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. 2021 Nov 23;13(12):2349.
doi: 10.3390/v13122349.

Investigation of P1/HC-Pro-Mediated ABA/Calcium Signaling Responses via Gene Silencing through High- and Low-Throughput RNA-seq Approaches

Yen-Hsin Chiu  1   2 , Yu-Ling Hung  3 , Hsin-Ping Wang  1 , Wei-Lun Wei  1 , Qian-Wen Shang  1 , Thanh Ha Pham  1 , Chien-Kang Huang  4 , Zhao-Jun Pan  1 , Shih-Shun Lin  1   5   6
Affiliations

Investigation of P1/HC-Pro-Mediated ABA/Calcium Signaling Responses via Gene Silencing through High- and Low-Throughput RNA-seq Approaches

Yen-Hsin Chiu et al. Viruses. .

Abstract

The P1/HC-Pro viral suppressor of potyvirus suppresses posttranscriptional gene silencing (PTGS). The fusion protein of P1/HC-Pro can be cleaved into P1 and HC-Pro through the P1 self-cleavage activity, and P1 is necessary and sufficient to enhance PTGS suppression of HC-Pro. To address the modulation of gene regulatory relationships induced by turnip mosaic virus (TuMV) P1/HC-Pro (P1/HC-ProTu), a comparative transcriptome analysis of three types of transgenic plants (P1Tu, HC-ProTu, and P1/HC-ProTu) were conducted using both high-throughput (HTP) and low-throughput (LTP) RNA-Seq strategies. The results showed that P1/HC-ProTu disturbed the endogenous abscisic acid (ABA) accumulation and genes in the signaling pathway. Additionally, the integrated responses of stress-related genes, in particular to drought stress, cold stress, senescence, and stomatal dynamics, altered the expressions by the ABA/calcium signaling. Crosstalk among the ABA, jasmonic acid, and salicylic acid pathways might simultaneously modulate the stress responses triggered by P1/HC-ProTu. Furthermore, the LTP network analysis revealed crucial genes in common with those identified by the HTP network in this study, demonstrating the effectiveness of the miniaturization of the HTP profile. Overall, our findings indicate that P1/HC-ProTu-mediated suppression in RNA silencing altered the ABA/calcium signaling and a wide range of stress responses.

Keywords: ABA signaling; HTP-Seq; LTP-Seq; P1/HC-ProTu; calcium signaling; stress response.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Network genes among the three comparison datasets obtained from the HTP RNA-seq profiling: Col-0 vs. P1/HC-ProTu, Col-0 vs. P1Tu, and Col-0 vs. HC-ProTu comparative sets. (A) Venn diagram showing the distributions of shared and unique network genes. (B) Functional classification of unique genes in the P1Tu-only, HC-ProTu-only, and P1/HC-ProTu-only sections.
Figure 2
Figure 2
The P1/HC-ProTu altered ABA-mediated immune responses. (A) P1/HC-ProTu-only genes in the P1/HC-ProTu-only section of the HTP datasets that were functionally annotated to categories and subcategories of genes involved in (i) ABA biosynthesis, (ii) catabolism, (iii) transport, (iv) signaling regulation, (v) posttranscriptional regulation, (vi) calcium signaling, and (vii) ABA responses. (B) Heatmap showing the expression patterns of the genes in the P1/HC-ProTu-only section that are involved in the ABA signaling pathway. ‘+’ and ‘−’ represent genes that positively or negatively regulate the ABA signaling pathway. The symbols in the right panel indicate the functions of genes in various biotic and abiotic stress responses and developmental processes.
Figure 3
Figure 3
Endogenous ABA detection and ABA sensitivity assay. (A) Determination of the endogenous ABA amounts. The mean values ± SD were obtained from three biological repeats. Comparisons between two groups were performed using a Student’s t test. *** p < 0.001. (B) ABA sensitivity assay. The germination phenotypes were classified into four phases: I, rupture of the seed coat; II, radicle protrusion; III, fully-opened cotyledons; and IV, true leaf development.
Figure 4
Figure 4
Calcium signaling pathway in response to P1/HC-ProTu. (A) Calcium signaling pathway in the P1/HC-ProTu-only section of the HTP profiles in the Col-0 vs. P1/HC-ProTu, Col-0 vs. P1Tu, and Col-0 vs. HC-ProTu comparison sets obtained from the HTP profiles. (B) Heatmap showing the expression patterns of the P1/HC-ProTu-only genes involved in the calcium signaling pathway.
Figure 5
Figure 5
Phenotypes of the P1/HC-ProTu and P1/HC-ProZy plants. Ten-day-old seedlings of (A) Col-0, (B) P1/HC-ProZy, and (C) P1/HC-ProTu transgenic plants. Scale bars = 0.5 cm.
Figure 6
Figure 6
The qRT-PCR-based validation of the gene expressions in the P1/HC-Pro-related plants obtained from the HTP profiles. (A–D) DEGs in the ABA signaling pathway. (E–G) DEGs in the Ca2+ signaling pathway. The mean values ± SD were from three biological repeats. Comparisons between two groups were performed with Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 7
Figure 7
Regulatory mechanism controlling stomatal dynamics in response to P1/HC-ProTu. (A) Genes involved in the regulation of stomatal guard cell dynamics and development found in the P1/HC-ProTu-only section. The genes involved in stomatal closure, stomatal opening, starch degradation, and stomatal development are labeled in blue, red, green, and brown, respectively. (B) Heatmap showing the expression patterns of the P1/HC-ProTu-only genes involved in the stomatal closure, opening, and development.
Figure 8
Figure 8
Heatmaps showing the expression patterns of (A) cold response- and (B) senescence-related genes in the P1/HC-ProTu-only section of the HTP datasets.
Figure 9
Figure 9
Comparison of gene expression and networks obtained with the HTP and LTP profiles. (A) PCA plot of gene expression profiles from the HTP and LTP datasets of Col-0, P1Tu, HC-ProTu, and P1/HC-ProTu samples. The top two principal components (X-axis, PC1; Y-axis, PC2) are shown. Each data point represents one biological sample. (B) Venn diagram showing the shared and unique genes in the Col-0 vs. P1/HC-ProTu comparison samples between the HTP and LTP datasets. Network-based comparison of the 75 shared genes in the Col-0 vs. P1/HC-ProTu datasets between (C) the HTP and (D) the LTP datasets. The red and green lines represent positive and negative correlations, respectively. The dark red and green lines indicate conserved connections between the HTP and LTP networks, respectively.
Figure 10
Figure 10
Heatmaps showing the expression patterns of the 75 overlapped genes in the Col-0 vs. P1/HC-ProTu comparison identified from both the HTP and LTP profiles.
Figure 11
Figure 11
Network genes among the three comparison sets obtained from the LTP profiles: Col-0 vs. P1/HC-ProTu, Col-0 vs. P1Tu, and Col-0 vs. HC-ProTu comparison sets. (A) Venn diagram showing the distributions of shared and unique network genes. (B) Functional classification of unique genes in the P1Tu-only, HC-ProTu-only, and P1/HC-ProTu-only sections.
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