Functional validation of pathogenicity genes in rice sheath blight pathogen Rhizoctonia solani by a novel host-induced gene silencing system

Abstract

Rice sheath blight, caused by the soilborne fungus Rhizoctonia solani, causes severe yield losses worldwide. Elucidation of the pathogenic mechanism of R. solani is highly desired. However, the lack of a stable genetic transformation system has made it challenging to examine genes' functions in this fungus. Here, we present functional validation of pathogenicity genes in the rice sheath blight pathogen R. solani by a newly established tobacco rattle virus (TRV)-host-induced gene silencing (HIGS) system using the virulent R. solani AG-1 IA strain GD-118. RNA interference constructs of 33 candidate pathogenicity genes were infiltrated into Nicotiana benthamiana leaves with the TRV-HIGS system. Of these constructs, 29 resulted in a significant reduction in necrosis caused by GD-118 infection. For further validation of one of the positive genes, trehalose-6-phosphate phosphatase (Rstps2), stable rice transformants harbouring the double-stranded RNA (dsRNA) construct for Rstps2 were created. The transformants exhibited reduced gene expression of Rstps2, virulence, and trehalose accumulation in GD-118. We showed that the dsRNA for Rstps2 was taken up by GD-118 mycelia and sclerotial differentiation of GD-118 was inhibited. These findings offer gene identification opportunities for the rice sheath blight pathogen and a theoretical basis for controlling this disease by spray-induced gene silencing.

Keywords: Rhizoctonia solani; host-induced gene silencing; pathogenicity gene; rice disease control strategy; rice sheath blight.

FIGURE 1

Disease index values in host‐induced gene silencing (HIGS) assays in Nicotiana benthamiana for 33 candidate pathogenicity genes in Rhizoctonia solani. Highly resistant lines, moderately resistant lines, and slightly resistant lines are highlighted in red, blue, and yellow, respectively. Error bars represent standard errors of biological triplicates. *p < .05, one‐way analysis of variance with Tukey's post hoc test

FIGURE 2

The effects of host‐induced gene silencing (HIGS) for Rstps2 on Nicotiana benthamiana. (a) The phenotype of pTRV2::Rstps2‐infiltrated plants at 14 days after infiltration. (b) Reverse transcription PCR was used to monitor the expression levels of target genes in infiltrated plants. M: 2‐kb DNA ladder; 1: pTRV2::GFP; 2: pTRV2::PDS; 3: pTRV2::Rstps2. (c) Necrotic symptoms on leaves inoculated with GD‐118 at 5 days postinoculation (dpi). (d) Relative leaf area that was visibly infected at 5 dpi. (e) Relative amounts of GD‐118 DNA. Fungal biomass was measured by quantitative PCR and normalized to EF1α transcript levels of N. benthamiana. (f) Reactive oxygen species detection by 3,3′‐diaminobenzidine (DAB) staining in tobacco leaves. (g) Quantified leaf areas with brown spots by DAB staining. (h) Gene expression levels of Rstps2. The data were normalized to the GAPDH transcript levels of GD‐118. Error bars represent SE (n = 3). **p < .01, ***p < .001, Student's t test

FIGURE 3

Molecular analysis and morphological characteristics of homozygous T₂ transgenic rice. (a,b) PCR analysis of T₂ transgenic plants using Rstps2 gene‐specific primers. (c) Plant height. (d) Number of tillers. (e) Southern blot analysis showing the integration of the transgene in the T₂‐Tps2 lines. Data are shown as the mean ± SE of triplicates. ns: no significant changes observed, one‐way analysis of variance and Tukey's test

FIGURE 4

Evaluation of homozygous T₂ transgenic rice lines against the rice sheath blight pathogen Rhizoctonia solani. (a) Disease symptoms in R. solani‐infected detached leaves of T₂ homozygous transgenic lines at 60 and 120 hr postinoculation (hpi). (b) Quantification of the visibly infected area at 60 and 120 hpi shown as the percentage of the total leaf area. *p < .05, **p < .01, two‐way analysis of variance and Tukey's test. (c) Relative amounts of fungal DNA at 120 hpi as determined by quantitative PCR. The data were normalized to the 18S rRNA gene transcript levels of rice and are shown as the mean ± SE of triplicates. **p < .01, one‐way analysis of variance and Tukey's test. (d) Inoculation of plants by placing R. solani sclerotia in sheath tissue. (e) Highest relative lesion height (HRLH) in T₂ transgenic and nontransgenic (WT) controls at 5 and 10 days postinoculation (dpi). **p < .01,***p < .001, two‐way analysis of variance Tukey's test [Correction added on 02 September 2021, after first online publication: Figure 4 has been updated in this version.]

FIGURE 5

siRNA targeting Rstps2 contributes to gene silencing and virulence inhibition. (a) The trehalose content of Rhizoctonia solani‐(TPS‐3‐2/6) was significantly decreased. Data are shown as the mean ± SE of triplicates. **p < .01, one‐way analysis of variance (ANOVA) and Tukey's test. (b) Relative gene expression of Rstps2 in standardized subcultured R. solani‐(TPS‐3‐2). G1–G5: generation 1 to generation 5. The data were normalized to the GAPDH transcript levels of GD‐118 and are shown as the mean ± SE of triplicates. Different labels represent statistical significance (p < .01), one‐way ANOVA and Tukey's test. (c) Each read length transcripts per million (TPM) sum of Rstps2‐derived specific siRNAs is the sum of all the TPM values of that read length. Data are shown as the mean ± SE of triplicates. Error bars represent SE (n = 3). *p < .05, Student's t test

FIGURE 6

Uptake of dsRNA synthesized in vitro by hyphae of Rhizoctonia solani and the target gene silenced. (a) Quantitative gene expression pattern of Rstps2 after supplementation of the dsRNA. The data were normalized to the GAPDH transcript levels of GD‐118 and are shown as the mean ± SE of triplicates. Different labels represent statistical significance (p < .05), one‐way analysis of variance and Tukey's test. (b) Sclerotial differentiation (SDIF) of sclerotia of R. solani AG‐1 IA strain GD‐118 treated with dsRNA for 10 days. Data are shown as the mean ± SE of triplicates. *p < .05, Student's t test. (c) Microscopic observation of the uptake of dsRNA with fluorescently labelled dsRNA and without fluorescently labelled dsRNA of Rstps2 by GD‐118 cells