Ustilaginoidea virens Nuclear Effector SCRE4 Suppresses Rice Immunity via Inhibiting Expression of a Positive Immune Regulator OsARF17

Abstract

Rice false smut caused by the biotrophic fungal pathogen Ustilaginoidea virens has become one of the most important diseases in rice. The large effector repertory in U. virens plays a crucial role in virulence. However, current knowledge of molecular mechanisms how U. virens effectors target rice immune signaling to promote infection is very limited. In this study, we identified and characterized an essential virulence effector, SCRE4 (Secreted Cysteine-Rich Effector 4), in U. virens. SCRE4 was confirmed as a secreted nuclear effector through yeast secretion, translocation assays and protein subcellular localization, as well as up-regulation during infection. The SCRE4 gene deletion attenuated the virulence of U. virens to rice. Consistently, ectopic expression of SCRE4 in rice inhibited chitin-triggered immunity and enhanced susceptibility to false smut, substantiating that SCRE4 is an essential virulence factor. Furthermore, SCRE4 transcriptionally suppressed the expression of OsARF17, an auxin response factor in rice, which positively regulates rice immune responses and resistance against U. virens. Additionally, the immunosuppressive capacity of SCRE4 depended on its nuclear localization. Therefore, we uncovered a virulence strategy in U. virens that transcriptionally suppresses the expression of the immune positive modulator OsARF17 through nucleus-localized effector SCRE4 to facilitate infection.

Keywords: Ustilaginoidea virens; auxin response factor 17; rice false smut; secreted cysteine-rich effector 4; transcription inhibition.

Figure 1

Identification of the secreted effector SCRE4 in U. virens. (a) Expression pattern of SCRE4 during U. virens infection. The expression of SCRE4 was detected in the inoculated panicles via quantitative RT-PCR at the indicated time points after the susceptible rice variety LYP9 was inoculated with U. virens. The α-tubulin gene was used as an internal reference. The representative data from three independent experiments are presented as mean ± standard error (SE) (n = 3). (b) Functionality of the putative signal peptide of SCRE4 confirmed by the yeast secretion system. b1–4: untransformed YTK12, SP^Avr1b-SUC2-, Mg87-SUC2- and SP^SCRE4-SUC2-transformed YTK12 strains were cultured on CMD-W medium, respectively; b5–8: the above-mentioned strains were cultured on YPRAA medium with raffinose as sole carbon source, respectively. SP^Avr1b-SUC2, the signal peptide-encoding sequence of P. sojae Avr1b in fusion with the truncated SUC2 gene. Mg87-SUC2, the N-terminal peptide-encoding sequence of non-secreted Mg87 in M. oryzae in fusion with the truncated SUC2 gene. SP^SCRE4-SUC2, the putative signal peptide-encoding sequence of SCRE4 fused to the truncated SUC2 gene. (c) Green fluorescence from SCRE4-GFP and Avr-Pia-GFP (a positive control) observed in BICs during M. oryzae infection. Leaf sheaths were inoculated with M. oryzae strains transformed with pYF11-ProRP27:SCRE4-GFP, pYF11-ProRP27:Avr-Pia-GFP or pYF11-ProRP27:GFP. The images were captured by confocal microscopy at 30 h after inoculation. BICs are indicated by white triangles. GFP, green fluorescent protein; BF, bright field; Merge, the overlay of GFP and BF images; Images on the right are enlarged from the blocks in broken squares in the GFP panels. Scale bar = 5 μm.

Figure 2

SCRE4 is an essential virulent factor in U. virens. (a) The average number of diseased grains per inoculated panicle. The U. virens wild−type (P1), Δscre4 knockout mutant and complemented strains (SCRE4−C1 and C2) were injection-inoculated into young panicles of the susceptible rice cultivar LYP9 (n = 14, 15, 15, 15, 12, 13). (b) Disease symptoms and diseased grains on rice panicles of the wild−type (Nip), SCRE4−OE−17 and −24 lines after inoculation of the U. virens isolate JS60−2 (n = 12, 13, 10). Left images showed disease symptoms on the representative rice panicles after U. virens inoculation. (c) Diseased grains on U. virens−inoculated panicles of the wild−type (Nip), SCRE4−IE and SCRE4^NM−IE lines. The wild−type and different transgenic lines were treated with 30 µM DEX and mock solution followed by injection inoculation with JS60−2 (n = 12, 11, 8, 8, 8, 8, 10, 10, 10, 9). In panels (a–c), diseased grains were counted at 4 weeks after inoculation. The representative data from three independent experiments are shown as mean ± SE. Different letters (a–c) indicate significant differences in the average number of diseased grains on rice panicles after inoculation of different strains (a), on the panicles of the wild−type and SCRE4−OE transgenic lines (b), and on the SCRE4−IE panicles after DEX and mock treatments (c) (p < 0.05, Duncan’s multiple range test). (d) Chitin−triggered MAPK activation in the wild−type and SCRE4−overexpressing transgenic lines. Total protein loading is indicated by Ponceau S staining. Right panel, the chitin−activated MAPK phosphorylation levels normalized to total proteins from three independent experiments are shown as mean ± SE (n = 3). Band intensity was determined by densitometry using ImageJ. Different letters (a vs. b) indicate a significant difference in the MAPK phosphorylation level (p < 0.05, Duncan’s multiple range test). (e) Chitin−triggered ROS burst in the wild−type and SCRE4−overexpressing transgenic lines. Right panel, relative ROS production in Nipponbare and SCRE4−overexpressing transgenic lines induced by chitin from three independent experiments are shown as mean ± SE. The total ROS levels were quantified by measurement of peak areas under the curve of ROS burst using GraphPad Prism5. Different letters (a vs. b) indicate a significant difference in the relative ROS level in the wild−type (Nip) and SCRE4−overexpressing transgenic lines (p < 0.05, Duncan’s multiple range test).

Figure 3

SCRE4 is internalized into plant cells during infection and is localized in nucleus. (a) Green fluorescence from SCRE4-GFP and GFP observed in rice cell nuclei during M. oryzae infection. The M. oryzae strains carrying pYF11-ProRP27:SCRE4-GFP and pYF11-ProRP27:GFP were inoculated onto detached rice sheaths. Green fluorescence was observed in epidermal cells of rice sheaths at 42 h after inoculation. BF, bright field; DAPI, the nuclei were stained with the dye DAPI; GFP, green fluorescent protein; Merge, the overlay of BF, GFP and DAPI images; Cell nuclei are indicated by white triangles, and appressoria are indicated by white arrows. Scale bar = 5 μm; (b) The subcellular localization of SCRE4-RFP and RFP expressed in Nicotiana benthamiana cells. SCRE4-RFP was transiently expressed in N. benthamiana leaves through Agrobacterium-mediated transient expression. The images were captured at 48 h after agro-infiltration under confocal microscopy. BF, bright field; RFP panels, red fluorescence from SCRE4-RFP; DAPI panels, the nuclei were stained with the dye DAPI; Merge, the overlay of GFP and DAPI images; Scale bar = 20 μm. (c) The expression of SCRE4-GFP and SCRE4^NM -GFP in rice protoplasts detected by immunoblotting with anti-GFP antibody (α-GFP). Protein loading is indicated by Ponceau S staining. (d) Subcellular localization of SCRE4-GFP and SCRE4^NM-GFP expressed in rice protoplasts. RFP-NLS was transiently co-expressed with SCRE4-GFP or SCRE4^NM-GFP in rice protoplasts. GFP and RFP signals were visualized under confocal microscope. RFP-NLS was used as a marker for nuclear localization. Scale bar = 7.5 μm.

Figure 4

The transcriptional expression of OsARF17 is suppressed by the Ustilaginoidea virens effector SCRE4. (a) The GFP expression level driven by the OsARF17 promoter when it was individually co−expressed with SCRE4, UV8b_03835 and UV8b_03279 in rice protoplasts. The ProOsARF17:GFP construct was transfected alone or co−transfected with different putative effector gene constructs into rice protoplasts. The blots were detected via immunoblotting using anti−GFP, anti−β−Actin and anti−FLAG antibodies. The band intensity was quantified using ImageJ. Relative GFP abundance was normalized to the level of β−Actin. Right panel, data from three independent experiments are shown as mean ± SE. Different letters (a vs. b) indicate a significant difference in the GFP expression level (p < 0.05, Duncan’s multiple range test). (b) The dual−LUC assay to show the relative LUC/REN activity when the ProOsARF17:LUC construct was co−transformed with different putative effector gene constructs in N. benthamiana. Upper diagram shows the features of the constructs used in the dual−LUC assay. The relative LUC/REN activity was measured after LUC, and putative effectors were transiently co−expressed in N. benthamiana leaves. Different letters (a vs. b) indicate a significant difference in the relative activity (LUC/REN) when LUC was co−expressed with different proteins (p < 0.05, Duncan’s multiple range test).

Figure 5

OsARF17 positively regulates rice defense against U. virens. (a) The chitin−induced MAPK phosphorylation levels in the wild−type and OsARF17−overexpressing lines. MAPK phosphorylation was detected by immunoblot analyses with anti−phospho−44/42 MAPK antibody (α−pMAPK). Protein loading is indicated by Ponceau S staining. (b) Chitin−triggered ROS burst in the wild−type and OsARF17−overexpressing lines. The leaves of the wild−type and transgenic lines OE17−2−5 and OE17−3−2 were treated with chitin (10 µg mL⁻¹) or mock control. ROS burst was detected immediately after treatment within 25 min. (c) Chitin−induced MAPK activation detected in the wild−type and osarf17 mutant lines. Chitin treatment and detection of MAPK phosphorylation were performed as described in (a). (d–f) The average number of diseased grains on inoculated panicles of the wild−type (ZH11) and different transgenic lines. The virulent U. virens isolate PJ52 was injection-inoculated into the wild−type and knockout lines osarf17−5, osarf17−6 and osarf17−8 (n = 12, 14, 10, 10) (d), the wild−type and mutant lines osarf17−2−1 and osarf17−5−2 generated with another sgRNA site (n = 12, 5, 8) (e), and the wild−type, OsARF17−overexpressing OE17−2−5 and OE17−3−2 lines (n = 12, 12, 12) (f). False smut balls were counted on the inoculated panicles 4 weeks after inoculation. The representative data from three independent experiments are shown as mean ± SE. Different letters (a vs. b) indicate a significant difference in the average number of diseased grains between the wild−type and different transgenic lines (p < 0.05, Duncan’s multiple range test).

Figure 6

The ability of SCRE4 to suppress rice immunity is dependent on nuclear localization. (a) Expression of OsARF17 in the wild−type, SCRE4−IE and SCRE4^NM−IE transgenic lines after DEX and mock treatments. OsGAPDH expression was used as an internal reference. Asterisk indicates significant difference in the expression level of OsARF17 in the SCRE4−IE−1 line between DEX and mock treatments (Student’s t-test, * p < 0.05). (b) The GFP expression level driven by the OsARF17 promoter when the ProOsARF17: GFP construct was co-transfected with the SCRE4 or SCRE4^NM constructs into rice protoplasts. Total proteins were subject to Western blot analyses probed with anti−GFP, anti−β−Actin and anti−FLAG antibodies. Upper panel, the band intensity was quantified with Image J. Data from three independent assays are shown as mean ± SE (n = 4). Different letters ((a) vs. (b)) indicate a significant difference in relative GFP abundance between SCRE4− and SCRE4^NM−expressing protoplasts (Duncan’s multiple range test, p < 0.05). (c) Diseased grains on rice panicles after inoculation of different U. virens strains. The U. virens wild−type (P1), Δscre4 knockout mutant and complemented strains (SCRE4−C1 and C2, SCRE4^NM−C2 and C5) were injection-inoculated into young panicles of the susceptible rice cultivar LYP9 (n = 14, 10, 13, 15, 15, 14, 15). (d) Disease symptoms on the representative rice panicles after U. virens inoculation. The images were captured 4 weeks after rice panicles of LYP9 were inoculated by the wild−type (P1), different scre4 knockout and complemented strains (SCRE4−C1 and C2, SCRE4^NM−C2 and C5). False smut balls are indicated by white triangles.

Figure 7

A working model of SCRE4 suppressing rice immunity. The auxin response factor OsARF17 functions as a positive regulator of PTI responses and positively regulates rice resistance to false smut. During U. virens infection, the essential virulence effector SCRE4 is secreted and translocated into rice nuclei. Through an unidentified mechanism, SCRE4 transcriptionally suppresses OsARF17 expression in the nucleus and subsequently inhibits MAPK activation and ROS production. Therefore, immune responses in rice are disarmed, thus promoting U. virens infection.