The wheat calcium-dependent protein kinase TaCPK7-D positively regulates host resistance to sharp eyespot disease

Abstract

Sharp eyespot, caused mainly by the necrotrophic fungus Rhizoctonia cerealis, limits wheat production worldwide. Here, TaCPK7-D, encoding a subgroup III member of the calcium-dependent protein kinase (CPK) family, was identified from the sharp eyespot-resistant wheat line CI12633 through comparative transcriptomic analysis. Subsequently, the defence role of TaCPK7-D against R. cerealis infection was studied by the generation and characterization of TaCPK7-D-silenced and TaCPK7-D-overexpressing wheat plants. Rhizoctonia cerealis inoculation induced a higher transcriptional level of TaCPK7-D in the resistant wheat line CI12633 than in the susceptible cultivar Wenmai 6. The expression of TaCPK7-D was significantly induced after exogenous application of 1-aminocyclopropane-1-carboxylic acid (an ethylene biosynthesis precursor). The green fluorescent protein signal distribution assays indicated that TaCPK7-D localizes to the plasma membrane in both onion epidermal cells and wheat protoplasts. Following R. cerealis inoculation, TaCPK7-D-silenced wheat CI12633 plants displayed more severe sharp eyespot symptoms than control CI12633 plants. Four defence-associated genes (β-1,3-glucanase, chitinase 1, defensin and TaPIE1) and an ethylene biosynthesis key gene, ACO2, were significantly suppressed in the TaCPK7-D-silenced wheat plants compared with control plants. Conversely, TaCPK7-D-overexpressing wheat lines showed increased resistance to sharp eyespot compared with untransformed recipient wheat Yangmai 16. Furthermore, the transcriptional levels of these four defence-related genes and ACO2 gene were significantly elevated in TaCPK7-D-overexpressing plants compared with untransformed recipient wheat plants. These results suggest that TaCPK7-D positively regulates the wheat resistance response to R. cerealis infection through the modulation of the expression of these defence-associated genes, and that TaCPK7-D is a candidate to improve sharp eyespot resistance in wheat.

Keywords: CPK gene TaCPK7-D; overexpression; resistance; sharp eyespot; virus-induced gene silencing; wheat.

Figure 1

Chromosome location and transcriptional analyses of TaCPK7‐D in wheat lines infected with Rhizoctonia cerealis. (A) Chromosome location of TaCPK7‐D. Polymerase chain reaction (PCR) assay using TaCPK7‐D gene‐specific primers showed that TaCPK7‐D is located on chromosome 2D. (B) Quantitative reverse transcription‐PCR (qRT‐PCR) analysis between resistant wheat line CI12633 and susceptible wheat cultivar Wenmai 6 on challenge with R. cerealis. Total RNA was extracted from the stems of wheat plants at 4, 7 and 21 days post‐inoculation (dpi) with R. cerealis. (C) Fold change of TaCPK7‐D in wheat tissues at 21 dpi with R. cerealis. Total RNA was extracted from root, stem, leaf and spike tissues of CI12633. (D) qRT‐PCR analysis of TaCPK7‐D in five wheat cultivars with different defence responses to sharp eyespot at 21 dpi with R. cerealis. Total RNA was extracted from the stems of five wheat cultivars. DI indicates the disease index of sharp eyespot. The transcript levels were normalized to the wheat actin gene. Significant differences were analysed based on the results of three replications (Student's t‐test: *P < 0.05, **P < 0.01). Error bars indicate standard error.

Figure 2

Structural scheme and phylogenetic tree analysis of TaCPK7‐D. (A) Genomic structure of TaCPK7‐D; dark grey and white portions indicate exons and introns, respectively. The numbers in the boxes indicate the nucleotide numbers of each portion. ATG and TGA are the start codon and stop codon, respectively. (B) Protein structure of TaCPK7‐D has the typical domains of plant calcium‐dependent protein kinase (CPK), including an N‐terminal variable domain, a kinase catalytic domain and a calmodulin (CaM)‐like domain with four calcium‐binding motifs (rhombi in the calmodulin domain). The N‐terminal amino acid sequence of TaCPK7‐D is indicated at the top. (C) Reconstruction of the phylogenetic tree based on the amino acids of putative TaCPK7‐D and known CPK members involved in defence responses from several plant species.

Figure 3

Expression of TaCPK7‐D in leaves of wheat seedlings in response to exogenous applications of hormones and an ethylene biosynthesis inhibitor. Wheat Yangmai 16 plants at the three‐leaf stage were sprayed with 1.0 mm salicylic acid (SA), 50 μm l‐aminocyclopropane‐l‐carboxylic acid (ACC, an ethylene biosynthesis precursor), 0.1 mm CoCl₂ (an ethylene biosynthesis inhibitor), 0.1 mm methyl jasmonate (MeJA) and 0.1% Tween‐20 (as a control), and the samples were collected at 0, 1, 3, 6 and 12 h after treatment. The transcript levels of TaCPK7‐D in wheat plants treated with Tween‐20 (A) or collected at 0 h (B, C) were set to unity. Values represent the average ± standard error of three replicates (Student's t‐test: *P < 0.05, **P < 0.01).

Figure 4

Plasma membrane localization of TaCPK7‐D. (A) Onion epidermal cells were transformed with TaCPK7‐D‐GFP or green fluorescent protein (GFP) alone via particle bombardment. Confocal images were taken at 12 h after bombardment. After the onion epidermal cells transformed with TaCPK7‐D‐GFP had been plasmolysed by 30% sucrose treatment for 15 min, confocal images showed the shrinkage of the protoplast. The Hechtian strands (h) attaching the plasma membrane (pm) to the cell wall (cw) clearly appear in the higher magnification of a plasmolysed onion cell. (B) Wheat protoplasts were transformed with TaCPK7‐D‐GFP or GFP alone via the polyethylene glycol (PEG)‐mediated method. TaCPK7‐D is localized to the plasma membrane in wheat protoplasts. Images were captured using the following wavelengths (excitation, 488 nm; emission, 509 nm) and chlorophyll autofluoresence (excitation, 448 nm; emission, 647 nm) Bars, 50 μm.

Figure 5

Responses to Rhizoctonia cerealis inoculation of TaCPK7‐D‐silenced wheat CI12633 plants. (A) Phenotypes of wheat CI12633 plants infected with BSMV:TaCPK7‐D or BSMV:GFP (control) showing typical striped mosaic virus symptoms. The Barley stripe mosaic virus (BSMV) coat protein gene (cp) was detected by reverse transcription‐polymerase chain reaction (RT‐PCR); wheat 18S rRNA was used as an internal control for normalization. (B) Quantitative RT‐PCR analysis of TaCPK7‐D in TaCPK7‐D‐silenced plants and control CI12633 plants. Total RNAs were extracted from the stems of CI12633 infected with BSMV. The relative transcript level of TaCPK7‐D in silenced lines was compared with that in control CI12633 plants (set to unity). (C) Typical sharp eyespot phenotypes of TaCPK7‐D‐silenced CI12633 plants and control CI12633 plants at 20 days after R. cerealis inoculation. IT indicates infection type of R. cerealis. (D) Quantitative RT‐PCR analysis of R. cerealis actin gene in TaCPK7‐D‐silenced plants and control CI12633 plants representing the biomass of R. cerealis. The RNA samples were the same as those used for TaCPK7‐D quantification in (B). Values represent the average ± standard error of three replicates (Student's t‐test: *P < 0.05, **P < 0.01).

Figure 6

Molecular characterization of TaCPK7‐D‐overexpressing wheat plants and responses to Rhizoctonia cerealis inoculation. (A) Scheme of the TaCPK7‐D expression cassette of the transformation vector pA25‐myc‐TaCPK7‐D. Ubi promoter, maize ubiquitin promoter; Tnos, terminator of Agrobacterium tumefaciens nopaline synthase gene. The arrows indicate the amplified regions of transgenic wheat plants by polymerase chain reaction (PCR). (B) PCR patterns of the TaCPK7‐D‐overexpressing wheat lines in the T₁ and T₂ generations using the primers specific to the TaCPK7‐D‐Tnos cassette. P, transformation vector pA25‐myc‐TaCPK7‐D; WT, untransformed Yangmai 16. (C) Relative expression levels of TaCPK7‐D in the three overexpressing wheat lines and untransformed Yangmai 16. The relative transcript levels of TaCPK7‐D in the three transgenic wheat lines were compared with that of untransformed Yangmai 16 (set to unity). (D) Western blotting analysis of the three TaCPK7‐D‐overexpressing wheat lines and untransformed Yangmai 16 using an anti‐myc antibody. (E) Typical sharp eyespot symptoms of TaCPK7‐D‐overexpressing plants and untransformed Yangmai 16 plants at 55 days after R. cerealis inoculation. Values represent the average ± standard error of three technical replicates (Student's t‐test: *P < 0.05, **P <0.01).

Figure 7

Expression of defence‐associated genes and ethylene signalling‐related genes in TaCPK7‐D‐overexpressing wheat lines and TaCPK7‐D‐silenced wheat plants by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). The transcript levels of these genes in TaCPK7‐D‐overexpressing wheat lines in the T₂ generation are relative to those in untransformed Yangmai 16 at 55 days post‐inoculation (dpi) with Rhizoctonia cerealis, whereas the levels in TaCPK7‐D‐silenced wheat plants are relative to those in the control plants [infected with Barley stripe mosaic virus:green fluorescent protein (BSMV:GFP)] at 10 dpi with R. cerealis. Values represent the average ± standard error of three technical replicates (Student's t‐test: *P < 0.05, **P < 0.01).