Spray-induced gene silencing for disease control is dependent on the efficiency of pathogen RNA uptake

Abstract

Recent discoveries show that fungi can take up environmental RNA, which can then silence fungal genes through environmental RNA interference. This discovery prompted the development of Spray-Induced Gene Silencing (SIGS) for plant disease management. In this study, we aimed to determine the efficacy of SIGS across a variety of eukaryotic microbes. We first examined the efficiency of RNA uptake in multiple pathogenic and non-pathogenic fungi, and an oomycete pathogen. We observed efficient double-stranded RNA (dsRNA) uptake in the fungal plant pathogens Botrytis cinerea, Sclerotinia sclerotiorum, Rhizoctonia solani, Aspergillus niger and Verticillium dahliae, but no uptake in Colletotrichum gloeosporioides, and weak uptake in a beneficial fungus, Trichoderma virens. For the oomycete plant pathogen, Phytophthora infestans, RNA uptake was limited and varied across different cell types and developmental stages. Topical application of dsRNA targeting virulence-related genes in pathogens with high RNA uptake efficiency significantly inhibited plant disease symptoms, whereas the application of dsRNA in pathogens with low RNA uptake efficiency did not suppress infection. Our results have revealed that dsRNA uptake efficiencies vary across eukaryotic microbe species and cell types. The success of SIGS for plant disease management can largely be determined by the pathogen's RNA uptake efficiency.

Keywords: RNA interference; double-stranded RNA; small RNA; spray-induced gene silencing; uptake efficiency.

Figure 1

Examination of dsRNA uptake efficiencies in multiple fungi using fluorescein‐labelled YFP‐dsRNA. (a, b) Fluorescein‐labelled YFP‐dsRNA was added to the spores of Botrytis cinerea, Verticillium dahliae, Aspergillus niger, C. gloeosporoides and Trichoderma virens, and to the hyphae of Sclerotinia sclerotiorum and Rhizoctonia solani. Micrococcal nuclease (MNase) treatment was performed 30 min before acquiring images using the confocal microscopy laser scanner (CMLS). Fluorescence signals were detected inside of B. cinerea, S. sclerotiorum, R. solani, A. niger and V. dahliae cells, but not C. gloeosporoides cells. A weak signal was observed in T. virens cells. Pictures were taken at 10 hours post‐treatment (hpt) for B. cinerea, S. sclerotiorum, and R. solani, A. niger and V. dahliae, and at 24 hpt for C. gloeosporoides and T. virens. Scale bars = 20 µm except for B. cinerea image (Scale bars = 10 µm). (c) C. gloeosporioides spores are unable to take up dsRNA. Fluorescence signals were only visible on the outer surface of C. gloeosporioides cells before MNase treatment, but disappeared after MNase treatment. Scale bars = 15 µm.

Figure 2

Topical application of pathogen gene‐targeting dsRNAs inhibited the virulence of B. cinerea and S. sclerotiorum. (a) Tomato fruits, lettuce leaves, rose petals and grape fruits were inoculated with B. cinerea spores after treating with controls (water or YFP‐dsRNA) or Bc‐VPS51+DCTN1+SAC1‐dsRNA or Bc‐DCL1/2‐dsRNA (20 ng/µL). (b) The relative lesion sizes were measured 3 days post‐inoculation (dpi) on lettuce leaves, rose petals and 5 dpi on grapes and tomato fruits using ImageJ software. Error bars indicate the SD of 10 samples and three biological repeats were conducted for the relative lesion sizes. Statistical significance (Student’s t‐test): **, P < 0.01. (c) qRT‐PCR analysis of Bc‐VPS51, Bc‐DCTN1 and Bc‐SAC1 expression in Bc‐VPS51+DCTN1+SAC1‐dsRNA treated B. cinerea cells. The expression levels of target genes were normalized to expression of B. cinerea Actin. Error bars represent the SD from three technical replicates. Statistical significance (Student’s t‐test): *, P < 0.05; **, P < 0.01 between the treatment and the water control. (d) Lettuce and collard green leaves were inoculated with S. sclerotiorum mycelium plugs after treating with controls (water or YFP‐dsRNA) or Ss‐VPS51+DCTN1+SAC1‐ and Ss‐DCL1/2‐dsRNA (40 ng/µL). Pictures were taken at 3 dpi. (e) The relative lesion sizes were measured at 3 dpi for lettuce and collard greens using ImageJ software. Error bars indicate the standard deviations (SD) of 10 samples. Asterisks (**) indicate statistically significant differences (P < 0.01, Student’s t‐test) between the treatment and the water control. (f) The mRNA expression levels of SsVPS51, SsDCTN1 and SsSAC1 were detected in Ss‐VPS51+DCTN1+SAC1‐dsRNA treated S. sclerotiorum by qRT‐PCR analysis. The expression levels of target genes were normalized to expression of S. sclerotiorum Actin. Asterisks (**) indicate statistically significant differences (P < 0.01, Student’s t‐test) between the treatment and the water control. Similar results were observed from three biological replicates in (c) and (f).

Figure 3

Topical application of pathogen gene‐targeting dsRNAs inhibited Aspergillus niger virulence. (a) Tomatoes, apples and grapes were inoculated with A. niger spores after treating with controls (water or YFP‐dsRNA), AnpgxB‐ or An‐VPS51+DCTN1+SAC1‐dsRNA (20 ng/µL). Pictures were taken at 5 dpi. (b) The relative lesion sizes were measured at 5 dpi using ImageJ software. Error bars indicate the SD of 10 samples. (c) The mRNA expression levels of AnVPS51, AnDCTN1, AnSAC1 and AnpgxB were detected in An‐VPS51+DCTN1+SAC1‐ or AnpgxB‐dsRNA treated A. niger. The expression levels of target genes were normalized to expression of A. niger ActinA. Error bars indicate the SD of three technical replicates. Statistical significance (Student’s t‐test) between the treatment and the water control: *, P < 0.05; **, P < 0.01. Similar results were observed from three biological replicates.

Figure 4

Topical application of pathogen gene‐targeting dsRNAs inhibited Rhizoctonia solani virulence. (a) Rice leaves were inoculated with R. solani mycelium plugs after treating with controls (water and YFP‐dsRNA), Rs‐DCTN1+SAC1‐dsRNA or Rs‐PG‐dsRNA (40 ng/µL). Pictures were taken at 3 dpi. (b) Relative biomass of R. solani was calculated by examining the expression of Rs‐Actin by qRT‐PCR, which is normalized to Os18S rRNA; error bars represent the SD of three replicates. Asterisks (**) indicate statistically significant differences (P < 0.01, Student’s t‐test) between the treatment and the water control. (c) The mRNA expression levels of RsDCTN1, RsSAC1, and Rs‐PG were detected in Rs‐DCTN1+SAC1‐ or Rs‐PG‐dsRNA treated R. solani by qRT‐PCR analysis. The expression levels of target genes were normalized to expression of R. solani Actin 1. Asterisks (**) indicate statistically significant differences (P < 0.01, Student’s t‐test). Similar results were observed from three biological replicates.

Figure 5

RNA is not stable in the soil and pretreatment of roots with pathogen gene‐targeting dsRNA reduced the infection of Verticillium dahliae. (a) Northern blot analysis of Vd‐DCL1/2 dsRNAs was performed over a time course after they were mixed with soil. (b) Arabidopsis plants were uprooted and incubated in V. dahliae spore suspensions (10⁶ spores/mL) with controls (water and YFP‐dsRNA), Vd‐DCL1/2‐dsRNA, and Vd‐DCTN1+SAC1‐dsRNA (40 ng/µL) treatments. Pictures were taken at 14 dpi. (c) Relative biomass of V. dahliae was calculated by examining the expression of Vd‐actin by qRT‐PCR, which was normalized to At‐actin2; error bars represent the SD of three replicates. Asterisks (**) indicate statistically significant differences (P < 0.01, Student’s t‐test) between the treatment and the water control. (d) The mRNA expression levels of VdDCL1 and VdDCL2 were detected in Vd‐DCL1/2‐dsRNA treated V. dahliae, and the mRNA expression levels of VdDCTN1 and VdSAC1 were detected in Vd‐DCTN1+VdSAC1‐dsRNA treated V. dahliae by qRT‐PCR analysis. The expression levels of target genes were normalized to expression of Vd‐  Actin. Asterisks (**) indicate statistically significant differences (P < 0.01, Student’s t‐test) between the treatment and the water control. Similar results were observed from three biological replicates.

Figure 6

Different cell types of Phytophthora infestans take up external fluorescein‐labelled YFP‐dsRNA with different efficiencies. (a) Weak fluorescent signals were observed in P. infestans hyphae from plug‐inoculated cultures treated with fluorescein‐labelled YFP‐dsRNA, whereas no signal was observed in germinated cysts (hyphae germinated from zoospores) treated with fluorescein‐labelled YFP‐dsRNA after culturing on rye agar medium for 10 h. Scale bars = 20 µm. A weak fluorescence signal was observed in P. infestans sporangia (b) and zoospore cysts (c) treated with fluorescein‐labelled YFP‐dsRNA for 10 h after culturing on rye agar medium. Scale bars = 10 µm.

Figure 7

Examining the longevity of dsRNA‐mediated plant protection. (a) Tomato leaves were inoculated with Botrytis cinerea spores after spraying with controls (water or YFP‐dsRNA) or Bc‐VPS51+DCTN1+SAC1‐dsRNA or Bc‐DCL1/2‐dsRNA (100 ng/µL) at 1, 3, 7 and 14 dpt. The relative lesion sizes were measured 4 dpi (d). Error bars indicate the SD of at least three independent biological replicates with total of 45 leaflets in each replicate, and the statistical significance (Student’s t‐test) by *, P < 0.05; ***, P < 0.001 between the treatment and the water control.