The Pathogen-Induced MATE Gene TaPIMA1 Is Required for Defense Responses to Rhizoctonia cerealis in Wheat

Abstract

The sharp eyespot, mainly caused by the soil-borne fungus Rhizoctonia cerealis, is a devastating disease endangering production of wheat (Triticum aestivum). Multi-Antimicrobial Extrusion (MATE) family genes are widely distributed in plant species, but little is known about MATE functions in wheat disease resistance. In this study, we identified TaPIMA1, a pathogen-induced MATE gene in wheat, from RNA-seq data. TaPIMA1 expression was induced by Rhizoctonia cerealis and was higher in sharp eyespot-resistant wheat genotypes than in susceptible wheat genotypes. Molecular biology assays showed that TaPIMA1 belonged to the MATE family, and the expressed protein could distribute in the cytoplasm and plasma membrane. Virus-Induced Gene Silencing plus disease assessment indicated that knock-down of TaPIMA1 impaired resistance of wheat to sharp eyespot and down-regulated the expression of defense genes (Defensin, PR10, PR1.2, and Chitinase3). Furthermore, TaPIMA1 was rapidly induced by exogenous H₂O₂ and jasmonate (JA) treatments, which also promoted the expression of pathogenesis-related genes. These results suggested that TaPIMA1 might positively regulate the defense against R. cerealis by up-regulating the expression of defense-associated genes in H₂O₂ and JA signal pathways. This study sheds light on the role of MATE transporter in wheat defense to Rhizoctonia cerealis and provides a potential gene for improving wheat resistance against sharp eyespot.

Keywords: Rhizoctonia cerealis; TaPIMA1; defense; multi-antimicrobial extrusion family; wheat (Triticum aestivum).

Figure 1

The expression profiles of TaPIMA1 in wheat with R. cerealis infection. (A) The expression pattern of TaPIMA1 in RNA-seq data of RILs. The sharp eyespot resistant/susceptible RILs were infected with R. cerealis and sampled at 4 and 10 dpi. (B) Transcript profiles of TaPIMA1 in sharp eyespot-resistant cultivar CI12633 and the susceptible wheat cultivar Wenmai6 at 1, 4, and 10 dpi with R. cerealis. (C) The expression patterns of TaPIMA1 in four wheat cultivars. All transcript data of TaPIMA1 were compared with those in Yangmai16. (D) Transcriptional analyzes of TaPIMA1 in different organs of CI12633 plants. The significant differences determined by one-way ANOVA (* p < 0.05, ** p < 0.01). Error bars indicates standard deviation.

Figure 2

Sequence and phylogenetic analyses of TaPIMA1. (A) The genomic structure of TaPIMA1 in wheat CI12633 plant. The grey frames, green frames, and lines represent exons, UTRs, and introns regions, respectively. (B) Schematic of TaPIMA1 protein. There are two MatE domains (no. 50–210 aa and no. 271–434 aa, respectively) and a transmembrane region (12 TMHs, no. 445–464 aa) in TaPIMA1 protein. (C) Phylogenetic analysis of the TaPIMA1 and other MATE proteins by the maximum likelihood method. Asterisk indicates this TaPIMA1 protein. The phylogenetic tree was constructed using MEGA 11 software. The sequences were referred to EnsemblPlants, GenBank, and Phytozome database.

Figure 3

Subcellular localization of TaPIMA1 in wheat protoplasts. The GFP (control) and fused TaPIMA1-GFP were transiently expressed in wheat chloroplasts, respectively. The confocal images were taken using 488 nm wavelengths. Scale bars are each 5 μm.

Figure 4

Silencing of TaPIMA1 by BSMV-VIGS reduced resistance to R. cerealis in CI12633 plants. (A) The mild chlorotic mosaic symptoms were displayed on the leaves of BSMV-infected CI12633 plants at 10 dpi. (B) RT-PCR analysis of BSMV CP in the CI12633 wheat plants. The TaActin (TraesCS5B02G124100.1) was set as an internal control. (C) Analysis of the expression of TaPIMA1 in BSMV-infected CI12633 plants by qRT-PCR at 10 dpi. The sharp eyespot symptoms on stems of BSMV-infected CI12633 plants at 10 (D) and 30 (F) dpi with R. cerealis. Disease severity was indicated by infection types (ITs). (E) qRT-PCR analysis of the biomass of R. cerealis in BSMV-infected CI12633 plants. (G) Disease lesion size of R. cerealis in TaPIMA1-silencing and control CI12633 plants at 30 dpi. The lesion length and width represent the lesion size of sharp eyespot. (H) The ITs of the BSMV-infected CI12633 plants in two batches. dpi, days post inoculation. The significant differences determined by one-way ANOVA (** p < 0.01). Error bars indicates standard deviation.

Figure 5

The expression levels of PR genes in TaPIMA1-silenced CI12633 plants at 10 dpi with R. cerealis. The PR-1.2 (A, GenBank accession no. AJ007349), PR10 (B, GenBank accession no. CA613496), chitinase3 (C, GenBank accession no. LOC542780), and defensin (D, GenBank accession no. CA630387), were regulated by TaPIMA1. The significant differences determined by one-way ANOVA (** p < 0.01). TaActin was used as internal control. Error bars indicates standard deviation.

Figure 6

The expression profiles of TaPIMA1 and PR genes in H₂O₂/MeJA-treated CI12633 plants. The expression profiles of TaPIMA1 in CI12633 plants after exogenous application of H₂O₂ (A) and MeJA (B). Expression of PR1.2, PR10, and Chitinase3 in H₂O₂ (C–E)- and MeJA (F–H)-treated CI12633 wheat plants. The CI12633 plants were sprayed with 10 mM H₂O₂/0.1 mM MeJA and 0.1% Tween-20 (mock) at the four-leaf stage, respectively. The significant differences were determined by Student’s t-test (* p < 0.05, ** p < 0.01). Error bars indicate standard deviation. TaActin was used as internal control.