Host-Induced Gene Silencing of Effector AGLIP1 Enhanced Resistance of Rice to Rhizoctonia solani AG1-IA

doi:10.1016/j.rsci.2024.04.005

摘要/Abstract

. [J]. Rice Science, 2024, 31(4): 463-474.

Fig. 1. Bioinformatics analysis for sequences of 10 489 proteins of Rhizoctonia solani AG1-IA. A, Length distribution of protein sequences with signal peptides in R. solani AG1-IA. B, Subcellular localizations of 484 proteins with signal peptides. C, Length distribution of candidate effector sequences with signal peptides in R. solani AG1-IA. D, Frequency of amino acids of signal peptides in R. solani AG1-IA candidate effectors.

Fig. 2. Disease index for Nicotiana benthamiana infiltrated with tobacco rattle virus (TRV) vectors of 45 candidate effector genes of Rhizoctonia solani AG1-IA. The Red bar indicates disease resistance, the yellow bar indicates moderate resistance, the blue bar indicates susceptibility, and the gray bar indicates high susceptibility. RNAi-GFP was used as a negative control. The data are presented as Mean ± SE (n = 3). Statistical significance is denoted as P < 0.05 (*), P < 0.01 (**), or P < 0.001 (***), respectively, according to one-way analysis of variance.

Fig. 3. Transient silencing of AGLIP1 enhanced resistance of Nicotiana benthamiana to Rhizoctonia solani AG1-IA. A, Phenotype of infiltrated N. benthamiana plants after a 14-day inoculation period. Phytoene desaturase (PDS) gene was used as a positive control for tobacco rattle virus-host induced gene silencing (TRV-HIGS) efficiency. Infiltration with pTRV:PDS caused photobleaching at 14 d post- infiltration, indicating successful gene silencing. Meanwhile, the AGLIP1-silenced tobacco plants exhibited no noticeable phenotypic differences compared with the negative control (pTRV:GFP). B, RT-PCR reflected the expression of target genes (AGLIP1, GFP, and PDS) in infiltrated N. benthamiana plants at 14 d after inoculation. M, Marker; Lanes 1, 2 and 3 represent the AGLIP1, GFP, and PDS genes, respectively. C, Symptom development on N. benthamiana leaves inoculated with GD-118 mycelial suspension at 5 d post-infection (dpi). D, Quantification of visible infected area at 5 dpi is shown as a percentage of the total N. benthamiana leaf area. E, Relative amounts of fungal DNA as determined by qRT-PCR. The samples were N. benthamiana leaves at 5 dpi and the data were normalized to the EF1α transcript level of N. benthamiana. F, 3,3ʹ-Diaminobenzidine (DAB) staining reflecting reactive oxygen species accumulation in N. benthamiana leaves. G, Quantification of visible brown spot area is shown as a percentage of the total N. benthamiana leaf area. H, Gene-specific expression levels of AGLIP1 (RNAi-15) were measured by qRT-PCR. The samples were N. benthamiana leaves at 5 dpi and the data were normalized to the GAPDH transcript level of R. solani. The data are presented as Mean ± SE (n = 3). Statistical significance is denoted as P < 0.05 (*), P < 0.01 (**), or P < 0.001 (***), respectively, according to the Student’s t-test.

Fig. 4. Molecular analysis and morphological characteristics of homozygous T2 transgenic rice. A, PCR analysis of T2-AGLIP1-Line 7 (L7) and T2-AGLIP1-Line 9 (L9) transgenic plants using a hygromycin gene-specific primer. M, Marker; Lanes 1‒25 represent transgenic lines. B, Southern blot analysis of AGLIP1 transgenic plants. WT, Wild type; L1, L7, L4, and L9 represent T2-AGLIP1-Line 1, -Line 7, -Line 4, and -Line 9, respectively. C‒E, Comparisons of plant height (C), flag leaf length (D), and the number of effective tillers per plant (E) between the WT and RNAi homozygous T2 transgenic lines. The data are present as Mean ± SE (n = 3). ns indicates no significant changes were observed using one-way analysis of variance with Tukey’s test.

Fig. 5. Evaluation of T2-AGLIP1 lines against sheath blight disease pathogen Rhizoctonia solani AG1-IA. A, Disease symptoms in R. solani AG1-IA infected detached leaves of T2 homozygous transgenic lines at 72 h post-infection (hpi). B, Quantification of visible infected area is shown as a percentage of the total rice leaf area at 72 hpi. C, Relative amounts of fungal DNA were determined by qRT-PCR. The samples were rice leaves at 72 hpi and the data werenormalized to the 18S rRNA transcript level of rice. D, Gene-specific expression levels of AGLIP1 were measured by qRT-PCR. The samples were japonica rice leaves at 72 hpi, and the data were normalized to the GAPDH transcript level of R. solani. WT, Wild type; L7, T2-AGLIP1-Line 7 transgenic plants; L9, T2-AGLIP1-Line 9 transgenic plants. The data are presented as Mean ± SE (n = 3). Statistical significance is denoted as P < 0.001 (***) according to one-way analysis of variance with Tukey’s test.

Fig. 6. AGLIP1-siRNA from rice reduced growth rate of Rhizoctonia solani AG1-IA. A, Length distribution and abundance of AGLIP1-specific siRNAs in T2-AGLIP1-Line 9. B, Relative expression levels of ALIP1 gene in R. solani-isolated rice plants. C, Colony diameter of re-isolated GD-118 from infected rice leaves. WT, Wild type; L7, T2-AGLIP1-Line 7 transgenic plant; L9, T2-AGLIP1-Line 9 transgenic plant. The data are presented as Mean ± SE (n = 3). Statistical significance is denoted as P < 0.05 (*), P < 0.01 (**), or P < 0.001 (***), respectively, one-way analysis of variance with Tukey’s test.

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