Wheat Leaf Rust Effector Pt48115 Localized in the Chloroplasts and Suppressed Wheat Immunity

Abstract

Wheat leaf rust caused by Puccinia triticina (Pt) is a prevalent disease worldwide, seriously threatening wheat production. Pt acquires nutrients from host cells via haustoria and secretes effector proteins to modify and regulate the expression of host disease resistance genes, thereby facilitating pathogen growth and reproduction. The study of effector proteins is of great significance for clarifying the pathogenic mechanisms of Pt and effective control of leaf rust. Herein, we report a wheat leaf rust candidate effector protein Pt48115 that is highly expressed in the late stages of infection during wheat-Pt interaction. Pt48115 contains a signal peptide with a secretory function and a transit peptide that can translocate Pt48115 to the host chloroplasts. The amino acid sequence polymorphism analysis of Pt48115 in seven different leaf rust races showed that it was highly conserved. Pt48115 inhibited cell death induced by Bcl-2-associated X protein (BAX) from mice or infestans 1 (INF1) from Phytophthora infestans in Nicotiana benthamiana and by DC3000 in wheat, and its 145-175 amino acids of the C-terminal are critical for its function. Furthermore, Pt48115 inhibited callose deposition and reactive oxygen species accumulation in the wheat cultivar Thatcher, demonstrating that it is an effector that enhances Pt virulence by suppressing wheat defense responses. Our findings lay a foundation for future studies on the pathogenesis of Pt during wheat-fungus interaction.

Keywords: Puccinia triticina; effector protein; host and fungus interaction; pathogenicity; wheat leaf rust.

Figure 1

The structure and homology analysis of Pt48115. (A) The structural diagram of Pt48115 was made by GraphPad Prism 9.5.0. (B) the homology of Pt48115 was analyzed with MEGA7 software; The red dot is Pt48115. (C) a three-stage structural model of Pt48115.

Figure 2

Transcription profile of Pt48115 at different time points. Pt48115 was highly expressed during the late stages of infection. Relative expression was calculated by the comparative 2^−ΔΔCt method. Standard deviation and the mean fold changes were calculated with results from three independent biological replicates. Asterisks indicate significant differences (**** p < 0.0001, unpaired two-tailored Student’s t-test).

Figure 3

Sequence polymorphism analysis of Pt48115 among different physiological species of rusts. ‘*’ indicates positions which have a single, fully conserved residue.

Figure 4

Functional validation of the Pt48115 signal peptide using the yeast invertase secretion assay. (A) The sequence of Pt48115 signal peptide was fused in frame to the invertase sequence in the pSUC2 vector and then transformed into the yeast strain YTK12. YTK12 carrying pSUC2-Avr1b served as positive control, and YTK12 and YTK12 carrying pSUC2-Mg87 were used as negative control. Only the yeast strains capable of secreting invertase grew on both SD-Trp and YPRAA medium. (B) Secreted invertase can catalyze the reduction of 2,3,5-triphenyltetrazolium chloride (TTC) to form insoluble red 1,3,5-triphenyl formazan (TPF). The presence of a red color confirms the occurrence of invertase activity.

Figure 5

Pt48115 inhibits programmed cell death in tobacco and wheat. (A) schematic diagram of tobacco infiltration; (B) Pt48115 was transiently expressed in Nicotiana benthamiana, and INF1 was injected 24 h later. The same leaf was examined before (left) and after (right) staining with decolorizing solution. The red circles highlight the Pt48115 could inhibit the PCD better than the black ones. (C) Pt48115 delivered via P. fluorescent EtHAn into leaves of wheat cultivar Thatcher suppresses necrosis triggered by P. syringae DC3000. DC3000 and pEDV6 served as a positive and negative control, respectively.

Figure 6

Pt48115 accumulates in the chloroplast. Leaf tissues of Nicotiana benthamiana transiently co-expressing Pt48115-GFP, Pt48115^ΔSP-GFP, and GFP were examined by epifluorescence microscopy. Green means the fluorescence of the GFP. C, chloroplast. Bars = 10 μm.

Figure 7

Pt48115 toxic functional domain analysis. The mutants of Pt48115 were constructed and verified on N. benthamiana. The amino acid sequence of the 145–175 position at the C-terminus of the effector protein Pt48115 is key for its function. SP: signal peptide; N: N terminal; C: C-terminal. The ovals represent the corresponding phenotypic observations of the deletion mutants on N. benthamiana leaves. Each color represents a different gene sequence. Orange, blue, green, red, mint green, yellow represent a segment of Pt48115 signal peptide amino acid sequence, 22–52, 53–82, 83–112, 113–144, 145–175 amino acid sequence.

Figure 8

Overexpression of Pt48115 in Thatcher suppressed callose deposition and H₂O₂ accumulation. (A) Callose deposition after aniline blue staining. Wheat leaf samples were collected 24 h after infiltration of wheat cultivar Thatcher with EtHAn. After decolorization, the leaves were stained overnight with 0.05% aniline blue. pEDV6:dsRED was used as a positive control, and EtHAn and MgCl₂ served as blank controls. Images were captured under a fluorescence microscope. Bar = 100 µm. (B) Statistical average number of callose deposits/mm². The mean values and standard deviations were obtained from nine 1 mm² areas of 3 biological replicates. Asterisks indicate significant differences (**** p < 0.0001, one-way ANOVA). (C) Wheat leaves were infiltrated with pEDV6:dsRED and pEDV6:Pt48115 and 24 h later were inoculated with the virulent Pt isolate THTT. pEDV6:dsRED was used as a positive control. Wheat leaf samples were collected 24 h post-inoculation with THTT. After DAB (1 mg/mL) staining, the accumulation of H₂O₂ was observed under a microscope. Images were captured under a microscope. Bar = 100 µm. (D) Statistics of H₂O₂ accumulation area/mm² Asterisks indicate significant differences (**** p < 0.0001, unpaired two-tailored Student’s t-test).

Figure 8

Overexpression of Pt48115 in Thatcher suppressed callose deposition and H₂O₂ accumulation. (A) Callose deposition after aniline blue staining. Wheat leaf samples were collected 24 h after infiltration of wheat cultivar Thatcher with EtHAn. After decolorization, the leaves were stained overnight with 0.05% aniline blue. pEDV6:dsRED was used as a positive control, and EtHAn and MgCl₂ served as blank controls. Images were captured under a fluorescence microscope. Bar = 100 µm. (B) Statistical average number of callose deposits/mm². The mean values and standard deviations were obtained from nine 1 mm² areas of 3 biological replicates. Asterisks indicate significant differences (**** p < 0.0001, one-way ANOVA). (C) Wheat leaves were infiltrated with pEDV6:dsRED and pEDV6:Pt48115 and 24 h later were inoculated with the virulent Pt isolate THTT. pEDV6:dsRED was used as a positive control. Wheat leaf samples were collected 24 h post-inoculation with THTT. After DAB (1 mg/mL) staining, the accumulation of H₂O₂ was observed under a microscope. Images were captured under a microscope. Bar = 100 µm. (D) Statistics of H₂O₂ accumulation area/mm² Asterisks indicate significant differences (**** p < 0.0001, unpaired two-tailored Student’s t-test).