabidopsis using the AurumTM Total RNA mini kit (BioRad).

Techniques: Expressing, Quantitative RT-PCR

Journal: Journal of experimental botany

Article Title: Evidence for a positive regulatory role of strawberry (Fragaria x ananassa) Fa WRKY1 and Arabidopsis At WRKY75 proteins in resistance.

doi: 10.1093/jxb/erp152

Figure Lengend Snippet: Fig. 9. Relative gene expression of defence-related genes in wild-type, wrky75At22 mutant and transgenic lines overexpressing Fa WRKY1, wrky75At22/Fa WRKY1 (A) and wild-type/Fa WRKY1 (B), during Pst DC3000 and Pst DC3000 avrRpm1 infection. Samples were harvested at 0, 3, 6, 8, 16, and 24 h post-inoculation for preparation of total RNA. Accumulation of transcripts was monitored by qRT-PCR. Expression levels were normalized with respect to the internal control ACTIN2 and displayed relative to the expression in mock-treated wild-type samples, as described in Fig. 2. Data bars represent the mean levels of transcript quantified from three independent biological experiments (6SD). Error bars for most data points are too small to see them on the graph. In (A), white circle indicates no significant differences (ANOVA, P >0.05) between wrky75At22 and wrky75At22/Fa WRKY1 values, although both are statistically significant relative to the wild-type values; black circle indicates no significant differences (ANOVA, P >0.05) between wild- type and wrky75At22 values, although both are statistically significant relative to the wrky75At22/Fa WRKY1 values. In (B), black circle indicates no significant differences (P >0.05, Student’s t test) between wild-type and wild-type/Fa WRKY1 values. The whole experiment (A, B) was performed twice with similar results.

Article Snippet: DAB staining were carried out as previously described (Alvarez et al., 1998). qRT-PCR analysis For strawberry, total RNA extraction was performed as previously described (Medina-Escobar et al., 1997) and for Arabidopsis using the AurumTM Total RNA mini kit (BioRad).

Techniques: Gene Expression, Mutagenesis, Transgenic Assay, Infection, Quantitative RT-PCR, Expressing, Control

Journal: Viruses

Article Title: Exploiting the Achilles' Heel of Viral RNA Processing to Develop Novel Antivirals.

doi: 10.3390/v17010054

Figure Lengend Snippet: Figure 3. 5342191 inhibits HCoV-229E and HCoV-OC43 replication. (A) A schematic of the exper- imental protocol. Huh7 cells were infected with indicated coronavirus strains for 1 h; cells were washed and then treated with either DMSO or 5342191. Media were harvested and levels of viral RNA release into media were determined by RTqPCR. Cells were lysed for RNA extraction to measure levels of intracellular viral RNA using RTqPCR, and viral protein production via Western blotting. (B) Effect of 5342191 concentrations on HCoV-229E RNA accumulation in media (by RTqPCR) and cell viability (assessed using Alamar blue). (C) A correlation of viral RNA release and infectious virus was produced. Infected cells were treated with EC50 (1.2 µM) or EC90 (2.5 µM) doses of 5342191 for 24 h, then media were harvested, and viral RNA accumulation in media was measured by RTqPCR, infectious virus by TCID50 assay. (D) Cells were infected with the HCoV-OC43 virus at an MOI of 2 and then treated with either DMSO or 2.5 µM 5342191 for 24 h. Culture supernatants were harvested and analyzed by RTqPCR to measure the release of HCoV OC43 into the culture media. (E) Top: a schematic of the coronavirus virus genome and subgenomic RNAs generated. Shown are the positions of primer sets used to measure genomic or total viral RNAs. Bottom: infected cells were treated with DMSO or 2.5 µM 5342191 for 24 h. Extracellular viral RNA accumulation was measured by RTqPCR. Total RNA was extracted from cells and the abundance of genomic or total viral RNA (genomic and nested RNAs) was measured by RTqPCR. (F) Representative immunoblots showing the impact of 5342191 on the production of the nucleocapsid (N) protein of the HCoV-229E virus (top) and the N protein and spike (S) protein of the HCoV-OC43 virus (bottom). ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Total RNA was extracted from cells using the BioRad Aurum Total RNA Lysis Kit (BioRad) as per the manufacturer’s instructions.

Techniques: Infection, RNA Extraction, Western Blot, Virus, Produced, TCID50 Assay, Generated

Journal: Viruses

Article Title: Exploiting the Achilles' Heel of Viral RNA Processing to Develop Novel Antivirals.

doi: 10.3390/v17010054

Figure Lengend Snippet: Figure 8. 5342191 inhibits PR8 influenza A virus replication. (A) A549 cells were infected at an MOI of 2 with PR8 for 1 h, and media were removed and replaced with media containing DMSO or indicated doses of 5342191. At 24 hpi, media were harvested, and levels of viral RNA released were determined by RTqPCR. (B) To correlate the level of viral RNA released to the infectious virus, infected cells were treated with doses of 5342191 required to reduce PR8 RNA accumulation in media by 90% (EC90, 0.104 µM). Media were assessed in parallel to determine the effect of the compound addition on the level of infectious virus present as determined by TCID50 assay. (C–E) A549 cells were infected with PR8 at an MOI of 2 for 1 h, and media were removed and replaced with media containing DMSO or 0.104 µM 5342191. At 24 hpi, cells were harvested, (C) expression of viral NP or NS1 was assessed by the western blot or (D) total RNA extracted, and intracellular levels of indicated viral RNAs were determined by RTqPCR. (E) Ratio of M2 versus M1 or NS2 versus NS1 to assess the impact of 5342191 on viral RNA splicing. Shown are the results from n > 3 independent assays. **, p < 0.01, ***, p < 0.001, ****, p < 0.0001.

Article Snippet: Total RNA was extracted from cells using the BioRad Aurum Total RNA Lysis Kit (BioRad) as per the manufacturer’s instructions.

Techniques: Virus, Infection, TCID50 Assay, Expressing, Western Blot

Journal: Viruses

Article Title: Exploiting the Achilles' Heel of Viral RNA Processing to Develop Novel Antivirals.

doi: 10.3390/v17010054

Figure Lengend Snippet: Figure 9. 5342191 acts post-entry to inhibit influenza replication. A549 cells were infected at an MOI of 2 with PR8 for 1 h, and then media were replaced. Media were harvested at various times post-infection and (A) the level of viral RNA in media was determined by RTqPCR; (B,C) cells were harvested and (B) protein lysates were used for western blot to detect PR8 NP or NS1 proteins, or (C) total RNA extracted and the intracellular abundance of viral RNAs (M1, NP, and NS1) was determined by RTqPCR. (D) To assess whether 5342191 could inhibit virus replication at times after infection, cells were infected with PR8, MOI 2, for 1 h, then 0.104 µM of 5342191 was added 1, 6, 9, or 12 hpi. At 24 hpi, media was harvested, and levels of viral RNA were determined by RTqPCR. ****, p < 0.0001.

Article Snippet: Total RNA was extracted from cells using the BioRad Aurum Total RNA Lysis Kit (BioRad) as per the manufacturer’s instructions.

Techniques: Infection, Western Blot, Virus