CRISPR-targeted mutagenesis of mitogen-activated protein kinase phosphatase 1 improves both immunity and yield in wheat

Abstract

Plants have evolved a sophisticated immunity system for specific detection of pathogens and rapid induction of measured defences. Over- or constitutive activation of defences would negatively affect plant growth and development. Hence, the plant immune system is under tight positive and negative regulation. MAP kinase phosphatase1 (MKP1) has been identified as a negative regulator of plant immunity in model plant Arabidopsis. However, the molecular mechanisms by which MKP1 regulates immune signalling in wheat (Triticum aestivum) are poorly understood. In this study, we investigated the role of TaMKP1 in wheat defence against two devastating fungal pathogens and determined its subcellular localization. We demonstrated that knock-down of TaMKP1 by CRISPR/Cas9 in wheat resulted in enhanced resistance to rust caused by Puccinia striiformis f. sp. tritici (Pst) and powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt), indicating that TaMKP1 negatively regulates disease resistance in wheat. Unexpectedly, while Tamkp1 mutant plants showed increased resistance to the two tested fungal pathogens they also had higher yield compared with wild-type control plants without infection. Our results suggested that TaMKP1 interacts directly with dephosphorylated and activated TaMPK3/4/6, and TaMPK4 interacts directly with TaPAL. Taken together, we demonstrated TaMKP1 exert negative modulating roles in the activation of TaMPK3/4/6, which are required for MAPK-mediated defence signalling. This facilitates our understanding of the important roles of MAP kinase phosphatases and MAPK cascades in plant immunity and production, and provides germplasm resources for breeding for high resistance and high yield.

Keywords: CRISPR/Cas9; MAP kinase phosphatase 1; MPK3; MPK4; MPK6; wheat defence responses.

Figure 1

Identification of bread wheat MKP1 (TaMKP1) homologues and expression patterns. (a) Multiple alignment of the amino acid sequences of TaMKP1a, TaMKP1b, TaMKP1d, and AtMKP1. The green box indicates the DSP (dual‐specificity phosphatase) domain, the red box represents the Gelsolin repeat region, and the yellow region represents the FXF motif. (b) Phylogenetic analysis between TaMKP1 and other plants. (c) The expression level of TaMKP1 induced by Pst CYR34. TaMKP1 contains DSP and Gelsolin domains, and the horizontal line under the domain indicates the TaMKP1 RT‐qPCR amplified fragment.

Figure 2

The silencing of TaMKP1 enhances the resistance of wheat to pathogens. (a) Wheat leaf symptoms after virus inoculation and phenotype of wheat leaves silenced for TaMKP1 gene infected by Pst. (b) Relative transcript levels of TaMKP1 in control and TaMKP1‐silenced plants. (c) The percentage of Pst urediniospores on wheat leaves. (d) The standard curve for MKP1 enzyme activity. (e) Changes in MKP1 enzyme activity in silenced plants.

Figure 3

TaMKP1 plays a negative regulatory role in the interaction between plants and pathogens. (a) Selection of TaMKP1 mutation targets and identification of mutation types in TaMKP1 homologues. Deleted nucleotides are represented by “‐”. Inserted nucleotides are highlighted in red. The numbers on the right indicate the number of nucleotides involved in the indel events, represented by “+” or “−”. (b) Leaf phenotypes of aaBBDD, AAbbDD, AABBdd and aabbddd compared with Fielder at 14 dpi with Pst CYR34, and Pst biomass statistics. (c) ROS production was observed in Pst‐infested leaves at 24 hpi. (d) Histological observation of host cell death on the Tamkp1 leaves at 120 hpi with CYR34. Student's t‐tests were used to assess the differences between knockdown plants and control plants. Significance levels were denoted as * for P < 0.05, ** for P < 0.01, *** for P < 0.001 and ****for P < 0.001. SV refers to substomatal vesicle. (e) Characterization of Tamkp1 plants for disease resistance at the adult plant stage. S represents susceptible, MS represents moderately susceptible, MR represents moderately resistant, and R represents resistant. (f) Observations on the infection phenotype in overexpressed TaMKP1 Nicotiana benthamiana inoculated with Sclerotinia sclerotiorum. (g) Statistical analysis of lesion diameter following inoculation with S. sclerotiorum. ** indicate a significant difference (P < 0.01) according to Student's t‐test. (h) Western blot was performed to confirm the expression of TaMKP1 in N. benthamiana.

Figure 4

Tamkp1 plants demonstrate broad‐spectrum disease resistance. (a) Leaf phenotypes of Tamkp1 plants compared with Fielder at 7 dpi with Bgt, and Bgt biomass statistics. (b) The leaves of T2 generation of Tamkp1 plants were infected, and 7 dpi, they were stained with trypan blue to make fungal structures visible. (c) Statistical analysis of Bgt microcolony formation on Fielder and Tamkp1 leaves. (d) Host cell death on the Tamkp1 leaves with Bgt.

Figure 5

Nucleus‐localized TaMKP1 contributes to plant susceptibility against pathogens. (a) Observation of TaMKP1 nuclear localisation and nuclear export signals. Scale bar: 20 μm. (b) TaMKP1‐nuclear localisation and nuclear export signal region of the lesion phenotype and statistical analysis of lesion diameter **** indicate a significant difference (P < 0.0001) according to Student's t‐test. (c) Western blot analysis to verify the expression of TaMKP1‐NES and TaMKP1‐NLS in plants.

Figure 6

TaMKP1 interacts with, dephosphorylates, and activates TaMPK3/4/6. (a) Yeast Two‐Hybrid (Y2H) assay was used to validate the interaction between TaMKP1 and TaMPK3/4/6 in yeast cells. (b) In vivo BiFC analysis of TaMKP1 interaction with TaMPK3/4/6. (c) The LUC assay confirmed the interaction between TaMKP1 and TaMPK3/4/6. (d) In vivo co‐immunoprecipitation of TaMKP1 with TaMPK3, TaMPK4 or MPK6. (e) TaMKP1 affects phosphorylation levels of TaMPK3/4/6 in vivo. Immunoblot assay for MAPK phosphorylation using anti‐phospho‐MAPK antibodies. The Tamkp1 plants and Filder were treated with Pst CYR34. Proteins were extracted from Tamkp1 mutant and Fielder leaves at 0, and 24 h. CBB staining is shown as a protein loading control. (f) The relative transcript levels of TaMPK3, TaMPK4, and TaMPK6 in Tamkp1 plants.

Figure 7

TaMPK4 interacts with TaPAL. (a) Y2H assay was used to validate the interaction between TaPAL and TaMPK4. (b) The LUC assay confirmed the interaction between TaPAL and TaMPK4. (c) BiFC analysis of TaPAL interaction with TaMPK4. Scale bar: 20 μm. (d) The relative transcript levels of TaPAL, TaWRKY33, and TaERF3 in Tamkp1 plants.

Figure 8

Field performances of the different Tamkp1 plants. (a) The growth conditions of Tamkp1 plants and Fielder in the field. (b) Mature paddy wheat grains of Fielder and Tamkp1. (c) Spike length statistics of Tamkp1 plants and Fielder. (d) plant length statistics of Tamkp1 plants and Fielder. (e) 1000 grain weight statistics of Tamkp1 plants and Fielder.