Results

Overview

We’ve done the experimental part in our program, although we’ve failed in some process of experiments. We constructed engineered E. coli biofactories that utilize E. coli HT115 to produce dsRNA, and crops inoculated with dsRNA showed significant resistance to disease when they encountered infestation with the Rhizoctonia solani, which is highly detrimental to agricultural production. When we interviewed Dr. Tan, he cautioned us to be patient with our experiments, to concentrate, and to treat each experiment fairly and equally, and that is exactly how we conducted our experiments.

Construction of Expression Vectors

We synthesized a sequence of the catalase gene RsCAT on a cloning vector at Genscript Biotech Corporation, 310bp, and we need to amplify the fragment to ligate it into the expression vector L4440.

DNA Amplification-PCR

We transferred the synthesized original plasmid containing this sequence of RsCAT into E. coli TOP10, preserved the bacteria and extracted the new plasmid to be used as an amplification template for PCR.

Fig. 1. PCR amplification of the 300 bp DNA sequence.

Fig. 1A shows the bacteria containing RsCAT template plasmid. Fig. 1B illustrates the agarose gel electrophoresis experiment conducted after our initial PCR amplification. The purpose of this experiment was to ascertain whether our gene of interest was successfully amplified. In the gel image, there is a faint signal emanating from the region around 300bp on the right side. This suggests the presence of the target gene, albeit with minimal amplification, indicating only a small degree of amplification was achieved. We conducted a second attempt, lowering the annealing temperature to 55°C while maintaining all other experimental conditions constant. As showed in Fig. 1C, the target area appeared even more faint than in the previous attempt. In our third attempt, we switched the PCR primers, as shown in Fig. 1D. This change proved successful, as the target band was amplified, suggesting that the initial primer set was suboptimal for efficient amplification.

Restriction Endonuclease Digestion

We used the restriction enzymes EcoRI and BamHI to digest the vector L4440 and the 300bp PCR product. Subsequently, agarose gel electrophoresis was performed on the digested products, followed by a further gel purification of the fragments (Fig. 2&3).

Fig. 2. L4440 blank plasmid and RsCAT gene fragments' BamH1-EcoR1 double digestion. M: Marker；1: Plasmid L4440 BamH1-EcoR1 double digestion；2: RsCAT gene fragments' BamH1-EcoR1 double digestion — Fig. 2. L4440 blank plasmid and RsCAT gene fragments’ BamH1-EcoR1 double digestion. M: Marker；1: Plasmid L4440 BamH1-EcoR1 double digestion；2: RsCAT gene fragments’ BamH1-EcoR1 double digestion

Fig. 3. The purified product is the result of restriction endonuclease digestion the vector L4440 and the 300 bp fragment.

DNA Ligation and Transformation

The purified enzyme-digested fragments were subsequently subjected to a DNA ligation reaction utilizing T4 DNA ligase, as detailed in the laboratory notebook. The ligated DNA was subsequently transformed into E.coli Top10 competent cells, and upon incubation on culture plates, a few colonies were observed (Fig. 4).

Fig. 4. The ligation product was transformed into E. coli TOP10 competent cells and coated with bacterial culture medium for overnight culture.

Colony PCR Identification and Sequencing

Conducted colony PCR identification, as depicted in the Fig. 5, the PCR amplification revealed the presence of the target band (Fig. 5). Selected three colones for shaking culture, extracted plasmids, and submitted for enzyme digestion verification and sequencing. The results of electrophoresis gel image (Fig. 6) and sequence alignment (Fig. 7B) confirmed the successful integration of the target fragment into the expression vector L4440.

Fig. 5. Colony PCR identification of TOP10 cells. M: Marker, 1-4: Four distinct bacterial colonies

Fig. 6. Recombine RsCAT-L4440 recombinate plasmids' BamH1-EcoR1 double digestion validation. M: Marker；1-3: enzyme digestional validation of BamHI-EcoRI recombinate plasmid from three disinct single bacterial colonies. — Fig. 6. Recombine RsCAT-L4440 recombinate plasmids’ BamH1-EcoR1 double digestion validation. M: Marker；1-3: enzyme digestional validation of BamHI-EcoRI recombinate plasmid from three disinct single bacterial colonies.

Fig. 7. Gene sequence testing. (A) Sequencing peak plot. (B) Sequence alignment results.

Expression of dsRNA in Engineered Bacteria

Obtained HT115 (DE3)-expressing Bacteria

Transferred the sequencing-verified plasmids from Top 10 into the expression strain HT115 (DE3), and conducted PCR validation on four colonies to ensure that the bacteria for subsequent induction and expression were those harboring the correct expression vector (Fig. 8).

DsRNA Extraction from E.coli HT115

After IPTG induction, the bacterial precipitate was collected. We extracted dsRNA using alcohol precipitation method and verified it by agarose gel electrophoresis and micro-spectrophotometer (Fig. 9).

Fig. 9 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.

Culture of Rhizoctonia Solani

Fig. 10A is the original fungal plate, we’ve removed a small fragment of rhizoctonia solan into a new petri dish. Fig. 10B is the removal fragment after 2 days in incubator, its diameter was about 3.5~4 cm long.

Co-culture of dsRNA with Rhizoctonia Solani

This process was managed to check out if RsCAT-dsRNA affects the development of Rhizoctonia solani. We’ve prepared two petri dishes, one of them was smeard by RsCAT-dsRNA, the another was smeared by pure water. Two fragements of cultured Rhizoctonia solani was prepared, and each fragment was innoculated into each petri dish. Here is the fungal colonies after five days (Fig. 11A). In addition, we weighed the mass, and measured the diameters of both two fungal colonies. Results are shown in graphs (Fig. 11B & 11C). Apparently, data proved that RsCAT-dsRNA will not influence the growth of fungal colonies, speed of growing was not effectively influenced by dsRNA (Fig. 11B). As same as diameter, weight change of two samples almost shared no difference (Fig. 11C). We detected the transcription level of the RsCAT gene in mycelium by quantitative PCR and found that the RsCAT gene was obviously silenced in the mycelium with the addition of RsCAT-dsRNA (Fig. 11D).

Fig. 11. Co-culture of dsRNA and Rhizoctonia solani. H2O and RsCAT-dsRNA were evenly spread on the PDA plate, and then inoculated with Rhizoctonia solani and cultured for 5 days. (A) Colony morphology. (B) Colony diameter. (C) Changes in mycelium weight. (D) Expression level of RsCAT gene. The error bars in the Fig. are mean ± s.d., according to t-test, asterisks represent significant differences, ****, P < 0.0001, n.s represent no significant differences, P > 0.05. — Fig. 11. Co-culture of dsRNA and Rhizoctonia solani. H2O and RsCAT-dsRNA were evenly spread on the PDA plate, and then inoculated with Rhizoctonia solani and cultured for 5 days. (A) Colony morphology. (B) Colony diameter. © Changes in mycelium weight. (D) Expression level of RsCAT gene. The error bars in the Fig. are mean ± s.d., according to t-test, asterisks represent significant differences, ****, P < 0.0001, n.s represent no significant differences, P > 0.05.

Tobacco Cultivation

Instead of maize, we cultured some tobacco due to its high survival rate, universality in experiments, and conivience in staining comparing to corn.

Although our product is mainly sprayed on corn, but tobacco leaves could be a suitable substitute in our experiments. We sowed tobacco seeds into the pot, as soon as they developed into the seedlings (Fig. 12A), we separated them into multi-pots. In later time, they developed into adult plants (Fig. 12B). Tobacco shares some similarity with maize, broader leaves of tobacco facilitate the process of observing distinctions on staining leaves, thus we utilized this advantage to simplify DAB staining experiment.

DAB Staining of Tobacco

To validate if catalase expressed by RsCAT is able to obliterate ROS accumulated during fungal invasion to assist fungal infection. Spray 50 ng/μL RsCAT-dsRNA on the tobacco leaves, inoculate Rhizoctonia solani after 24 hours. Fetch the leaves after 17 hours, immerse the leaves in DAB solution in 23 hours, observe the brown stains after the chlorophyll is totally taken off (Fig. 13). The results showed that dsRNA enhanced the accumulation of ROS induced by fungi in plants.

Maize Cultivation

In order to test our product, we should definitely culture some corn. Firstly, we were trying to pre-germinate seeds in petri dish, covered the base with humid tissue (Fig. 14A). In two days, maize seeds started to germinate (Fig. 14B), in later three days, the seeds continued to develop their sprouts (Fig. 14C), that time, it’s ready to transplant the seedlings into a flowerpot.

As the maize was being transplanted into the flowerpot, we left it in an area, waiting for it to grow its leaves. Nine days later, our maize has grown their leaves healthily and successfully (Fig. 14D).

It was able to innoculate fungi or carry out other experiments.

Validation of Functions in Prevention and Treatment of Sheath Blight in Maize

In order to test if our product will effectively influence the infection of sheath blight in maize. Two leaves were inoculated with Rhizoctonia solani, and one leaf was sprayed by pure water, the another was sprayed with RsCAT-dsRNA (Fig. 15).

Fig. 15. Functional verification of RsCAT-dsRNA in preventing and controlling corn sheath blight. First, RsCAT-dsRNA was evenly sprayed on corn leaves, and Rhizoctonia solani was inoculated 24 hours later. The phenotype of the leaves was observed 5 days post infection(dpi).

The leaf sprayed with pure water was dominately infected five days later, the whole leaf was wilt and yellowing. Meanwhile, the sample sprayed by dsRNA was remian at a healthy state, no dominant infected features. The results showed that spraying dsRNA could help corn resist Rhizoctonia solani.