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. 2017 Nov 24:8:2322.
doi: 10.3389/fmicb.2017.02322. eCollection 2017.

Overexpression of OsCIPK30 Enhances Plant Tolerance to Rice stripe virus

Zhiyang Liu  1   2 , Xuejuan Li  1   2 , Feng Sun  1   2 , Tong Zhou  1   2 , Yijun Zhou  1   2
Affiliations

Overexpression of OsCIPK30 Enhances Plant Tolerance to Rice stripe virus

Zhiyang Liu et al. Front Microbiol. .

Abstract

Rice stripe virus (RSV) causes a severe disease in Oryza sativa (rice) in many Eastern Asian countries. The NS3 protein of RSV is a viral suppressor of RNA silencing, but plant host factors interacting with NS3 have not been reported yet. Here, we present evidence that expression of RSV NS3 in Arabidopsis thaliana causes developmental abnormalities. Through yeast two-hybrid screening and a luciferase complementation imaging assay, we demonstrate that RSV NS3 interacted with OsCIPK30, a CBL (calcineurin B-like proteins)-interaction protein kinase protein. Furthermore, OsCIPK30 was overexpressed to investigate the function of OsCIPK30 in rice. Our investigation showed that overexpression of OsCIPK30 in rice could delay the RSV symptoms and show milder RSV symptoms. In addition, the expression of pathogenesis-related genes was increased in OsCIPK30 transgenic rice. These results suggest that overexpression of OsCIPK30 positively regulates pathogenesis-related genes to enhance the tolerance to RSV in rice. Our findings provide new insight into the molecular mechanism underlying resistance to RSV disease.

Keywords: NS3; OsCIPK30; Rice stripe virus; interaction; pathogenesis-related genes.

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Figures

FIGURE 1
FIGURE 1
NS3 causes developmental abnormalities in Arabidopsis. (A) Morphological defects of Arabidopsis transgenic plants expressing RSV NS3. Photographs were taken 30 days after sowing. (B) Western blot analysis of 35S-Flag-6Myc-NS3 transgenic lines. The hyphen indicates the locations of the tagged NS3 proteins (23.87 KDa NS3 plus 7.2 KDa 6Myc). +: 35S-Flag-6Myc-SERRATE; –: Col-0; 1–8: individual NS3 transgenic lines.
FIGURE 2
FIGURE 2
NS3 and OsCIPK30 interactions are detected in the yeast two-hybrid assay. Yeast strain Gold was co-transformed with the indicated plasmids shown on the left side of the panels. (A) The co-transformants were spread onto the SD/Leu-Trp (DDO) selection medium and (B) the SD/-Ade/-His/-Leu/-Trp (QDO) selection medium. AD-T+BD-53 were used as positive control, AD-T+BD-lam were used as negative control.
FIGURE 3
FIGURE 3
Phylogenetic analysis of OsCIPK proteins and the interaction between NS3 and OsCIPK5, OsCIPK25, OsCIPK26, OsCIPK29, OsCIPK30, and OsCIPK32 in yeast cells and plant cells. (A) Phylogenetic analysis of all 33 OsCIPK proteins. The analysis was performed using MEGA 4 software. The ellipses indicate the proteins chosen for the interaction assay. (B) The interaction between NS3 and OsCIPK5, OsCIPK25, OsCIPK26, OsCIPK29, OsCIPK30, and OsCIPK32 by yeast two-hybrid assay. The co-transformants were spread onto the SD/-Ade/-His/-Leu/-Trp (QDO) selection medium. (C) The interaction between NS3 and OsCIPK5, OsCIPK25, OsCIPK26, OsCIPK29, OsCIPK30, and OsCIPK32 by a luciferase complementation imaging (LCI) assay. Schematic representation of the LCI assay shows the different combinations of infiltrated constructs that were fused either to the N-terminal (NLuc) or C-terminal (CLuc) regions of luciferase. The infiltration positions of the constructs (red arrows) and luminescence signals resulting from the protein–protein interaction in a leaf are shown.
FIGURE 4
FIGURE 4
Expression of OsCIPK genes infected by RSV. The assay was performed by real-time RT-PCR. RNA was isolated from RSV-infected rice plants at 7 days post-inoculation. The relative mRNA levels were calculated with respect to the expression level of the corresponding transcript in the wild type (WT) rice plants without RSV infection. All the experiments were repeated three times, and similar results were obtained. The data represent the means ± SD of triplicate measurements. Asterisks above the columns represent significance based on an unpaired, two-tailed Student’s t-test relative to the WT. ∗∗P < 0.01; ∗P < 0.05.
FIGURE 5
FIGURE 5
Effect of OsCIPK30 overexpression on RSV infection in rice. (A) Growth of the WT and Ubi-OsCIPK30 transgenic rice (T1 generation). (B) WT and Ubi-OsCIPK30 T1 transgenic rice were inoculated with RSV and photographed at 21 dpi. The lower panel of (B) shows the symptomatic leaves of the representative plants. (C) Temporal changes of RSV symptom development in WT and Ubi-OsCIPK30 T1 transgenic rice (OE-1, OE-2, OE-3). Thirty plants were used for each treatment in the experiment. (D) The expression level of OsCIPK30 in T1 transgenic rice (OE-1, OE-2, OE-3) by real-time RT-PCR at 21 dpi with RSV. The relative mRNA levels were calculated with respect to the expression level of the corresponding transcript in the WT rice plant. (E) The expression level of NP in T1 transgenic rice (OE-1, OE-2, OE-3) by real-time RT-PCR at 21 dpi with RSV. The relative mRNA levels were calculated with respect to the expression level of the corresponding transcript in the WT rice plant. All the experiments were repeated three times, and similar results were obtained. The data represent the means ± SD of triplicate measurements. The asterisks above the columns represent significance based on an unpaired, two-tailed Student’s t-test relative to the WT. ∗∗P < 0.01; ∗P < 0.05.
FIGURE 6
FIGURE 6
Transcriptionally modulated OsCIPK30 influenced the expression of a set of pathogenesis-related genes. (A) The expression levels of PR10 were significantly higher in OsCIPK30-overexpressing rice plants (OE-1, OE-2, OE-3) than WT plants after RSV infection. (B) The expression levels of PR1 were higher in OsCIPK30-overexpressing rice plants (OE-1, OE-2, OE-3) than WT plants after RSV infection. (C) The expression levels of PRB1-2 were significantly higher in OsCIPK30-overexpressing rice plants (OE-1, OE-2, OE-3) than WT plants after RSV infection. (D) The expression levels of PRB1-3 were significantly higher in OsCIPK30-overexpressing rice plants (OE-1, OE-2, OE-3) than WT plants after RSV infection. The un-inoculated plants were used as control. All experiments were repeated three times, and similar results were obtained. The data represent the means ± SD of triplicate measurements. Asterisks above the columns represent significance based on an unpaired, two-tailed Student’s t-test relative to the WT plants. ∗∗P < 0.01; ∗P < 0.05.
FIGURE 7
FIGURE 7
Expression of pathogenesis-related genes infected by RSV. The assay was performed by real-time RT-PCR. RNA was isolated from RSV-infected rice plants at 7 days post-inoculation. The relative mRNA levels were calculated with respect to the expression level of the corresponding transcript in the WT rice plants without RSV infection. All the experiments were repeated three times, and similar results were obtained. The data represent the means ± SD of triplicate measurements. Asterisks above the columns represent significance based on an unpaired, two-tailed Student’s t-test relative to the WT. ∗∗P < 0.01; ∗P < 0.05.
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