Loose Plant Architecture 1-Interacting Kinesin-like Protein KLP Promotes Rice Resistance to Sheath Blight Disease

Abstract

Background: Sheath blight disease (ShB) is a destructive disease affecting rice production. Previously, we have reported that Loose Plant Architecture 1 (LPA1) promotes resistance to ShB. However, the mechanisms by which LPA1 confers resistance against this disease have not been extensively investigated. Notably, interactors that regulate LPA-1 activity remain elusive.

Findings: In this study, we identified the interaction of kinesin-like protein (KLP) with LPA1 in the nucleus of rice cells by yeast two-hybrid, bimolecular fluorescent complimentary (BiFC), and co-immunoprecipitation (co-IP) assays. To investigate the role of KLP in promoting resistance to ShB, wild-type, klp mutant, and KLP overexpressor (KLP OX) rice plants were inoculated with Rhizoctonia solani AG1-IA. The results indicated that, compared with the wild-type control, klp mutants were more susceptible while KLP OX plants were less susceptible to ShB. Since LPA1 transcriptionally activates PIN-FORMED 1a (PIN1a), we examined the expression of 8 related PIN genes. The results showed that only the expression of PIN1a and PIN3b coincided with KLP expression levels. In addition, a chromatin immunoprecipitation (ChIP) assay showed that KLP bound directly to the promoter region of PIN1a but not of PIN3b. Transient expression assays confirmed that LPA1 and KLP transcriptionally activate PIN1a, and that coexpression of KLP and LPA1 had an additive effect on the activation of PIN1a, suggesting that KLP enhances LPA1 transcriptional activation activity.

Conclusions: Taken together, our results show that KLP is a novel LPA1 interactor that promotes resistance of rice to ShB.

Keywords: Defense; KLP; Rice; Sheath blight disease; Transcription activation.

Fig. 1

Interaction between KLP and LPA1. a The interaction between LPA1 and KLP or IDD13 was analyzed by yeast two hybrid (Y2H) assay. BD: GAL4-DNA binding domain; AD: activation domain; −TL: SD medium without tryptophan and leucine; −TLH: SD medium without tryptophan, leucine, and histidine. b LPA1-nYFP + − cYFP or LPA1-nYFP + KLP-cYFP were coexpressed in rice protoplasts to detect YFP protein reconstruction. Bars = 10 μm. c The interaction between LPA1 and KLP was analyzed in tobacco leaves by co-IP. LPA1-GFP+ KLP-Myc or LPA1-GFP were transformed into tobacco leaves using Agrobacterium-mediated transformation. Western blot analysis used an anti-Myc or anti-GFP antibody. Anti-GFP antibody was used to immunoprecipitation. d Relative expression patterns of LPA1 and KLP were examined at 0, 24, 48, and 72 h post-inoculation (hpi) with R. solani AG1-IA. The error bars indicate the mean ± SE (n = 3). Different letters indicate significant differences at P < 0.05

Fig. 2

KLP promotes resistance of rice to ShB. a Genomic structure of KLP mutants. White and black boxes indicate untranslated regions (UTR) and open reading frame (ORF) region, respectively. The lines between black boxes indicate introns. The red triangles indicate T-DNA insertion sites. The labels inside triangle indicate mutant numbers from SALK (http://signal.salk.edu/cgi-bin/RiceGE). klp-1 and klp-2 are the individual KLP insertional mutants. b Expression levels of KLP in wild-type (WT) and KLP mutants (klp-1 and klp-2). The error bars indicate the mean ± SE (n = 3). Different letters indicate significant differences at P < 0.01. c Expression level of KLP was analyzed in WT and KLP overexpressors (OX #1, #2, #4, and #5). The error bars indicate the mean ± SE (n = 3). Different letters indicate significant differences at P < 0.05. d Wild-type (WT), klp mutants (#1 and #2) and KLP OX (#2 and #5) plants were inoculated with R. solani AG1-IA. e Percentage of leaf area covered with lesions in the plant lines shown in (d). Data represent the means ± standard error (n > 15). The error bars indicate the mean ± SE (n = 3). Different letters indicate significant differences at P < 0.05

Fig. 3

Expression levels of PIN genes in wild-type, klp, and KLP OX plants. Shown are the expression levels of PIN1a, PIN1b, PIN1c, PIN1d, PIN3a, PIN3b, PIN5a, and PIN5b in one-month-old, klp-1 and KLP OX-5 plant leaves relative to WT plants. The error bars indicate the mean ± SE (n = 3). Different letters indicate significant differences at P < 0.05

Fig. 4

LPA1 and KLP directly activate PIN1a. a Schematic diagram showing the location of the probes (P1-P3 and P4-P6) used for chromatin immunoprecipitation (ChIP) assay within the 1.5 kb promoter regions of PIN1a and PIN3b, respectively. b The DNA fragments were immunoprecipitated from p35S:KLP:GFP transgenic plants calli, and the enrichment was analyzed by qPCR. Input DNA was used to normalize the data. Anti-GFP antibody was used for immunoprecipitation with pre-immune serum as control. Error bars represent the mean ± SE (n = 3). Different letters indicate significant differences at P < 0.05. c Schematic diagram indicating the constructs used in the transient assay. 1.5 kb of PIN1a promoter was used to drive ß-glucuronidase (GUS) gene coding sequences. 35S promoter was used to drive LPA1, KLP or luciferase (Luc) gene OFR sequences. d Plasmids corresponding to p35S:LPA1, p35S:KLP, p35S:KLP + p35S:LPA1 were co-transformed with the vector expressing the GUS under the control of the PIN1a promoter (pPIN1a) in protoplasts. The luciferase expression level was utilized to normalize the GUS expression. Error bars represent the mean ± SE (n = 3). Different letters indicate significant differences at P < 0.05