The essential effector SCRE1 in Ustilaginoidea virens suppresses rice immunity via a small peptide region

Abstract

The biotrophic fungal pathogen Ustilaginoidea virens causes rice false smut, a newly emerging plant disease that has become epidemic worldwide in recent years. The U. virens genome encodes many putative effector proteins that, based on the study of other pathosystems, could play an essential role in fungal virulence. However, few studies have been reported on virulence functions of individual U. virens effectors. Here, we report our identification and characterization of the secreted cysteine-rich protein SCRE1, which is an essential virulence effector in U. virens. When SCRE1 was heterologously expressed in Magnaporthe oryzae, the protein was secreted and translocated into plant cells during infection. SCRE1 suppresses the immunity-associated hypersensitive response in the nonhost plant Nicotiana benthamiana. Induced expression of SCRE1 in rice also inhibits pattern-triggered immunity and enhances disease susceptibility to rice bacterial and fungal pathogens. The immunosuppressive activity is localized to a small peptide region that contains an important 'cysteine-proline-alanine-arginine-serine' motif. Furthermore, the scre1 knockout mutant generated using the CRISPR/Cas9 system is attenuated in U. virens virulence to rice, which is greatly complemented by the full-length SCRE1 gene. Collectively, this study indicates that the effector SCRE1 is able to inhibit host immunity and is required for full virulence of U. virens.

Keywords: Ustilaginoidea virens; SCRE1; fungal effector; immunosuppressive peptide; rice immunity.

Figure 1

SCRE1 is a secreted protein in Ustilaginoidea virens. (A) The putative signal peptide of SCRE1 is functional to guide invertase secretion into culture medium (CM). YTK12 is an invertase secretion‐deficient yeast strain. The secretion of invertase is indicated by the growth of YTK12 on YPRAA plates with raffinose as sole carbon source. The signal peptide of Avr1b and the first 25 amino acids of Mg87 were used for positive and negative controls, respectively. (B) SCRE1‐HA was detected in CM and in the total cell lysate (TCL) via western blot (WB) analysis. The non‐secreted β‐tubulin protein UV_1410‐HA and β‐actin, as negative controls, were only detected in the TCL, but not in the CM. α‐HA, anti‐haemagglutinin antibody; α‐β‐actin, anti‐β‐actin antibody. CCB, Coomassie brilliant blue staining; SCRE1‐HA, U. virens transformed with pCAMBIA1301‐RP27::SCRE1‐HA; UV_1410‐HA, U. virens transformed with pCAMBIA1301‐RP27::UV_1410‐HA.

Figure 2

SCRE1 is an effector in Ustilaginoidea virens as revealed by expression and translocation assays. (A) SCRE1 expression was dramatically induced and peaked at 5 days post‐inoculation (dpi) and gradually decreased thereafter during U. virens infection. SCRE1 expression was normalized to the internal reference tubulin gene. (B), (C) Red fluorescence was clearly observed in barley cell nuclei and invasive hyphae of Magnaporthe oryzae. The fluorescence (B) and light (C) microscopy images were taken at 30 h after barley leaves were inoculated with M. oryzae carrying pKS‐RP27::SCRE1‐mCherry‐NLS. The barley nuclei stained with DAPI are indicated by arrowheads, while M. oryzae invasive hyphae are indicated by arrows. The images in lower panels are enlarged from the regions in broken squares in upper panels. (D) Red fluorescence was only observed in M. oryzae hyphae, but not in barley nuclei. The fluorescence (upper panel) and light (lower panel) microscopy images were taken 30 h after barley leaves were inoculated with M. oryzae carrying pKS‐RP27::mCherry‐NLS. The nuclei stained with DAPI are indicated by arrowheads, while M. oryzae hyphae are indicated by arrows. (E) Green fluorescence was clearly observed in biotrophic interfacial complexes during the infection of M. oryzae strains transformed with pYF11‐RP27::SCRE1‐GFP and pYF11‐RP27::AVR‐Pia‐GFP. The transformed M. oryzae Guy11 strains were inoculated into rice sheaths. The images were captured by confocal microscopy 30 h after inoculation. The images in the uppermost panels are enlarged from the regions in broken squares in the GFP panels. GFP, green fluorescent protein; BF, bright field; Merge, the overlay of GFP and BF images; IH, invasive hyphae; CO, conidia; BIC, biotrophic interfacial complex. Scale bar: 10 μM.

Figure 3

SCRE1 is required for full virulence of Ustilaginoidea virens to rice. The panicle incidence rate (%) (A) and average diseased grains per inoculated panicle (B) after injection inoculation of the wild‐type, different scre1 mutant and complementary strains in U. virens. The complementary strain was generated through transforming plasmid‐borne full‐length SCRE1 gene into the scre1‐1‐10 mutant. Different letters above error bars indicate statistically significant differences among the wild‐type, scre1 mutant and complementary strains of U. virens. Data shown are combined from three independent inoculation experiments. Error bars represent means ± standard error (SE).

Figure 4

SCRE1 suppresses different types of immunity‐associated hypersensitive cell death in Nicotiana benthamiana. (A) Co‐expression of SCRE1 or truncated SCRE1 without signal peptide (SCRE1‐SP) suppressed BAX‐ (left panel) and INF1‐ (right panel) triggered hypersensitive cell death in N. benthamiana. (B) SCRE1/SCRE1‐SP expression, in contrast to green fluorescent protein (GFP) expression, significantly suppressed BAX‐ and INF1‐induced electrolyte leakage in N. benthamiana. Different letters over error bars indicate significant difference in BAX‐ and INF1‐induced electrolyte leakage of N. benthamiana leaves between SCRE1/SCRE1‐SP co‐expression and GFP co‐expression (P < 0.05). (C) SCRE1‐FLAG and GFP‐FLAG co‐expression imposed no evident effect on BAX‐FLAG expression in N. benthamiana leaves. Upper panel: the proteins isolated from the agroinfiltrated leaves were detected via immunoblotting with anti‐FLAG antibody (α‐FLAG). The bands of SCRE1‐FLAG, GFP‐FLAG and BAX‐FLAG are indicated by arrows. Lower panel: protein loading is indicated by Ponceau S staining.

Figure 5

Ectopic expression of SCRE1 suppresses pattern‐triggered immunity and promotes pathogen infection in rice. (A), (B) Dexamethasone (DEX)‐induced expression of SCRE1 inhibited the oxidative burst triggered by flg22 (A) and chitin (B) in the Z195 and Z196 transgenic rice plants. Leaf disks collected from the wild‐type, Z195 and Z196 transgenic plants were immersed in DEX (10 µM) and mock solution for 16 h followed by treatment of flg22 (10 µM) and hexa‐N‐acetyl‐chitohexaose (8 µM). The area under the curve for 30‐min oxidative burst (relative luminescence units × time, see Fig. S4B–E) was determined for the wild‐type and transgenic plants after different treatments. Asterisks indicate statistical significance in oxidative burst from flg22‐ and chitin‐treated transgenic rice leaves with and without DEX treatment (P < 0.05). Histogram shows means ± SE for nine leaf disks from different plants. RLU, relative luminescence unit. (C), (D) DEX‐induced expression of SCRE1 significantly inhibited OsPR10a expression triggered by flg22 and chitin in the Z178 (C) and Z196 (D) transgenic lines. The wild‐type and transgenic seedlings were treated with DEX (10 µM) and mock solution for 24 h followed by flg22 (1 µM) and chitin (10 µg/mL). OsPR10a expression was detected by RT‐qPCR and normalized to the reference gene OsActin. Different letters above error bars indicate statistically significant differences in the OsPR10a transcript levels in the wild‐type and transgenic lines under different treatments (P < 0.05). (E), (F) SCRE1 expression in the transgenic lines significantly promoted pathogen infection after inoculation of rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae (E) and rice blast fungus Magnaporthe oryzae (F). The wild‐type and transgenic plants were challenged with pathogens at 24 h after spraying with DEX (10 µM) and mock solution. At least 12 leaves were measured for lesion lengths at 12 days after bacterial inoculation. Lesion areas on 20 inoculated leaves were photographed at 6 days after fungal inoculation and calculated using Abode Photoshop. Asterisks indicate statistically significant differences in disease lesions on the inoculated leaves with and without DEX treatment. Error bars represent means ± SE. Nip, Nipponbare; Z178, Z195 and Z196, three independent transgenic lines; DEX−, no dexamethasone treatment; DEX+, with dexamethasone treatment.

Figure 6

SCRE1^45‐70 is an essential region that suppresses BAX‐ and INF1‐triggered cell death in Nicotiana benthamiana. (A) Transient expression of SCRE1^45‐70 had an even stronger ability than full‐length SCRE1 to inhibit BAX‐ and INF1‐triggered electrolyte leakage in N. benthamiana. BAX and INF1 were transiently expressed at 6 h after expression of green fluorescent protein (GFP), SCRE1 and SCRE1^45‐70 in N. benthamiana. Letters above error bars indicate statistically significant differences in BAX‐ and INF1‐triggered electrolyte leakage when co‐expressing with different proteins in N. benthamiana. (B) Diagrams of alanine‐scanning mutagenesis in the 45–70th residues of full‐length SCRE1. (C), (D) SCRE1^45AAAAA49, in which the 45–49th residues of SCRE1 were all replaced with Ala, significantly lost the ability to suppress BAX‐ (C) and INF1‐ (D) induced ion leakage in N. benthamiana. Different letters (a–d) indicate statistically significant differences in BAX‐ and INF1‐triggered electrolyte leakage in N. benthamiana. BAX and INF1 were expressed at 6 h after expression of the indicated proteins. (E), (F) The Ala replacement in the 45‐49th residues of SCRE1^1‐179 greatly disrupted the ability of in vitro‐purified SCRE1^1‐179 to suppress BAX‐ and INF1‐induced hypersensitive response symptoms (E) and ion leakage (F). In vitro‐purified proteins (1 µM) were co‐infiltrated with Agrobacterium strains carrying pGR107‐BAX and pGR107‐INF1 constructs into N. benthamiana leaves. Hypersensitive response‐like symptoms and electrolyte leakage in the infiltrated leaves were investigated at 5 days post‐infiltration.

Figure 7

SCRE1 compromises pattern‐triggered immunity in rice. (A), (B) The flg22‐ and chitin‐induced expression of OsPR10a (A) and OsPR1b (B) was significantly attenuated in SCRE1^1‐179‐pretreated rice seedlings, but not in mock‐ and SCRE1^{1‐179(45AAAAA49)}‐pretreated seedlings. Rice seedlings were incubated with 1 µM of SCRE1^1‐179, SCRE1^{1‐179(45AAAAA49)} or mock solution for c. 12 h followed by treatment with flg22 (1 µM) or chitin (10 µg/mL) for 6 h. The expression level of OsPR10a and OsPR1b was detected by quantitative RT‐PCR and normalized to that of the reference gene OsActin. Different letters above error bars indicate significant differences in the transcript levels of PR genes among SCRE1^1‐179, SCRE1^{1‐179(45AAAAA49)} and mock treatments. (C) The flg22‐ and chitin‐induced MAPK phosphorylation was greatly inhibited in the SCRE1^1‐179‐pretreated rice seedlings, but not in the SCRE1^{1‐179(45AAAAA49)}‐pretreated seedlings. The SCRE1^1‐179‐pretreated seedlings were treated by flg22 and chitin for 15 min and were then collected for protein extraction. MAPK activation was detected through immunoblotting with anti‐Phospho‐p44/42 MAPK antibody (α‐p44/42). Lower panel: protein loading is indicated by Coomassie brilliant blue (CBB) staining.