Ustilaginoidea virens-secreted effector Uv1809 suppresses rice immunity by enhancing OsSRT2-mediated histone deacetylation

Abstract

Rice false smut caused by Ustilaginoidea virens is a devastating rice (Oryza sativa) disease worldwide. However, the molecular mechanisms underlying U. virens-rice interactions are largely unknown. In this study, we identified a secreted protein, Uv1809, as a key virulence factor. Heterologous expression of Uv1809 in rice enhanced susceptibility to rice false smut and bacterial blight. Host-induced gene silencing of Uv1809 in rice enhanced resistance to U. virens, suggesting that Uv1809 inhibits rice immunity and promotes infection by U. virens. Uv1809 suppresses rice immunity by targeting and enhancing rice histone deacetylase OsSRT2-mediated histone deacetylation, thereby reducing H4K5ac and H4K8ac levels and interfering with the transcriptional activation of defence genes. CRISPR-Cas9 edited ossrt2 mutants showed no adverse effects in terms of growth and yield but displayed broad-spectrum resistance to rice pathogens, revealing a potentially valuable genetic resource for breeding disease resistance. Our study provides insight into defence mechanisms against plant pathogens that inactivate plant immunity at the epigenetic level.

Keywords: OsSRT2; Uv1809; histone acetylation; immunity; rice false smut.

Figure 1

Uv1809 is a key virulence effector. (a) RT‐qPCR analysis of the expression of Uv1809 in different infection stages on rice spikelets (1–20 dpi). Data are presented as mean ± SD (n = 3). The P values were determined by unpaired t‐tests compared with 0 dpi. (b) Virulence assays of the wild‐type HWD‐2, ΔUv1809 mutants, CΔUv1809‐1 and C∆Uv1809 ^∆SP ‐1 strains on rice cultivar Wanxian98 at 21 dpi. (c) Number of rice smut balls per panicle. Data were collected from three independent experiments for each treatment. The P values were determined by unpaired t‐tests compared with the wide‐type strain HWD‐2. (d) The yeast YTK12 and its transformants expressing the empty vector pSUC2 (negative control) or pSUC2‐Uv1809SP and pSUC2‐Avr1SP (positive control) were tested for growth on SD‐Trp or YPRAA medium and invertase activity in TTC medium. (e) Transient expression of Uv1809 suppressed programmed cell death of Nicotiana benthamiana leaves triggered by Bax. Representative leaves were photographed at 4 dpi. (f) Subcellular localization of Uv1809‐GFP in N. benthamiana leaves. N. benthamiana histone H2B protein was used as a nuclear localization marker protein. DIC, differential interference contrast; GFP, green fluorescent protein. Scale bar = 20 μm.

Figure 2

Heterologous overexpression of Uv1809 increases susceptibility to rice pathogens and HIGS of Uv1809 enhances rice resistance against Ustilaginoidea virens. (a) Left: Resistance assays of 35S‐Uv1809 and 35S‐EV transgenic rice lines to U. virens strain HWD‐2 infection at 25 dpi. Right: Mean number of rice smut balls measured in resistance assays. (b) Left: Disease symptoms at 14 dpi of 35S‐Uv1809 and 35S‐EV transgenic rice lines after inoculation with Xoo PXO99. Right: Mean lesion lengths at 14 dpi on the leaves of 35S‐Uv1809 and 35S‐EV transgenic rice lines after inoculation with Xoo PXO99. (c) Disease symptoms (Left), leaf lesion area (Middle) and the relative fungal biomass (Right) of 35S‐Uv1809 and 35S‐EV transgenic rice lines after spot‐inoculation with Magnaporthe oryzae ZB‐25 at 7 dpi. Relative fungal biomass was determined using quantitative reverse transcription (RT‐qPCR) for M. oryzae Pot2 and normalized to rice OsUBQ1. The leaf lesion area was measured using Image J software. (d) RT‐qPCR analysis of defence‐related genes at 1 dpi in 35S‐EV and 35S‐Uv1809 transgenic rice lines inoculated with U. virens. (e) Left: Resistance assays of Uv1809HIGS and EV transgenic rice lines against U. virens strain HWD‐2 at 25 dpi. Right: Mean number of rice smut balls measured in resistance assays. (f) Relative mRNA expression of Uv1809 of U. virens during infection in the T2 transgenic rice lines at 6 dpi. (g) Length distribution and abundance of siRNAs targeting Uv1809HIGS‐L1 in T2 transgenic rice plants. (h) Visualization of siRNAs targeting Uv1809 in infected Uv1809HIGS‐L1 rice spikelets at 6 dpi by FISH using a specific probe. An, anther; hy, U. virens hyphae. Scale bar = 20 μm. All data are presented as mean ± SD (n = 3 unless otherwise indicated) and analysed by Fisher's least significant difference (LSD) test. The P values were determined by unpaired t‐tests compared with the 35‐EV or EV.

Figure 3

Uv1809 physically interacts with OsSRT2. (a) Y2H analysis of the interaction between Uv1809 and OsSRT2. The interaction between BD‐53 and AD‐T was taken as the positive control, BD and AD‐OsSRT2 was taken as the negative control. SD‐3, SD‐Trp‐Leu‐His; SD‐4, SD‐Trp‐Leu‐His‐Ade; BD, pGBKT7; AD, pGADT7. (b) In vivo Co‐IP of Uv1809 interacts with OsSRT2. Co‐IP was performed on extracts of Nicotiana benthamiana leaves by co‐expression of OsSRT2‐Flag and Uv1809‐GFP. Uv1809‐GFP and OsSRT2‐Flag were detected by western blotting using anti‐GFP and anti‐Flag antibodies, respectively. (c) A GST pull‐down assay was used to detect the interaction between Uv1809^∆SP‐His and OsSRT2‐GST. Uv1809 and OsSRT2 were fused to His and GST tags, respectively, and expressed in Escherichia coli. OsSRT2‐GST or GST‐bound resin was incubated with E. coli crude extracts containing Uv1809^∆SP‐His and analysed by western blotting. Uv1809^∆SP‐His and OsSRT2‐GST were detected using anti‐His and anti‐GST antibodies, respectively. (d) BiFC assays for the interaction between Uv1809 and OsSRT2. N. benthamiana leaves were infiltrated with a mixture of Agrobacterium tumefaciens strains co‐expressing the OsSRT2‐nYFP and Uv1809‐cYFP constructs. YFP signals were observed at 2 dpi. Infiltration with Agrobacterium co‐expressing the OsSRT2‐nYFP and cYFP, nYFP and Uv1809‐cYFP constructs were used as the negative control. No YFP signals was observed in these negative controls. Scale bar = 20 μm.

Figure 4

OsSRT2 negatively regulates rice broad‐spectrum resistance against rice pathogens. (a) Morphology and agronomic traits of the wild‐type ZH11 and ossrt2 mutants rice lines at mature stage following growth in field conditions. (b) Left: Resistance assays of ZH11 and ossrt2 mutants rice lines against Ustilaginoidea virens HWD‐2 at 25 dpi. Right: Numbers of rice smut balls were calculated in resistance assays. (c) Left: Disease symptoms of ZH11 and ossrt2 mutants rice lines after spray inoculation with Magnaporthe oryzae P131 at 7 dpi. Right: Relative fungal biomass was determined using RT‐qPCR for the M. oryzae Pot2 gene normalized to rice OsUBQ1. (d) Disease symptoms (Left) and the relative fungal biomass (Right) of ZH11 and ossrt2 mutants rice lines after spray inoculation with M. oryzae ZB‐25 at 7 dpi. (e) Disease symptoms (Left) and leaf lesion area (Right) of ZH11 and ossrt2 mutants rice lines were spot inoculated with spore suspensions of M. oryzae P131 at 7 dpi. (f) Disease symptoms (Left) and leaf lesion area (Right) of ZH11 and ossrt2 mutants rice lines after spot‐inoculation with M. oryzae ZB‐25 at 7 dpi. (g) Left: Disease symptoms of ZH11 and ossrt2 mutants rice lines after inoculated with Xoo PXO99 at 14 dpi. Right: Lesion lengths on rice leaves of ZH11 and ossrt2 mutants rice lines after inoculated with Xoo PXO99 at 14 dpi. (h) Disease symptoms at 3 dpi of ZH11 and ossrt2 mutants rice lines after inoculation with Rhizoctonia solani HG81. Right: Leaf lesions on ZH11 and ossrt2 mutants rice lines after inoculation with R. solani HG81 at 3 dpi. (i) RT‐qPCR analysis of defence‐related genes at 1 dpi in ZH11 and ossrt2 mutants rice lines inoculated with U. virens. Data are presented as mean ± SD (n = 3 unless otherwise indicated). The P values were determined by unpaired t‐tests compared with the wild‐type ZH11.

Figure 5

Uv1809 modulates histone deacetylation activity of OsSRT2. (a) The levels of H3K9ac, H3K27ac, H3K36ac, H3K56ac, H4K5ac, H4K8ac, H4K12ac and H4K16ac in the wild‐type ZH11 and ossrt2 mutants rice spikelets were detected by western blotting. Relative quantified signals of each band are indicated with the first ZH11 loading set as 1.00. (b) Relative the levels of H3K9ac, H3K27ac, H3K36ac, H3K56ac, H4K5ac, H4K8ac, H4K12ac and H4K16ac in Nip, 35S‐EV and 35S‐Uv1809 transgenic rice plants as detected by western blotting. (c) In vitro lysine deacetylation activity of OsSRT2 by fluorometric assays. A HDAC Assay Kit (Fluorescent) (Active Motif) was used to determine HDAC activity of purified GST, Uv1809^∆SP‐His, Uv1809^23–245‐His, Uv1809^246–391‐His, OsSRT2‐GST, OsSRT2‐GST and Uv1809^∆SP‐His, OsSRT2‐GST and Uv1809^23–245‐His, OsSRT2‐GST and Uv1809^246–391‐His, HADCs inhibitor Nicotinamide and OsSRT2‐GST proteins. (d) In vitro histone H4K5 and H4K8 deacetylation activity of OsSRT2 by immunoblotting. For in vitro deacetylation assay, 20 μL rice histone protein, 2 μL the purified GST, Uv1809^∆SP‐His, Uv1809^23–245‐His, Uv1809^246–391‐His, OsSRT2‐GST, OsSRT2‐GST and Uv1809^∆SP‐His, OsSRT2‐GST and Uv1809^246–391‐His, OsSRT2‐GST and Uv1809^23–245‐His, OsSRT2‐GST and HDACi proteins were incubated in 20 μL reaction buffer at 30 °C for 4 h. The reaction products were analysed by western blotting with anti‐H4K5ac and anti‐H4K8ac antibodies. (e) In vivo H4K5ac and H4K8ac levels when OsSRT2 and Uv1809 co‐expression in Nicotiana benthamiana leaves. OsSRT2‐Flag, GFP, Uv1809‐GFP, Uv1809‐GFP and OsSRT2‐Flag, GFP and OsSRT2‐Flag fusion proteins were expression/co‐expression in N. benthamiana leaves as detected with anti‐H4K5ac and anti‐H4K8ac antibodies. (f) In vitro lysine deacetylation activity of nuclear proteins from (e) by fluorometric assays.

Figure 6

RNA‐seq and ChIP‐seq data analysis of ossrt2 and wild‐type rice spikelets. (a) Volcano plots of differential transcript levels in ossrt2 relative to wild type. Purple plots represent upregulated genes (Fold change > 2, P value < 0.05); cyan plots represent downregulated genes (Fold change > 2, P value < 0.05); grey plots represent genes with no significant difference. (b) Representative GSEA enriched pathways in ossrt2. (c) GO pathway analysis of the genes (n = 604) that were upregulated in ossrt2 mutants. (d) Peaks with H4K5ac and H4K8ac level changes in ossrt2 compared with wild type. Peaks with reduced H4K5ac or H4K8ac in the mutant are shown in cyan, and those with gained H4K5ac or H4K8ac are in pink (Fold change > 1.5, P < 0.05). (e) Venn diagrams of upregulated H4K5 and H4K8 acetylation peaks (left) or genes (right) in ossrt2. (f) GO pathways found in both H4K5ac and H4K8ac significantly upregulated genes in ossrt2 versus wild type.

Figure 7

Role of H4K5ac and H4K8ac in gene expression regulation in ossrt2 rice spikelets. (a) Correlation analysis of expression changes and H4K5ac or H4K8ac changes in ossrt2 versus wild type. Person Correlation Coefficient was shown. (b) Metaplots of H4K5 or H4K8 acetylation ChIP‐seq reads in transcriptionally upregulated genes. TSS, transcriptional start site. TES, transcriptional end site, RPM, reads count per million mapped reads. (c) Venn diagrams of H4K5 and H4K8ac hyper‐acetylated genes, and transcriptionally upregulated genes in ossrt2. (d) Representative GO pathways of H4K5ac, H4K8ac, and transcriptionally upregulated genes (N = 84) in ossrt2. (e) RT‐qPCR and ChIP‐qPCR analysis of H4K5ac‐ or H4K8ac‐marked defence‐related genes in ZH11 and ossrt2 rice spikelets. Asterisks indicate statistically significant differences compared to ZH11 at P < 0.05.

Figure 8

A working model illustrating how Uv1809 manipulates histone deacetylase OsSRT2 to suppress rice immunity during Ustilaginoidea virens infection. During infection, U. virens effector Uv1809 is secreted and translocated into host cells, and then physically interacts with OsSRT2. Uv1809 disrupts host immunity by recruiting enhancing OsSRT2‐modulated deacetylation, thereby reducing the levels of H4K5ac and H4K8ac in rice plants and interfering with defence gene activation.