TaPIMP2, a pathogen-induced MYB protein in wheat, contributes to host resistance to common root rot caused by Bipolaris sorokiniana

Abstract

MYB transcription factors (TFs) have been implicated in various biology processes in model plants. However, functions of the great majority of MYB TFs in wheat (Triticum aestivum L.) have not been characterized. The soil-borne fungal pathogens Bipolaris sorokiniana and Rhizoctonia cerealis are the causal agents of important destructive diseases of wheat. Here, the TaPIMP2 gene, encoding a pathogen-induced MYB protein in wheat, was isolated through comparative transcriptomic analysis, and its defensive role was studied. TaPIMP2 was proved to localize in nuclei. TaPIMP2 responded in a different extent and speed upon infections of B. sorokiniana or R. cerealis. TaPIMP2 displayed different expression patterns after exogenous application of phytohormones, including abscisic acid, ethylene, and salicylic acid. Silencing of TaPIMP2 repressed resistance of wheat cultivar Yangmai 6 to B. sorokiniana, but did not alter resistance of wheat line CI12633 to R. cerealis. TaPIMP2 overexpression significantly improved resistance to B. sorokiniana rather than R. cerealis in transgenic wheat. Moreover, TaPIMP2 positively modulated the expression of pathogenesis-related genes, including PR1a, PR2, PR5, and PR10. Collectively, TaPIMP2 positively contributes to wheat resistance to B. sorokiniana possibly through regulating the expression of defense-related genes, and TaPIMP2 plays distinct roles in defense responses to different fungal infection.

Figure 1

Multiple sequence alignment of TaPIMP2, TaRIM1, and TaPIMP1 from wheat. The R2 and R3 domains are indicate by solid lines.

Figure 2

A phylogenetic analysis of 18 known-functional MYB members from 8 plant species. TaPIMP2 from wheat was indicated by black box. The Genbank accession number of proteins including this analysis: Ntmyb1: AAB41101; TaRIM1: AMP18876; AtMYB96: AED97610; AtMYB30: Q9SCU7; AtMYB72: NP_176012; GhMYB108: ALL53614; AtMYB108: Q9LDE1; TaPIMP1: ABU93236; OsJAMyb: AAK08983; AtMYB3R3: NP_001078127; ZmMYB3R3: NP_001151448; TaMYB3R1: ADO32617; OsMYB3R2: NP_001044767; StMYB1R-1: NP_001275346; GmMYB176: ABH02865; TaLHY: ADW09013; CCA1: AEC10760.

Figure 3

Transcriptional patterns of TaPIMP2 homoeologous member response to fungal infection. Transcription of TaPIMP2 homoeologous member response to infection with Rhizoctonia cerealisin in wheat line CI12633 (a) and infection with Bipolaris sorokiniana in wheat cultivar Yangmai 6 (b) was examined by RT-qPCR. The samples were collected from stem of infected wheat plants. The relative gene expression was quantified using the comparative threshold (2^−ΔΔCT) method. The transcriptional level of TaPIMP2-1B from the untreated wheat plants was used as control. The mean and standard deviation were calculated using data from three independent biological replicates. Asterisks indicate statistically significant differences. (Student’s t-test: *P < 0.05).

Figure 4

Transcriptional patterns of TaPIMP2 homoeologous members after treatment with hormones. The wheat cultivar Yangmai 16 plants were sprayed with abscisic acid (ABA), 1-aminocyclopropane-1-carboxylic acid (ACC, precursor of ethylene), salycilic acid (SA), or Tween-20 solution. The transcriptional level of TaPIMP2-1B from the wheat plants treated with Tween-20 was used as control. The mean and standard deviation were calculated using data from three independent biological replicates. Asterisks indicate statistically significant differences. (Student’s t-test: *P < 0.05, **P < 0.01).

Figure 5

Subcellular localization of TaPIMP2 in wheat mesophyll protoplasts (a) and onion epidermal cells (b). TaPIMP2-GFP (green fluorescent protein) fusion protein is localized to nucleus. GFP alone is distributed in both cytoplasm and nucleus. The red fluorescence is from chloroplast autofluoresence. Images were captured using the following wavelengths, green fluorescence (excitation, 488 nm; emission, 509 nm) and red fluoresence (excitation, 448 nm; emission, 647 nm). Bars: 50 μm.

Figure 6

Responses of TaPIMP2-silenced wheat plants to infection with Rhizoctonia cerealis or Bipolaris sorokiniana. (a) The barley stripe mosaic virus coat protein (cp) gene was detected by RT-PCR. (b) Phenotypes of wheat leaves infected with BSMV:TaPIMP2 or BSMV:GFP (control). (c) Transcriptional analysis of TaPIMP2 in stems of TaPIMP2-silencing CI12633 wheat and control plants. The expression level of TaPIMP2 in control CI12633 plants was set to 1. (d) The typical sharp eyespot symptoms were displayed in the susceptible control wheat Wenmai 6. (e) Transcriptional analysis of TaPIMP2 in TaPIMP2-silencing Yangmai 6 wheat and control plants. The expression level of TaPIMP2 in control Yangmai 6 plants was set to 1. (f) The typical common root rot symptoms were displayed in the TaPIMP2-silencing Yangmai 6. Values represent the mean and standard deviation from three independent biological replicates (Student’s t-test: *P < 0.05).

Figure 7

Molecular characterization of TaPIMP2-overexpression wheat plants and responses to Bipolaris sorokiniana. (a) Scheme of the TaPIMP2 expression cassette on the transformation vector pUBI::myc-TaPIMP2. Ubi: maize ubiquitin promoter; Tnos: terminator of Agrobacterium tumefaciens nopaline synthase gene. The arrow indicates the amplified regions of transgenic plants by PCR. (b) PCR patterns of the TaPIMP2-transgenic wheat lines using the primers specific to the TaPIMP2-Tnos cassette. M: molecular marker; P: plasmid pUBI::myc-TaPIMP2; Y16: the recipient Yangmai 16. (c) Relative expression levels of TaPIMP2 in overexpressing wheat lines and untransformed Yangmai 16. The relative transcript level in the untransformed Yangmai 16 was set to 1. (d) Western blotting analysis of the TaPIMP2-overexpressing wheat lines and untransformed Yangmai 16 using an anti-myc antibody. (e) The typical common root rot symptoms of TaPIMP2-overexpression and untransformed Yangmai 16. Values represent the mean and standard deviation from three independent biological replicates (Student’s t-test: *P < 0.05).

Figure 8

Expression of defense-associated genes in TaPIMP2-overexpressing wheat lines and TaPIMP2-silenced wheat plants by RT-qPCR. The transcript levels of these genes in TaPIMP2-overexpressing wheat lines are relative to those in untransformed Yangmai 16, whereas the levels in TaPIMP2-silenced wheat plants are relative to those in the control plants infected with BSMV:GFP. Values represent the average standard error of three independent biological replicates (Student’s t-test: *P < 0.05).