A novel cysteine-rich receptor-like kinase gene, TaCRK2, contributes to leaf rust resistance in wheat

Abstract

Leaf rust, caused by Puccinia triticina, is one of the most destructive fungal diseases in wheat production worldwide. The hypersensitive reaction (HR) is an important defence response against P. triticina infection. In this study, the physiological races 165 and 260 of P. triticina were combined with a line derived from the bread wheat cultivar Thatcher with the leaf rust resistance locus Lr26 to form compatible and incompatible combinations, respectively. Based on an RNA-Seq database of the interaction systems, a new wheat cysteine-rich receptor-like kinase gene, TaCRK2, is specifically induced and up-regulated in the incompatible combination. We identified that TaCRK2 was regulated in a Ca²⁺ -dependent manner. Knockdown of TaCRK2 by virus-induced gene silencing and RNAi leads to a dramatic increase in HR area and the number of haustorial mother cells at the single infection site. In addition, urediniospores, a P. triticina-specific pathogenic marker in compatible combinations, were observed on leaf surfaces of silenced plants at approximately 15 days after inoculation in the incompatible combination. Moreover, transcription levels of TaPR1, TaPR2, and TaPR5 were obviously reduced in TaCRK2-silenced plants. TaCRK2 overexpression in Nicotiana benthamiana induced strong HR-like cell death. Finally, transient expression of green fluorescent protein fused with TaCRK2 in N. benthamiana indicated that TaCRK2 localizes in the endoplasmic reticulum. Thus, TaCRK2 plays an important role in the resistance to P. triticina infection and has a positive regulation effect on the HR cell death process induced by P. triticina.

Keywords: Puccinia triticina; VIGS; cysteine-rich receptor-like protein kinase; hypersensitive reaction; wheat.

FIGURE 1

Domain and amino acid sequence analysis of a cysteine‐rich receptor‐like kinase (TaCRK2) in wheat. (a) TaCRK2 protein domains showing the signal peptide (SP) and the N‐terminal unknown function domain (DUF26) followed by the transmembrane domain (TM) and the conserved kinase domain‐like (S‐TKc). (b) Amino acid sequence of TaCRK2 showing the signal peptide (indicated by underlining), two DUF26 domains (highlighted in the black box), important cysteine residues (indicated by ▲), the TM domain (highlighted in grey), and the kinase domain‐like (highlighted in black)

FIGURE 2

Phylogenetic relationships and motif compositions of cysteine‐rich receptor‐like kinases (CRKs). (a) Multiple sequence alignment of the protein encoded by TaCRK2 and its homologous proteins using MEGA 7.0, and an unrooted phylogenetic tree for the protein and its homologues was constructed. Note: TaCRK2 is displayed in red in a black box. (b) The conserved motifs of the CRK proteins. The different‐coloured boxes represent different motifs and their position in each CRK protein sequence. The length of a given protein can be estimated using the scale at the bottom

FIGURE 3

Relative transcript levels of TaCRK2 in different combinations assayed using quantitative reverse transcription PCR. Seven‐day‐old wheat seedlings (Thatcher Lr26) were inoculated with Puccinia triticina spore suspensions (race 165 or 260) by brushing spores on the surface of the first leaf. The inoculated leaves were collected at 0, 4, 8, 12, 16, 24, and 48 hr post‐inoculation (hpi) for RNA extraction. Error bars represent the standard error among three independent replicates. The different letters represent significant differences (p < .01 according to SPSS analysis of variance)

FIGURE 4

Transcription level of TaCRK2 after injection of ethylene glycol tetraacetic acid (EGTA) as measured by quantitative reverse transcription PCR. Seven‐day‐old wheat seedlings were used for injection of EGTA or deionized distilled H₂O and immediate Puccinia triticina infection. Seedlings were inoculated with the spore suspension by brushing it on the surface of the first leaf. The inoculated leaves were collected at 0, 4, 8, 12, 16, 24, 48, and 72 hr post‐inoculation (hpi) for RNA extraction. Error bars represent the standard error among three independent replicates. The different letters represent significant differences (*p < .05 and **p < .01)

FIGURE 5

Subcellular localization of TaCRK2 in epidermals cell of Nicotiana benthamiana leaves. (a) pSuper1300:TaCRK2‐GFP and pSuper1300:GFP were injected separately into N. benthamiana leaves. GFP fluorescence was detected by confocal laser scanning microscopy after 48 hr. Leaves injected with GFP alone showed characteristic diffuse cytoplasmic fluorescence and dense fluorescence in the nucleus. The TaCRK2‐GFP fusion protein is located in the structural network around the nucleus, which is similar to the endoplasmic reticulum (ER). (b) Co‐localization of TaCRK2‐GFP fusion protein and the ER marker HDEL‐mCherry in epidermal cells of N. benthamiana leaves. (c) The subcellular localization of TaCRK2^Y323V, TaCRK2^R326AR329A, and the triple mutant TaCRK2^{R326AK327AR329A} by confocal microscopy of C‐terminal GFP‐tagged proteins transiently expressed in N. benthamiana. All three mutant proteins coexpressed with HDEL‐mCherry. Bar = 50 µm. (d) Transient overexpression of TaCRK2 in N. benthamiana induced strong hypersensitive reaction (HR)‐like cell death. Agrobacterium tumefaciens containing AtRPS2, TaCRK2, GFP, TaCRK2^Y323V, TaCRK2^R326AR329A, and the triple mutant TaCRK2^{R326AK327AR329A} were infiltrated into the leaves of N. benthamiana. The HR symptoms were photographed at 72 hr after infiltration. The circular areas indicate the infiltrated site

FIGURE 6

Effects of silencing the TaCRK2 gene using the virus‐induced gene‐silencing (VIGS) technique on Thatcher (Tc) Lr26, which is resistant to Puccinia triticina infection. (a) Phenotypic assessment of the barley stripe mosaic virus (BSMV) infection symptoms. All BSMV‐inoculated plants exhibited BSMV symptoms at 12 days post‐inoculation (dpi). The third leaf of BSMV:PDS‐inoculated plants exhibited full leaf bleaching. CK, healthy control. (b) The relative transcription level of TaCRK2 in the third leaves of plants infected with BSMV:γ₀ or BSMV:TaCRK2 determined by quantitative reverse transcription PCR. Error bars represent the srandard error among three independent replicates. The different letters represent highly significant differences (p < .05). (c) A portion of the third leaf sampled at 120 hr after P. triticina inoculation was subjected to Rohringer fluorescence staining. Leaves inoculated with BSMV:γ₀ were used as controls. Confocal laser scanning microscopy was used for examination. (A)–(C) Plants inoculated with BSMV:γ₀ at 120 hr after P. triticina inoculation; (D)–(F) Plants inoculated with BSMV:TaCRK2 at 120 hr after P. triticina inoculation; (A),(D) Superimposed images of (B),(C) and (E),(F), respectively. Bar = 100 µm. (d) Relationship between hypersensitive reaction (HR) and haustorial mother cells (HMCs) at a single infection site. (A)–(C) Plants inoculated with BSMV:γ₀ at 120 hr after P. triticina inoculation; (D)–(F) Plants inoculated with BSMV:TaCRK2 at 120 hr after P. triticina inoculation; (A),(D) Superimposed images of (B),(C) and (E),(F), respectively. Bar = 50 µm. Note: The red arrows indicate P. triticina HMCs and the white arrows indicate HR cells. (e) Statistical results for HR areas (A) and the number of HMCs (B) of 50 single infection sites observed by fluorescence microscopy. Error bars represent the standard error among three independent replicates. The different letters represent significant differences (p < .05). (f) Phenotype of TaCRK2 gene‐silenced leaves at 15 days after P. triticina inoculation. Note: Plant 1–plant 5 refer to the third leaves of five different BSMV:TaCRK2‐silenced plants and the red arrows indicate P. triticina uredosori

FIGURE 7

Transcription levels of TaPR1, TaPR2, and TaPR5 estimated using quantitative reverse transcription PCR after silencing of TaCRK2. Error bars represent the standard error among three independent replicates. The different letters represent significant differences (p < .01)