Two members of TaRLK family confer powdery mildew resistance in common wheat

Abstract

Background: Powdery mildew, caused by Blumeria graminearum f.sp. tritici (Bgt), is one of the most severe fungal diseases of wheat. The exploration and utilization of new gene resources is the most effective approach for the powdery mildew control.

Results: We report the cloning and functional analysis of two wheat LRR-RLKs from T. aestivum c.v. Prins- T. timopheevii introgression line IGV1-465, named TaRLK1 and TaRLK2, which play positive roles in regulating powdery mildew resistance in wheat. The two LRR-RLKs contain an ORF of 3,045 nucleotides, encoding a peptide of 1014 amino acids, with seven amino acids difference. Their predicted proteins possess a signal peptide, several LRRs, a trans-membrane domain, and a Ser/Thr protein kinase domain. In response to Bgt infection, the TaRLK1/2 expression is up-regulated in a developmental-stage-dependent manner. Single-cell transient over-expression and gene-silencing assays indicate that both genes positively regulate the resistance to mixed Bgt inoculums. Transgenic lines over-expressing TaRLK1 or TaRLK2 in a moderate powdery mildew susceptible wheat variety Yangmai 158 led to significantly enhanced powdery mildew resistance. Exogenous applied salicylic acid (SA) or hydrogen peroxide (H2O2) induced the expression of both genes, and H2O2 had a higher accumulation at the Bgt penetration sites in RLK over-expression transgenic plants, suggesting a possible involvement of SA and altered ROS homeostasis in the defense response to Bgt infection. The two LRR-RLKs are located in the long arm of wheat chromosome 2B, in which the powdery mildew resistance gene Pm6 is located, but in different regions.

Conclusions: Two members of TaRLK family were cloned from IGV1-465. TaRLK1 and TaRLK2 contribute to powdery mildew resistance of wheat, providing new resistance gene resources for wheat breeding.

Fig. 1

The deduced amino acid sequences of TaRLK1 and TaRLK2 proteins. Red characters represent seven amino acid differences. The open boxed region represents the N-terminal region of TaRLK2 which comprises a putative twin arginine translocation (Tat) signal domain. Characters in the green brackets represent conserved pairs of cysteines spaced by six or seven amino acids. Roman numerals mark the 11 tandem copies of a 24-amino acid LRR. Blue characters represent nine glycosylation sites (N-X-S/T). Green characters represent a putative protein kinase catalytic domain with ATP binding site. Black arrow heads indicate a Ser/Thr kinase active site (D). Characters underlined as red represent the conserved motifs (VAVK, HRD and DFG) in the RD kinases. SP: signal peptide domain; LRR: leucine-rich repeat domain; TM: transmembrane domain, PK: Ser/Thr protein kinase domain

Fig. 2

Physical localization of TaRLK1/2. PCR was conducted using specific primer pair NAU-2 F and NAU-2R differentiating TaRLK1/2 on BB subgenome and its homologue genes on AA, DD and GG subgenome (a). The arrows point the amplified bands on 2A, 2B, 2D and 2G. TaRLK1/2 homologue gene on GG subgenome was physically mapped to the same chromosome region as reported markers CINAU123 and CINAU124 (b) using a set of introgression lines from T. timopheevii.(c). In Fig. 2b, the arrows point the specific bands from GG subgenome. In Fig. 2c, white boxes indicate the chromosome from Prins BB subgenome and black boxes indicate introgression fragments from T. timopheevii GG subgenome. White ovals indicate centromere region. Letter S, L and CEN represent chromosome short arm, long arm and centromere. At the bottom, Pm means powdery mildew responses, resistance (R) or susceptibility (S)

Fig. 3

Gene expression profiling of TaRLKs in Prins and IGV1-465. TaRLKs expression in Prins and IGV1-465 at the second and fourth leaf stages without Bgt inoculation (a), and response to Bgt infection at the second (b) and fourth (c) leaf growing stages, respectively. “*” indicates significant differences across different time point within each genotype at 0.05 levels, using 2nd leaf stage (a) and non-inoculated sample (b, c) as control. h: hours after Bgt inoculation. Data were from three independently replicated experiments with similar result

Fig. 4

The interaction of leaf epidermal cells challenged with Bgt and the statistics of Haustorium Index (HI) after single-cell transient over-expression assay. Representative of compatible (a and b) and incompatible interaction (c) wheat leaf epidermal cells challenged with Bgt, the haustorium index (HI) of Bgt after single-cell transient expression of TaRLK1 and TaRLK2 in the epidermal cells in Prins (d) and after transient induced gene silencing of TaRLK1/2 in IGV1-465 (e) co: conidia; ha: haustorium; hy: hyphae. Scale bars = 50 μm. Different letters indicate the significant difference at 0.05 levels. Data were from three independent replicated experiments

Fig. 5

The molecular identification of TaRLK1 and TaRLK2 transgenic plants and powdery mildew resistance evaluation. Using specific primer pair, we performed PCR to identify TaRLK1 and TaRLK2 T₀ positive transgenic plants (a) and qRT-PCR to study the gene expression level of TaRLK1 and TaRLK2 in T₀ positive transgenic plants, “*” indicates significant differences at 0.05 levels, compared with Yangmai 158. Powdery mildew resistance evaluation for TaRLK1 and TaRLK2 T₀ transgenic plants at seedling and adult leaf stage were conducted, using susceptible Yangmai 158 as control (c). The hyphae growing on the surface of leaves from transgenic lines for TaRLK2 gene at seedling growing stage (d) and transgenic lines for TaRLK1 and TaRLK2 at adult growing stage (e) were observed, using susceptible Yangmai 158 as control

Fig. 6

H₂O₂ accumulation in the leaves and endogenous H₂O₂ contents in different materials. Microscope observation of H₂O₂ accumulation in wheat leaves of Prins, Yangmai 158, IGV1-465 and the transgenic lines at 0 and 24 hai of Bgt (a) and endogenous H₂O₂ contents of wheat at second (e) and fourth (f) leaf stages after Bgt treatments. b was the representative image at Bgt interaction sites in susceptible genotypes. c and d were the representative images at Bgt interaction sites in resistant genotypes. H₂O₂ was detected at Bgt interaction sites in leave epidermal cells of resistant IGV1-465 and transgenic lines (c and d), while not obvious in those of susceptible Prins and Yangmai 158 (b). “*” indicates significant differences across different time point within each genotype at 0.05 levels, using non-inoculated sample as control. h: hours after Bgt inoculation. Results are replicated in three independent experiments of similar result

Fig. 7

Gene expression patterns for TaRLK1/2 in response to the treatments of exogenous phytohormones MeJA (a), SA (b), ABA (c) and signal molecule H₂O₂ (d) in Prins and IVG1-465. “*” indicates significant differences across different time point within each genotype at 0.05 levels, using non-inoculated sample as control. h: hours after treatment. N/A: data missing