Part:BBa_K5474000

dsRNA targeting CAT gene of Rhizoctonia solani

This is a DNA sequence designed based on the catalase gene (CAT) of Rhizoctonia solani. After being ligated into the multiple cloning site of the L4440 vector, it can produce dsRNA under IPTG induction. This dsRNA targets the CAT gene of Rhizoctonia solani to silence it, thereby achieving the purpose of controlling the pathogen.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Introduction

Our goal is to produce a double-strand RNA (dsRNA) fungicide to prevent corn sheath blight caused by Rhizoctonia solani, which has led to a significant reduction in global corn production(Di et al., 2023). We constructed a part, RsCAT-dsRNA (BBa_K5474000), to achieve this goal. The dsRNA was designed to silence the target gene RsCAT through the RNAi mechanism. Two T7 promoters are located on both sides of the dsRNA encoding sequence in opposite directions, so there is bidirectional transcription, which allows the RNA to form a double-stranded structure.

Design

We designed a prokaryotic expression system for producing dsRNA. Our part (BBa_K5474000) is a 310 bp sequence encoding RsCAT-dsRNA (Fig. 1A). The recombinant expression vector (Fig. 1B) was constructed by T4 ligation technique and then transformed into E. coli HT115 competent cells for large-scale expression of RsCAT-dsRNA.

Fig. 1 Design and assembly of our Part. (A) Model diagram of our BioBrick (ID: BBa_K5474000) (B) Recombinant vectors that make our BioBrick work.

Characterization

1.dsRNA expression and extraction

The expression of dsRNA was detected by agarose gel electrophoresis. 50 mL of bacterial culture was cultured each time and induced for 3 h at 37 ℃ with 0.5 mM IPTG. The bacterial precipitate was collected and dsRNA was extracted by alcohol precipitation method. The product was identified by agarose gel electrophoresis (Fig. 2A) and its concentration was determined by micro-spectrophotometer (Fig. 2B). Then the dsRNA was diluted to 50 ng/μL for subsequent functional verification experiments.

Fig. 2 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 promotes the accumulation of ROS in plants

2.1 Co-culture of Rhizoctonia solani and RsCAT-dsRNA

To determine whether RsCAT-dsRNA affects the growth of Rhizoctonia solani, we designed a co-culture experiment. Spread water and dsRNA on two PDA media respectively, and then inoculate the same size Rhizoctonia solani block in the center of the plate and culture it for five days (Fig. 3A). By measuring colony diameter and mass changes, we found that dsRNA did not affect the growth of Rhizoctonia solani (Fig. 3B & 3C). However, the results of quantitative PCR experiments showed that dsRNA significantly reduced the expression level of RsCAT (Fig. 3D).

Fig. 3 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.

2.2 DAB staining of tobacco leaves

Plants produce ROS signals and enhance their immune response when facing fungal infection(Zhang et al., 2023). In order to verify whether the downregulation of RsCAT gene expression induced by dsRNA would affect the accumulation of ROS in plants, we conducted a DAB staining experiment on tobacco leaves. First, we sprayed water and dsRNA on two leaves respectively, dried them in the shade for 24 hours, and then inoculated them with Rhizoctonia solani. The leaves were then cut off and DAB stained (Fig. 4). The results showed that dsRNA can promote the accumulation of ROS in leaves.

Fig. 4 DAB staining of tobacco leaves. Brown color indicates hydrogen peroxide accumulation in the leaves.

3. dsRNA can be used to prevent the Sheath Blight

To further verify the efficacy of dsRNA in controlling corn sheath blight, we applied water and dsRNA to corn leaves respectively and inoculated Rhizoctonia solani 24 hours later. Five days later, we found that the leaves in the dsRNA-treated group had a normal phenotype, while the leaves in the control group showed a wilt and yellow disease phenotype (Fig. 5), indicating that dsRNA is beneficial for controlling corn sheath blight.

Fig. 5 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.

Conclusion

The above results show that we have successfully constructed a prokaryotic expression system for dsRNA and obtained dsRNA products by inducing expression through IPTG. In addition, we conducted a functional verification experiment of dsRNA to control Rhizoctonia solani. The results showed that dsRNA promoted the accumulation of ROS in plants by silencing RsCAT, thereby helping corn resist sheath blight.

References

[1] Di, R., Liu, L., Shoaib, N., et al. (2023). Sheath Blight of Maize: An Overview and Prospects for Future Research Directions. Agriculture, 13(10), 2006. [2] Zhang, N., Lv, F., Qiu, F., et al. (2023). Pathogenic fungi neutralize plant-derived ROS via Srpk1 deacetylation. Embo j, 42(9), e112634.