Additional file 1: of The prediction of a pathogenesis-related secretome of Puccinia helianthi through high-throughput transcriptome analysis

Dataset of 35286 ORFs of the Puccinia helianthi transcriptome (XLSX 4749 kb

Additional file 2: of The prediction of a pathogenesis-related secretome of Puccinia helianthi through high-throughput transcriptome analysis

Dataset of 908 putative secretory proteins (XLSX 129 kb

W287G and Y71G show increased resistance to tryptic cleavage.

<p>CHO cells were transfected with wild-type, W287G or Y71G ASIC1a. Following biotinylation of surface proteins, cells were treated with 0, 1, or 10 µg/ml trypsin for 20 mins. Surface proteins were isolated and blotted for ASIC1a. Note that wild-type ASIC1a at the surface was cleaved by trypsin in a dosage-dependent manner. Also note that the amount of uncleaved mutants was comparable to that of uncleaved WT, although the mutants only had a small fraction of cleaved population (see the bottom image with level enhanced). The decrease in loading in trypsin treated lanes was likely due to the loss of some cells during the treatment. Blots shown are representative from three separate experiments.</p

Mutating Tyr71 and Trp287 reduces ASIC1a surface expression.

<p>(<b>A</b>) The effect of N- and C-terminal tags on surface expression of ASIC1a. CHO cells were transfected with untagged, N-terminal or C-terminal HA- or Flag-tagged ASIC1a as indicated. Surface and total proteins were blotted with a goat anti-ASIC1 and a mouse anti-tubulin antibody, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026909#s4" target="_blank">Methods</a>. Right panel shows the quantification of the surface:total ratio of untagged and tagged ASIC1a. The dotted line indicates that the controls for HA and Flag tagged experiments were different. Average surface:total ratio for WT was 4.4%±0.5%. For easy comparison, the ratio of WT was set arbitrarily to 1. (<b>B</b>) Typical traces and quantification of pH6-activated current recorded from cells expressing untagged (n = 6) or N-terminal HA-tagged (n = 7) ASIC1a. (<b>C</b>) Western blot and quantification showing surface:total ratio of HA-tagged wild-type ASIC1a, W287G, or Y71G mutants. Asterisks indicate a significant difference from controls (P<0.01, unbalanced ANOVA).</p

Lowering the culture temperature increases the current amplitude and slows the rate of desensitization of ASIC1a.

<p>CHO cells were co-transfected with wild-type, W287G or Y71G ASIC1a together with GFP. Cells were cultured overnight at 37°C followed by 24 hrs at 37°C or 27°C. All recordings were performed at the same temperature (∼23°C). (<b>A</b>) Representative pH 6-induced current from cells expressing wild-type or mutant ASIC1a. (<b>B & C</b>) Quantification of the effect of 27°C on current amplitude (B) and the rate of desensitization (C) of wild-type ASIC1a. Asterisks indicate significant differences from 37°C (P<0.01, student's t-test).</p

W287G and Y71G reduces ASIC1a glycosylation while a reduced culture temperature has an opposite effect.

<p>(<b>A & B</b>) Representative blots showing the effect of a reduced culture temperature on ASIC1a glycosylation. CHO cells were transfected with wild-type ASIC1a, W287G or Y71G mutant. Surface biotinylation was performed after culturing at 37°C (A) or 27°C (B) for 24 hrs. Lysates were treated with PNGase F or Endo H as indicated, followed by NeutrAvidin precipitation. Surface and total fraction were blotted for ASIC1a. The relative position of Endo H-resistant and -sensitive ASIC1a was indicated by arrows. Note that 27°C increased the percentage of Endo H-resistant wild-type ASIC1a in total lysate. Also note the appearance of Endo H-resistant W287G and Y71G at the cell surface at 27°C (arrows). (<b>C</b>) Quantification of Endo H-resistant wild-type ASIC1a at 37°C and 27°C. Asterisk indicates significant difference (P = 0.025); n.s. stands for not significant.</p

Performance of the ddPCR assay in detecting <i>Y</i>. <i>pestis</i> DNA from soil and mouse liver tissue samples.

(A–C) The ddPCR readouts of the detection of ypo2088, caf1, and pla, respectively. (D–F) Linearity and dynamic ranges of the ddPCR assay for quantifying Y. pestis in soil and liver tissue samples with ypo2088, caf1, and pla as the target gene, respectively. The x-axis refers to the bacterial concentration [Log10 (CFU/sample)], and the y-axis refers to the ddPCR readout [Log10 (copies/reaction)]. Each test was repeated five times.</p

Performance of the multi-target ddPCR and single-target qPCR assays in detecting genomic DNA samples of <i>Y</i>. <i>pestis</i>.

(A–C) Serial dilutions of Y. pestis ranging from 102 to 106 CFU/mL were subjected to DNA extraction and ddPCR detection. The x-axis refers to the bacterial concentration [Log10 (CFU/sample]), and the y-axis refers to the ddPCR readout [Log10 (copies/reaction)]. (D–F) The same batch of DNA samples were also tested by single-target qPCR assays. Each test was repeated in triplicate.</p

Primers and probes for detecting <i>Y</i>. <i>pestis</i>.

BackgroundPlague, caused by the bacterium Yersinia pestis, is a zoonotic disease that poses considerable threats to human health. Nucleic acid tests are crucial for plague surveillance and the rapid detection of Y. pestis. However, inhibitors in complex samples such as soil and animal tissues often hamper nucleic acid detection, leading to a reduced rate of identifying low concentrations of Y. pestis. To address this challenge, we developed a sensitive and specific droplet digital polymerase chain reaction (ddPCR) assay for detecting Y. pestis DNA from soil and animal tissue samples.MethodsThree genes (ypo2088, caf1, and pla) from Y. pestis were used to develop a multi-target ddPCR assay. The limits of detection (LoD), reproducibility, and specificity were assessed for bacterial genomic DNA samples. The ability of the assay to detect low concentrations of Y. pestis DNA from simulated soil and mouse liver tissue samples was respectively evaluated and compared with that of quantitative real-time PCR (qPCR).ResultsThe results showed that the ddPCR LoDs ranged from 6.2 to 15.4 copies/reaction for the target genes, with good reproducibility and high specificity for Y. pestis. By testing 130 soil and mouse liver tissue samples spiked with Y. pestis, the ddPCR assay exhibited a better sensitivity than that of the qPCR assay used in the study, with LoDs of 102 colony forming units (CFU)/100 mg soil and 103 CFU/20 mg liver. Moreover, the assay presented good quantitative linearity (R2 = 0.99) for Y. pestis at 103–106 CFU/sample for soil and liver samples.ConclusionThe ddPCR assay presented good performance for detecting Y. pestis DNA from soil and mouse tissue samples, showing great potential for improving the detection rate of low concentrations of Y. pestis in plague surveillance and facilitating the early diagnosis of plague cases.</div

The qPCR performance in detecting <i>Y</i>. <i>pestis</i> DNA from simulated samples.

The qPCR performance in detecting Y. pestis DNA from simulated samples.</p