Indeterminate Domain Proteins Regulate Rice Defense to Sheath Blight Disease

Abstract

Background: Loose Plant Architecture 1 (LPA1), an indeterminate domain (IDD) protein, exhibits almost no expression in the leaves, but the overexpression of LPA1 significantly increases the resistance of rice to sheath blight disease (ShB) via the activation of PIN-FORMED 1a (PIN1a).

Results: In this study, we determined that Rhizoctonia solani infection significantly induced LPA1 expression in the leaves, and lpa1 was more susceptible to R. solani compared with the wild-type and revertant plants. In addition, infection with R. solani altered the expression of IDD3, IDD5, IDD10, and IDD13, and yeast two-hybrid, split-GFP, and coimmunoprecipitation assays showed that LPA1 interacts with IDD3 and IDD13. IDD13 RNAi plants were more susceptible, while IDD13 overexpressors were less susceptible to ShB compared with the wild-type. In parallel, idd3 exhibited no significant differences, while IDD3 overexpressors were more susceptible compared to the wild-type response to ShB. Additional chromatin-immunoprecipitation and electrophoretic mobility shift assay experiments indicated that IDD13 and IDD3 bound to the PIN1a promoter, and the transient assay indicated that IDD13 and IDD3 positively and negatively regulate PIN1a expression, respectively. Moreover, IDD13, IDD3, and LPA1 form a transcription factor complex that regulates PIN1a. A genetic study showed that the LPA1 repressor lines were similar to lpa1/IDD13 RNAi and were more susceptible than the lpa1 and IDD13 RNAi plants in response to ShB. The overexpression of IDD13 increased resistance to ShB in the lpa1 background.

Conclusions: Taken together, our analyses established that IDD3, IDD13, and LPA1 form a transcription factor complex to regulate the defense of rice against ShB possibly via the regulation of PIN1a.

Keywords: Defense; Indeterminate domain protein; Rice; Sheath blight disease; Transcription activation.

Fig. 1

Rhizoctonia solani-mediated LPA1 expression patterns and lpa1 mutant response to sheath blight disease. aLPA1 expression level in the leaves at 0, 24, 48, and 72 h post-inoculation (hpi) of R. solani AG1-IA. The error bars are the mean ± SE (n = 3). b Response of lpa1 and revertant (Rev.) to R. solani AG1-IA compared with the wild-type (WT). c Percentage of the leaf area covered with lesions in lpa1 and revertant (Rev.) compared with the WT. Data represent means ± SE (n > 10). dR. solani-mediated expression of PIN1a in WT, lpa1, and Rev. leaves before and after 72 hpi of R. solani. Different letters indicate significant differences at P < 0.05

Fig. 2

Interaction between LPA1 and IDD3 or IDD13. aIDD3, IDD5, IDD10, and IDD13 expression levels in the leaves at 0, 24, 48, and 72 h post-inoculation (hpi) of Rhizoctonia solani AG1-IA. The error bars are the mean ± SE (n = 3). The statistical analysis was performed for each gene, and different letters indicate significant differences at P < 0.05. b A yeast two-hybrid assay was performed to analyze the interaction between LPA1 and IDD3, IDD10, or IDD13. BD: GAL4-DNA binding domain; AD: activation domain; −T: without tryptophan; −L: without leucine; −H: without histidine. c Reconstitution of GFP fluorescence from LPA1-nYFP + IDD3-cCFP, LPA1-nYFP + IDD13-cCFP, and LPA1-nYFP + cCFP. Bars = 10 μm. DIC: differential interference contrast. d A co-IP assay was performed to analyze the interaction between LPA1 and IDD3, IDD13, or IDD10 in tobacco leaves. IDD3-Myc, IDD13-Myc, IDD10-Myc + LPA1-GFP, or empty vector + LPA1-GFP were transformed into tobacco leaves using Agrobacterium-mediated transformation. Green fluorescent protein (GFP) antibody-immunoprecipitated proteins were analyzed using western blot analysis with the Myc antibody. IDD3-Myc, IDD13-Myc, IDD10-Myc, and LPA1-GFP levels were analyzed by western blot using Myc and GFP antibodies, respectively

Fig. 3

Response of IDD3 and IDD13 mutants to sheath blight (Rhizoctonia solani). aIDD3 expression level was examined in the wild-type, the idd3 mutants (idd3–1 and idd3–2), and IDD3 OX (#2, #3, #4, and #6) plants. The error bars are the mean ± SE (n = 3). b IDD13 expression level was detected in the wild-type, IDD13 RNAi (#1, #2, #4, and #5), and IDD13 OX (#2, #3, #5, and #7) plants. The error bars are the mean ± SE (n = 3). c Response of the idd3 mutants and IDD3 OX (#2 and #4) plants to R. solani AG1-IA compared with the wild-type (WT). d Percentage of leaf area covered with lesions in the lines shown in (c). Data represent the means ± SE (n > 10). e Response of IDD13 RNAi (#1 and #4) and IDD13 OX (#2 and #5) plants to R. solani AG1-IA compared with the wild-type (WT). f Percentage of leaf area covered with lesions in the lines shown in (e). Data represent the means ± SE (n > 10). Different letters indicate significant differences at P < 0.05

Fig. 4

IDD3 and IDD13 bind and activate the PIN1a promoter. a IDD3-GFP and IDD13-GFP were detected in the lateral roots. GFP signal and bright field are shown in the left and right, respectively. Bars = 20 μm. b Schematic diagram indicating the location of the putative IDD-binding motif (gray circle) within 1.5 kb of the PIN1a promoter and the probes (P) used for chromatin immunoprecipitation (ChIP) assays. c Relative ratios of immunoprecipitated DNA to input DNA were determined by qPCR. Input DNA was used to normalize the data. –Ab or + Ab: green fluorescent protein (GFP) antibody. Error bars represent the mean ± SE (n = 3). d An electrophoretic mobility-shift assay (EMSA) was conducted to evaluate GST-IDD3 and GST-IDD13 affinities to P2 and mutated probe mP2. e A transient expression assay was conducted by co-transfection with p35S:IDD3 or p35S:IDD13 and each of the vectors expressing GUS under the control of native (pPIN1a) and IDD-binding motif-mutated (mpPIN1a) PIN1a promoters in protoplast cells. The luciferase gene driven by the 35S promoter was used as an internal control to normalize GUS expression. Error bars represent the mean ± SE (n = 6). Different letters indicate significant differences at P < 0.05

Fig. 5

IDD3, IDD13, and LPA1 form a complex to regulate PIN1a transcription. a A transient expression assay was conducted by co-transfection with p35S:IDD3, p35S:IDD13, p35S:LPA1, p35S:IDD3 + p35S:LPA1, p35S:IDD13 + p35S:LPA1 and the vector expressing the GUS under the control of native (pPIN1a) PIN1a promoters in protoplast cells. The luciferase gene driven by the 35S promoter was used as an internal control to normalize the GUS expression. Error bars represent the mean ± SE (n = 6). Different letters indicate significant differences at P < 0.05. b IDD3-HA + IDD13-Myc, IDD3-HA + IDD13-Myc + LPA1-GFP, or IDD3-HA + empty vector were transformed into tobacco leaves using Agrobacterium-mediated transformation. HA antibody-immunoprecipitated proteins were analyzed using western blot analysis with the Myc antibody. IDD3-HA, IDD13-Myc, and LPA1-GFP levels were analyzed by a western blot using HA, Myc, and GFP antibodies, respectively

Fig. 6

IDD13 and LPA1 genetic combinations in response to sheath blight (Rhizoctonia solani). a Response of IDD13 RNAi, lpa1, IDD13 RNAi/lpa, LPA1 repressor plants to R. solani AG1-IA compared with the wild-type (WT). b Percentage of the leaf area covered with lesions in the lines shown in (a). Data represent the means ± SE (n > 10). c Response of the lpa1, IDD13 OX, and lpa1/IDD13 OX plants to R. solani AG1-IA compared with the wild-type (WT). d Percentage of the leaf area covered with lesions in the lines shown in (c). Data represent the means ± SE (n > 10). Different letters indicate significant differences at P < 0.05

Fig. 7

The tiller number, thousand-grain weight and number of spikelet per panicle in the wild-type and IDD13 OX plants. a The three-and-a-half-month-old wild-type and IDD13 OX plants (#2, #5, and #7), as well as their panicles, are shown. Tiller number b, tiller angles c, thousand-grain weight d, and the number of grain weight per panicle e from the wild-type and IDD13 OX plants (#2 and #5) were calculated. Data indicate the average ± SD (n > 15). Different letters indicate significant differences at P < 0.05