Engineering

Background

Corn sheath blight caused by Rhizoctonia solani is one of the most destructive fungal diseases, causing serious losses to corn grain production(Hooda et al., 2017). Although traditional chemical fungicides can effectively resist sheath blight, they also bring problems such as fungicide residues, environmental pollution and drug resistance. In order to solve these problems, double stranded RNA (dsRNA)-based fungicides have been developed. By spraying dsRNA on the surface of plants to activate the RNAi mechanism in fungi, the relevant pathogenic genes are silenced, that is, the spray-induced gene silencing (SIGS) method(Wang & Jin, 2017), and finally the effect of preventing sheath blight is achieved. The catalase encoded by RsCAT scavenges hydrogen peroxide, a type of reactive oxygen species (ROS), thereby reducing the plant’s immune response and facilitating infection by Rhizoctonia solani. Here, we designed a dsRNA sequence that can target and silence RsCAT, constructed its recombinant expression vector, and transformed E. coli HT115(DE3) to express the target dsRNA, namely RsCAT-dsRNA. Through a series of functional validation experiments, we found that RsCAT-dsRNA has the potential to prevent and treat corn sheath blight.

Design

The above picture shows the workflow of our project (Fig. 1). We designed a prokaryotic expression system to express dsRNA. Our part (BBa_K5474000) is a 300 bp sequence encoding RsCAT-dsRNA (Fig. 2A). The target gene fragment expressing dsRNA was obtained by PCR amplification and ligated to plasmid L4440 using T4 DNA ligase to generate a recombinant plasmid (Fig. 2B). L4440 contains two T7 promoters in opposite directions, and the gene fragments inserted into them can be transcribed to form dsRNA. E. coli HT115 (DE3) is an ideal expression strain that produces T7 RNA polymerase under IPTG (isopropyl β-D-1-thiogalactopyranoside)-inducing conditions to initiate transcription of dsRNAs, and it is an RNase III-deficient strain whose expressed dsRNAs are less susceptible to degradation. We chose E. coli HT115 (DE3) as the chassis cell.

Fig. 2 Plasmid design. (A) Constitution of RsCAT-dsRNA gene circuits. (B) Fusion plasmid RsCAT-dsRNA-L4440 construction strategy map.

Build

First, we amplified the target gene fragment with restriction enzyme sites at both ends by PCR (Fig. 3A). Then, we performed double restriction enzyme digestion reaction on L4440 and target gene fragment (Fig. 3B), connected the two products with T4 DNA ligase, and transformed them into E. coli TOP10 competent cells. Finally, the positive clones were identified by colony PCR (Fig. 3C), and the correct plasmid was extracted and sent for sequencing. Sequence alignment was performed using DNAMAN software, and the results showed that the RsCAT-dsRNA-L4440 recombinant vector was successfully constructed (Fig. 3D).

Fig. 3 Construction of fusion plasmid RsCAT-dsRNA-L4440. (A) Gene fragment of interest for RsCAT-dsRNA was amplified by PCR. M: DNA Marker; 1: PCR results of positive clone; Product size: 349 bp. (B) Identification of double enzyme digestion products. M: Marker; 1: Enzyme digestion product of empty plasmid L4440; 2: Enzyme digestion products of target gene. C Validation of Colony PCR. M: DNA Marker; 1 to 4: PCR results of positive clone. (D) Sequence alignment between RsCAT-dsRNA and positive clone sequencing (Drawing software: DNAMAN). — Fig. 3 Construction of fusion plasmid RsCAT-dsRNA-L4440. (A) Gene fragment of interest for RsCAT-dsRNA was amplified by PCR. M: DNA Marker; 1: PCR results of positive clone; Product size: 349 bp. (B) Identification of double enzyme digestion products. M: Marker; 1: Enzyme digestion product of empty plasmid L4440; 2: Enzyme digestion products of target gene. (C) Validation of Colony PCR. M: DNA Marker; 1 to 4: PCR results of positive clone. (D) Sequence alignment between RsCAT-dsRNA and positive clone sequencing (Drawing software: DNAMAN).

Test

1. Expression and extraction of RsCAT-dsRNA

The RsCAT-dsRNA-L4440 vector was transformed into HT115 (DE3) competent cells. Expression of dsRNA was induced with 0.5 mM IPTG and extracted using an ethanol method. The extracted dsRNA was analyzed by agarose gel electrophoresis (Fig. 4A) and its concentration was measured using a nanodrop (Fig. 4B). The concentration of the dsRNA is 1155.4 ng/μL.

Fig. 4 Identification of prokaryotic expressed dsRNA extracted by alcohol precipitation method. (A) Identification of dsRNA by agarose gel electrophoresis. M: Marker; 1: RsCAT-dsRNA. (B) dsRNA concentration was measured by micro-spectrophotometer.

2. RsCAT-dsRNA reduces the transcription level of RsCAT

To determine how dsRNA affects Rhizoctonia solani, we performed co-culture experiments. Rhizoctonia solani was inoculated on the PDA medium evenly coated with dsRNA (Fig. 5A). It was found that compared with the control group, there was no significant difference in the changes in mycelium diameter (Fig. 5B) and weight (Fig. 5C), but the transcription level of RsCAT was significantly reduced (Fig. 5D). The results showed that dsRNA did not affect the growth of Rhizoctonia solani, but significantly silenced RsCAT.

Fig. 5 Co-culture of RsCAT-dsRNA and Rhizoctonia solani. (A) Colony morphology. (B) Colony diameter. (C) Changes in mycelium weight. (D) Relative expression level of RsCAT gene in mycelium by qPCR. The experiment was performed with at least three biological replicates. The error bars in the figure are mean±s.d., according to t-test, asterisks represent significant differences, ****, P < 0.0001; n.s represent no significant differences, P > 0.05.

3. RsCAT-dsRNA promotes ROS accumulation in tobacco leaves

During fungal infections, plants generate ROS and activate their innate immune signaling pathways for defense (Zhang et al., 2020). To investigate the impact of RsCAT expression on the accumulation of ROS within plants, Nicotiana benthamiana was employed as the experimental material. dsRNA was sprayed onto Nicotiana benthamiana leaves, which were then allowed to air-dry before inoculation with Rhizoctonia solani. Subsequently, the leaves were stained with 3,3’-diaminobenzidine (DAB), a histochemical reagent that, in the presence of hydrogen peroxide, undergoes oxidation to form a brown, insoluble product. This reaction enables the visualization of hydrogen peroxide in plant tissues. Our observations revealed that leaves treated with dsRNA exhibited a larger area of brown staining compared to the control group (Fig. 6), suggesting that dsRNA enhances the accumulation of ROS induced by fungi within the plant.

Fig. 6 DAB staining of tobacco leaves. Brown color indicates hydrogen peroxide accumulation in the leaves.

4. RsCAT-dsRNA helps corn resist Rhizoctonia solani

To assess the efficacy of RsCAT-dsRNA in controlling sheath blight, dsRNA was initially sprayed onto maize leaves and then inoculated them with rhizobia 24 hours later. Five days post-inoculation, the leaf phenotypes were recorded. The results revealed that the dsRNA-treated leaves exhibited a normal phenotype, whereas the control group (inoculated with water) displayed symptoms of wilting and yellowing (Fig. 7), indicating that spraying dsRNA can aid maize in resisting sheath blight.

Fig. 7 Functional verification of RsCAT-dsRNA in controlling corn sheath blight. The phenotype of wilting and yellowing of leaves may indicate infection with Rhizoctonia solani. Dpi represent days post infection.

Learn

We developed a prokaryotic expression system for RsCAT-dsRNA. Following induction with IPTG, bacterial cells were harvested and dsRNA was extracted. A series of experiments demonstrated that the dsRNA silenced RsCAT, leading to a reduction in hydrogen peroxide levels, thereby promoting the accumulation of reactive oxygen species (ROS) in the host, which significantly enhanced the resistance of corn to diseases. This represents a viable antifungal strategy. However, it is well-known that dsRNA is unstable and prone to degradation. To address this issue, we plan to incorporate nanomaterials, such as layered double hydroxide (LDH), into future dsRNA applications to enhance its stability and prolong its action time (Chen et al., 2019).

References

[1] Chen, Z., He, J., Luo, P., et al. (2019). Production of functional double-stranded RNA using a prokaryotic expression system in Escherichia coli. MicrobiologyOpen, 8(7), e00787.

[2] Hooda, K., Khokhar, M., Parmar, H., et al. (2017). Banded leaf and sheath blight of maize: historical perspectives, current status and future directions. Proceedings of the National Academy of Sciences, India Section B: Biological Sciences, 87, 1041-1052.

[3] Wang, M., & Jin, H. (2017). Spray-Induced Gene Silencing: a Powerful Innovative Strategy for Crop Protection. Trends Microbiol, 25(1), 4-6.

[4] Zhang, Z., Chen, Y., Li, B., et al. (2020). Reactive oxygen species: A generalist in regulating development and pathogenicity of phytopathogenic fungi. Comput Struct Biotechnol J, 18, 3344-3349.