Suppressing chlorophyll degradation by silencing OsNYC3 improves rice resistance to Rhizoctonia solani, the causal agent of sheath blight

Abstract

Necrotrophic fungus Rhizoctonia solani Kühn (R. solani) causes serious diseases in many crops worldwide, including rice and maize sheath blight (ShB). Crop resistance to the fungus is a quantitative trait and resistance mechanism remains largely unknown, severely hindering the progress on developing resistant varieties. In this study, we found that resistant variety YSBR1 has apparently stronger ability to suppress the expansion of R. solani than susceptible Lemont in both field and growth chamber conditions. Comparison of transcriptomic profiles shows that the photosynthetic system including chlorophyll biosynthesis is highly suppressed by R. solani in Lemont but weakly in YSBR1. YSBR1 shows higher chlorophyll content than that of Lemont, and inducing chlorophyll degradation by dark treatment significantly reduces its resistance. Furthermore, three rice mutants and one maize mutant that carry impaired chlorophyll biosynthesis all display enhanced susceptibility to R. solani. Overexpression of OsNYC3, a chlorophyll degradation gene apparently induced expression by R. solani infection, significantly enhanced ShB susceptibility in a high-yield ShB-susceptible variety '9522'. However, silencing its transcription apparently improves ShB resistance without compromising agronomic traits or yield in field tests. Interestingly, altering chlorophyll content does not affect rice resistance to blight and blast diseases, caused by biotrophic and hemi-biotrophic pathogens, respectively. Our study reveals that chlorophyll plays an important role in ShB resistance and suppressing chlorophyll degradation induced by R. solani infection apparently improves rice ShB resistance. This discovery provides a novel target for developing resistant crop to necrotrophic fungus R. solani.

Keywords: chlorophyll; maize (Zea mays); resistant breeding; rice (Oryza sativa); sheath blight; transcriptomics.

Figure 1

YSBR1 shows high resistance to sheath blight (ShB) with a strong ability to suppress Rhizoctonia solani (R. solani) infection. (a) Evaluation of YSBR1 and Lemont in resistance to ShB disease in a field. Artificial inoculation was performed at booting stage for both varieties and ShB disease scores were rated at 30 days after heading. Photographs were taken at 30 days after heading. Values are means±s.d. Scale bar, 10 cm. (b) Lesion lengths and areas of adult plants were measured at different days post inoculation (DPI) in the growth chamber. Values are means ± SD. (c) Scanning electron microscopy (SEM) of R. solani hyphal behaviour on the surface of leaf sheaths of Lemont and YSBR1 at 24, 60 and 72 h after inoculation (HAI). 8‐week‐old plants grown in natural conditions were inoculated. The inoculated sheaths were collected at the indicated time points. Infection cushions are labelled with yellow arrows. All data are presented as mean ± SD, *, P < 0.05, **, P < 0.01 using Student’s t‐tests.

Figure 2

Resistant responses were activated by Rhizoctonia solani (R. solani) invasion as revealed by transcriptomics. (a) Numbers of R. solani‐ and circadian‐responsive genes detected in each comparison. (b) Hierarchical clustering analysis of 2632 differentially expressed genes (DEGs) in response to R. solani. DEGs were identified using |Log2‐Fold change (FC)| ≥0.75 and q‐value <0.1 as the cut‐offs. (c) Four‐way Venn diagram showing the number of common and unique R. solani‐responsive genes among four comparisons. (d) Comparison of YSBR1 and Lemont DEGs involved in pathogenesis‐related proteins (PRs) and xylanase inhibitor proteins (XIPs). (d) Comparison of YSBR1 and Lemont DEGs involved in pathogenesis‐related proteins (PRs) and xylanase inhibitor proteins (XIPs). (e) R. solani infection affects the hormone signal in both varieties. The DAVID (https://david.ncifcrf.gov/) and KEGG (https://www.kegg.jp/kegg/tool/) websites were used for gene annotation The scale bars display the Log2‐FC, shown in red when >0.75, in blue when <−0.75, and in white when −0.75 ~ 0.75.

Figure 3

Rhizoctonia solani triggered susceptible responses through the repression of chlorophyll and ROS metabolism. (a,b) Comparison of YSBR1 and Lemont DEGs involved in chlorophyll (a) and ROS metabolism (b). The scale bars display the Log2‐FC, shown in red when >0.75, in blue when <−0.75, and in white when −0.75 ~ 0.75. (c,d) Detection of hydrogen peroxide (H₂O₂) accumulation by 3,3′‐diaminobenzidine (DAB) staining (c) and cell death by trypan blue (TB) (d) in infected epidermal cells of leaf sheath at 20 HAI. Plants grown in natural conditions were inoculated at tilling stage. Scale bar, 20 μm in (c); 1 mm in (d).

Figure 4

Dark treatment reduces YSBR1 resistance to Rhizoctonia solani (R. solani). (a) Changes in transcript levels of chlorophyll biosynthesis (CHLM, CAO1) and degradation (SGR1, NYC3) genes in YSBR1 untreated (NYS) and treated (DYS) with darkness. All qPCR data were normalized to ubiquitin (Ubq) and were presented as mean ± SD. The experiment was repeated three times with similar trends among each other. (b,c) The whole plants (b) and the detached leaves (c) were used for inoculation, respectively. DYS: dark‐treated YSBR1; NYS: normal YSBR1; NLE: normal Lemont. Disease symptoms were investigated at 6 DPI. Scale bar, 10 cm in (b); 1 cm in (c). Values are means ± SD.

Figure 5

Rice and maize mutants with defects in chlorophyll biosynthesis were impaired in resistance to Rhizoctonia solani (R. solani). (a) Comparison of three rice mutants (ygl8, dvr and cao1) with abolished chlorophyll biosynthesis with wild type on disease symptoms post R. solani inoculation in detached leaf (upper) and whole plant (bottom). Pictures of inoculated leaves were taken at 3 and 6 DPI, which were used to calculate the lesion areas (upper), scale bar, 1 cm; pictures of inoculated whole plants were taken at 8 DPI, and the lesion lengths were measured at 4 and 8 DPI (bottom), scale bar, 10 cm. (b) DAB and TB staining of the three mutants and wild type at 3 DPI. Scale bar, 1 cm in left; 100 μm in right. (c) Comparison of a maize chlorophyll biosynthesis mutant ygl‐1 with wild type on disease symptoms post R. solani inoculation. Scale bar, 10 cm. All data are presented as mean ± SD, *, P < 0.05, **, P < 0.01 using Student’s t‐tests.

Figure 6

The effect of NYC3, a regulator of chlorophyll degradation, on rice ShB resistance and yield‐related traits. (a) NYC3 is more significantly induced by Rhizoctonia solani (R. solani) in variety 9522 than in YSBR1. Data are shown as means ± SD. The experiment was repeated three times with similar trends among each other. (b,c) NYC3‐RI and NYC3‐OX alter rice resistance to R. solani in detached leaf (b) and whole plant (c) inoculations. All transgenic lines are in the 9522 variety. Images were taken and data collected at 6 and 8 DPI, respectively. Scale bar, 2 cm in (b); 5 cm in (c). (d) DAB and TB staining for H₂O₂ accumulation and cell death of NYC3‐RI, NYC3‐OX and wild type 9522 at 3 DPI. Scale bar, 1 cm in left, 100 μm in right. (e–h) Effects of NYC3 silencing on the yield‐related agronomic traits. Pictures displayed the morphology of whole plant (e), grain length and width (f). (g) shown the 1000 grain weight and yield per plant, (h) shown the yield test. Scale bar, 10 cm in (e); 1 cm in (f). All data are presented as mean ± SEM; *P < 0.05; **P < 0.01; ns, not significant using Student’s t‐tests.