Salicylic acid-dependent immunity contributes to resistance against Rhizoctonia solani, a necrotrophic fungal agent of sheath blight, in rice and Brachypodium distachyon

Abstract

Rhizoctonia solani is a soil-borne fungus causing sheath blight. In consistent with its necrotrophic life style, no rice cultivars fully resistant to R. solani are known, and agrochemical plant defense activators used for rice blast, which upregulate a phytohormonal salicylic acid (SA)-dependent pathway, are ineffective towards this pathogen. As a result of the unavailability of genetics, the infection process of R. solani remains unclear. We used the model monocotyledonous plants Brachypodium distachyon and rice, and evaluated the effects of phytohormone-induced resistance to R. solani by pharmacological, genetic and microscopic approaches to understand fungal pathogenicity. Pretreatment with SA, but not with plant defense activators used in agriculture, can unexpectedly induce sheath blight resistance in plants. SA treatment inhibits the advancement of R. solani to the point in the infection process in which fungal biomass shows remarkable expansion and specific infection machinery is developed. The involvement of SA in R. solani resistance is demonstrated by SA-deficient NahG transgenic rice and the sheath blight-resistant B. distachyon accessions, Bd3-1 and Gaz-4, which activate SA-dependent signaling on inoculation. Our findings suggest a hemi-biotrophic nature of R. solani, which can be targeted by SA-dependent plant immunity. Furthermore, B. distachyon provides a genetic resource that can confer disease resistance against R. solani to plants.

Keywords: Brachypodium distachyon; Rhizoctonia solani; biotroph; disease resistance; necrotroph; rice; salicylic acid (SA); sheath blight.

Figure 1

Infectivity of Rhizoctonia solani Japanese isolates on detached shoots of Brachypodium distachyon standard accession Bd21. Bd21 shoots were detached and inoculated with the different R. solani isolates listed in Table 1. Photographs were taken at 4 d post‐inoculation (dpi). Asterisks indicate the location of inoculum agar plugs, and arrows indicate necrotic lesions or etiolated regions of leaf blades, similar to symptoms typically observed in rice grown in the field. Bars, 1 cm. The assay was performed twice with similar results.

Figure 2

Salicylic acid (SA) induces Rhizoctonia solani resistance in Brachypodium distachyon. (a, b) Lesion formation (a) and relative biomass (linear scale) (b) of R. solani AG‐1 on B. distachyon leaves treated with water (Mock) or SA, jasmonic acid (JA) or ethylene (ET) (1 mM each) at 3 d post‐inoculation (dpi). Bars, 1 cm. Data are represented as means ± SEM, n = 4; *, P < 0.05; **, P < 0.01 using Student's t‐tests. (c, d) Relative biomass of R. solani AG‐1 in B. distachyon leaves treated with 0, 1, 10, 100 and 1000 μM of SA (c) or JA (d) at 3 dpi. Data are represented as means ± SEM, n = 3; *, P < 0.05 using Student's t‐tests. (e, f) Lesion formation (e) and relative biomass (linear scale) (f) of R. solani AG‐5 in B. distachyon leaves treated with water (Mock) or 1 mM SA at 3 dpi. Bars, 1 cm. Data are represented as means ± SEM, n = 4; *, P < 0.05 using Student's t‐tests. All experiments were repeated three times with similar results.

Figure 3

Salicylic acid (SA) induces Rhizoctonia solani resistance in soil‐grown Brachypodium distachyon intact plants. (a, b) Disease symptoms (a) and fungal biomass (linear scale) in leaves (b) of R. solani AG‐1 in B. distachyon intact plants grown on soil treated with water (Mock) or 1 mM SA. Bd21 whole plants grown on soil for 3 wk were sprayed with water (Mock) or 1 mM SA for 48 h and inoculated with R. solani (MAFF305230) by a mycelial agar plug held on the stem with tape. Photographs were taken at 7 d post‐inoculation (dpi). Data are represented as means ± SEM, n = 4; **, P < 0.01 using Student's t‐tests. Similar results were obtained in three independent experiments.

Figure 4

Salicylic acid (SA) induces and is required for Rhizoctonia solani resistance in rice. (a, b) Lesion formation (a) and fungal biomass (linear scale) (b) of R. solani AG‐1 in rice leaves treated with water (Mock) or 1 mM SA. Photographs were taken at 3 d post‐inoculation (dpi). The graph shows the relative R. solani biomass in the inoculated leaves at 3 dpi. Bars, 1 cm. Data are represented as means ± SEM of values relative to the mock treatment, n = 12; **, P < 0.01 using Student's t‐test. (c, d) Lesion formation (c) and relative biomass (linear scale) (d) of R. solani AG‐1 in the leaves of wild‐type and NahG‐overexpressing transgenic rice at 3 dpi. Bars, 1 cm. Data are represented as means ± SEM, n = 12; ***, P < 0.001 using Student's t‐tests. All experiments were repeated three times with similar results.

Figure 5

Salicylic acid (SA) induces post‐invasion resistance to Rhizoctonia solani in Brachypodium distachyon. (a) Hyphal growth of R. solani AG‐1 on B. distachyon leaf surfaces treated with dimethyl sulfoxide (DMSO; Mock) or 1 mM SA. The inoculated leaves were collected at the indicated time points and R. solani hyphae were stained with trypan blue. Infection cushions were recognized as aggregates of convoluted hyphae. Bars, 100 μm. (b) Relative biomass (linear scale) of R. solani AG‐1 in B. distachyon leaves treated with DMSO (Mock) or 1 mM SA at the indicated time points. Data are represented as means ± SEM, n = 6; *, P < 0.05; ***, P < 0.001 using Student's t‐tests relative to the values of mock at 20 h post‐inoculation (hpi). The experiments were repeated at least twice with similar results.

Figure 6

Specific salicylic acid (SA) analogs, but not commercial plant defense activators, increased Rhizoctonia solani resistance in Brachypodium distachyon. (a, b) Lesion formation (a) and relative biomass (linear scale) (b) of R. solani AG‐1 at 3 d post‐inoculation (dpi) in B. distachyon leaves treated with dimethyl sulfoxide (DMSO; Mock), 1 mM SA, 200 μM probenazole (PBZ) or 200 μM tiadinil (TDN). Bars, 1 cm. Data are represented as means ± SEM, n = 4; *, P < 0.05 using Student's t‐tests. (c) Expression levels of an SA‐responsive marker gene BdWRKY45L1 (Bradi2 g30695). Bd21 seedlings were treated with water (Mock) or 500 μM chemical solutions for 24 h. Data are represented as means ± SEM, n = 3; **, P < 0.01; ***, P < 0.001 using Student's t‐tests. (d, e) Relative biomass (linear scale) of R. solani AG‐1 at 3 dpi (d) or 6 dpi (e) in B. distachyon leaves sprayed with DMSO (Mock) or 500 μM of SA, acetylsalicylic acid (Ac‐SA), 3,5‐dichloroanthranilic acid (DCA), 2,6‐dichlorisonicotianic acid (INA) or benzothiadiazole (BTH). Data are represented as means ± SEM, n = 4; *, P < 0.05; **, P < 0.01 using Student's t‐tests. The experiments were repeated three times with similar results.

Figure 7

Salicylic acid (SA)‐specific transcripts include secondary cell wall‐related genes in Brachypodium distachyon. (a) Proportional Venn diagrams showing the overlap of the up‐regulated (left) and down‐regulated (right) gene sets 24 h after treatment with SA or benzothiadiazole (BTH). (b, c) Functional classification of the genes specifically upregulated by SA with gene ontology (GO) categories related to biological process (b) and cellular component (c) using a REVIGO scatterplot. Circles denote significantly enriched GO terms with false discovery rate (FDR) < 0.05. The circle size represents the −log₁₀ transformed FDR in REVIGO analysis. (d) Expression levels of secondary cell wall‐related genes after 24 h of treatment with SA or BTH. Data are represented as means ± SEM relative to those of the dimethyl sulfoxide (DMSO) treatment, n = 3; *, P < 0.05 using Student's t‐tests. All experiments were repeated at least twice with similar results.

Figure 8

Brachypodium distachyon accessions Bd3‐1 and Gaz‐4 are resistant to Rhizoctonia solani. (a, b) Lesion formation (a) and relative biomass (linear scale) (b) of R. solani AG‐1 in the leaves of B. distachyon accessions Bd21, Bd3‐1 and Gaz‐4 at 3 d post‐inoculation (dpi). Bars, 1 cm. Data are represented as means ± SEM, n = 5; **, P < 0.01 using Student's t‐test. (c) Expression levels of B. distachyon marker genes BdWRKY45L1 and BdWRKY45L2 for SA and BdAOS for jasmonic acid (JA) in Bd21, Bd3‐1 and Gaz‐4 at 0, 5, 24 and 48 h post‐inoculation (hpi) with R. solani AG‐1. Data are represented as means ± SEM, n = 4; *, P < 0.05; **, P < 0.01; P < 0.001 using Student's t‐tests compared with the corresponding values of each accession at 0 dpi. All experiments were repeated at least three times with similar results.