Overview

To deal with rice sheath blight，SZU iGEMer developed a comprehensive prevention and cure strategy based on synthetic biology. Our modules cover the early detection of R.solani, genetic engineering of the biocontrol fungus Trichoderma, RNAi therapy, and so on. In order to verify our design, in addition to completing basic molecular biology experiments for verification (see Results page), we also conducted a variety of different confirmatory experiments to support our design as below.

Prevention
Trichoderma atroviride

In our project, we want to use our engineered T.atroviride(T.a) for the prevention of rice infection by R.solani, from preventing the spread of the sclerotia of R.solani at interfaces to making the modified T.atroviride more efficient in killing R. solani. Therefore, we plan to perform three proofs of concept for our engineered T.atroviride: for Epl 1, we will test whether its altered hydrophobicity can better block the spread of the sclerotia. For Prb 1, we will test its enzymatic activity and compare the inhibition ability to R.solani of the engineered T.a with that of the wild-type. For Snakin 1, we will compare the inhibition ability of the engineered T.a with that of the wild type.

1.Epl 1

Epl 1, a hydrophobic protein, was overexpressed to make trichoderma more hydrophobic and better able to block the nucleus of R.solani. We planned to perforate wild-type Trichoderma and engineered Trichoderma grown on cellophane for 4 days with a 5mm punch and remove four pieces from each and place them around the rice stalk to record the ability to block R.solani over two days. However, our proof of concept of it is incomplete due to the time and the failure of the proposed supernate to grow in the second selection. We will continue to delve into its related functional features after the competition.

2.Prb 1

2.1 Enzyme activity test

Prb 1 protein is a kind of serine protease, so we first test the enzyme activity of wild-type T.a (WT) and engineered T.a. Our Trichoderma were grown in Mini medium coupled with the R.solani cell wall for 2 days and the supernate was used to test the enzyme activity. We used AKP Activity Assay Kit to measure the Prb 1 activity. Control is the enzyme activity of wild-type T.a lysate. One unit of enzyme activity (1 U) is defined as catalyze to produce 1 umol of Tyrosine per minute per mg of Prb 1 at 40 °C. The enzyme activity of wild-type (control) T.a in supernate is 7.3829 U and for Prb 1 transformant the enzyme activity has been enhanced to 12.6496U.

Figure 1-1 : Enzyme activity of Prb 1.Wild -type represents the Prb 1 enzyme activity of wild-type T.a and it is the control group. Prb 1+ represents the Prb 1 enzyme activity of Prb 1 integrated T.a. Prb 1 activity of wild-type is 7.3829U and 12.6496U for transformed T.a, which means our transformed T.a has a better Prb 1 enzyme activity.

2.2 Inhibition test

Otherwise, we compared the inhibition effect of wild-type Trichoderma and Prb 1 engineered Trichoderma. We conducted a three-day confrontation experiment，and after using the algorithm to calculate the area of the R.soalni and calculating the area according to the following formula:

We got Figure 1-2, the comparison of inhibition rate between wild-type T.a and Prb 1 integrated T.a. According to the graph, the Prb 1 transformant has a higher inhibition rate than wild-type T.a, which means transformant has a better inhibition ability.

Figure 1-2. The inhibition test of wild-type T.a and Prb 1 integreatd T.a. Wild -type represents the inhibition rate of wild-type T.a and Prb 1+ refers to the inhibition rate of Prb 1 integrated T.a.

3. Snakin-1

3.1 Inhibition test

Snakin 1 is a potato derived cysteine-rich antimicrobial peptide which has a significant inhibition effect on R.solani. We want our T.atroviride express Snakin 1 to increase its ability to kill R.solani, so we compared the inhibition effect of wild type Trichoderma and Snakin 1 engineered Trichoderma.

We also conducted a three-day confrontation experiment. Both R.solani and T.atroviride were grown in PDA for 3 days for activation and were placed in a new PDA through a 5mm hole puncher for a three-day confrontation experiment. After using the algorithm to calculate the area of the R.soalni and calculating the area according to the following formula:

We got Figure 1-3, the comparison of inhibition rate between wild-type T.a and Snakin 1 integrated T.a. The Snakin 1 transformant has an 10% increase in inhibiting R.solani compared with wild-type T.a.

Figure 1-3. The inhibition test of wild-type T.a and Snakin 1 integreatd T.a. Wild -type represents the inhibition rate of wild-type T.a and Snakin 1 + refers to the inhibition rate of Snakin 1 integrated T.a.

Detection
LAMP-LFD test

Implementation of LAMP in LFD for the detection of R. solani

1.DNA crude extraction of isolated rice leaves infected with R.solani

Before LAMP-LFD detection, we collected 10 groups of isolated experimental rice leaves infected with R.solani, extracted DNA from each group of leaves by crude extraction method (Fig. 2-1), weighed the leaves and measured the extracted DNA concentration (Table 2-1).

Figure 2-1. Isolated rice leaves infected with R.solani. The figure shows the five experimental groups with the lightest collected leaf mass among all the experimental groups.

Table 2-1. Weight of collected leaves and DNA concentration of each experimental groups.

2.LAMP-LFD test

We used crude extraction of DNA from isolated rice leaves infected with R.solani, amplified DNA templates with biotin-labeled primers(each amplification product was running in 1.3% agarose gel), incubated the amplification products with probes labeled with fluorescein amidite, then loaded final products onto LFD (Fig. 2-2). For the isolated diseased rice leaves extracted in this experiment, ladders-like bands were found in experimental group CE1 and CE2. No ladders-like bands were found in other experimental groups, which may show rice DNA and primer dimers, and no ladders-like bands were found in the control group (Trichoderma DNA extract) (Fig. 2-2A). However, on lateral flow devices(LFD), all the experimental group showed positive results, and even a control group showed a false positive result (Fig. 2-2B).

Figure 2-2. LAMP-LFD test. CE1~CE10: crude extraction of DNA from isolated rice leaves infected with R.solani. T.a.III: genome DNA of Trichoderma atroviride extracted by easy method III, which is a negative control group. T.a.IV: genome DNA of Trichoderma atroviride extracted by easy method IV, which is a negative control group.

It is speculated that cross contamination of laboratory samples have led to false positives. This can be avoided if researchers bring equipments to the field for testing. If LAMP-LFD test is performed in the laboratory, researchers should pay attention to conducting the experiment in different zones in order to avoid contamination among DNA samples. Final results should be analysis combined with the control group, gel imaging and LFD imaging. For instance, for the DNA of isolated rice leaves extracted this time, both CE1 and CE2 experimental groups showed ladders-like bands, and the LFD showed positive results, indicating that the corresponding rice leaves had been infected with R.solani.

Treatment
RNAi therapy

Spraying experiment

1. Inhibition of R. solani on PDA medium

The shRNA we designed can target and silence genes necessary for the survival of Rhizoctonia solani, so we want to verify whether it can inhibit the growth of Rhizoctonia solani in the PDA medium. Punch holes at the same radius of the same PDA medium full of Rhizoctonia solani with a hole punch with a diameter of 0.5cm, inoculate them to the center of new PDA medium respectively, and at the same time, spray 100uL solution containing 50ug shRNA on the plate. On the third day after spraying, it can be clearly observed that our shRNA reduces the radius of the colony of Rhizoctonia solani (Fig. 3-1), and the effect of spraying shRNA bound with CNT is more obvious (Fig. 3-2). You can click measurement to view more details.

Figure.3-1 Effect of spraying shRNA on the growth of R.solani.

Figure.3-2 Effect of shRNA after spraying bundled CNT on the growth of R.solani.

2. Inhibition of rice leaf infection

2.1 Microscope experiment

2.1.1 Observation on mycelia of Rhizoctonia solani

The hyphae of R.solani with a magnification of 100 times were observed under the optical microscope (Fig. 3-3). After the rice leaves were inoculated with R.solani for infection, the invasion of Rhizoctonia solani hyphae in rice leaves was also observed (Fig. 3-4).

Figure 3-3. R.solani hyphae magnified by 100 times.

Figure 3-4. Infection of hyphae in leaves. (a) The mycelium is in the leaf (40×). (b) The mycelium is in the leaf(100×). (c) The mycelium is in the leaf (400×).

2.1.2 shRNA can be absorbed by R.solani

Spraying Cy5 dye labeled shRNA (gfp-shRNA) targeting green fluorescent protein gene on infected leaves and observed under laser confocal microscope. As shown in Figure 3-5, shRNA can effectively enter the mycelia during the process of infection of leaves by mycelia, which provides basic evidence for shRNA silencing in R.solani.

Figure 3-5. Adding GFP shRNA of Cy5 labeled bundled CNT to R.solani on leaves. The white arrows point to the mycelium. Chlorophyll will automatically emit fluorescence.

Scratch micro mycelia on the plate, add the gfp shRNA labeled with Cy5 dye after binding CNT to the mycelia, and observe the distribution of shRNA in the mycelia through laser confocal microscope after treatment. The results showed that shRNA could also effectively enter the cells of Rhizoctonia solani (Fig. 3-6).

Figure 3-6. Adding GFP shRNA of Cy5 labeled bundled CNT to R.solani.

2.1.3 Inhibition of hyphae distribution under microscope

Take the penultimate leaf of rice, set a 10cm long area at the same position of the leaves as the shRNA smear area, and treat each leaf with 100uL of 10ug shRNA solution. Punch holes (3mm in diameter) at the same radius on a plate full of Rhizoctonia solani, and connect the bacterial cake to the center of the shRNA coating area of each leaf.

After the colony is inoculated to the center of the leaf, R.solani will grow on it, and its mycelia will extend from the center to both sides. After a period of time after the infection experiment was carried out, we observed the growth of the mycelium on the rice leaves under the stereo microscope, and obtained the extension length of the mycelium on the leaves under different shRNA treatments (Fig. 3-7).

Figure 3-7. Effect of spraying different shRNA on mycelial elongation without binding CNT.

Similarly, after binding CNT, we also tested the effect of shRNA spraying on hyphal elongation (Fig. 3-8).

Figure 3-8. Effect of spraying different shRNA after binding CNT on mycelial elongation.

The results showed that the inhibition of shRNA on the growth of Rhizoctonia solani was not strong or significant compared with the control group when CNT was not bound; However, compared with the control group and the group without CNT binding, the mycelia bound with CNT and smeared with CNT showed obvious inhibition. To sum up, our CNT promoted the inhibition of shRNA against R.solani.

In addition, in order to better explain the inhibition effect of shRNA on mycelial growth, we made statistics on the length of mycelial extension and put the statistical results on the page measurement.

2.2 Distribution of disease spots

With the increase of infection time, rice leaves will begin to appear disease spots, and the number and area of disease spots will gradually increase (Figure 3-9).

Figure 3-9. Distribution of disease spots on infected rice.

From the distribution of disease spots, spraying siRNA can reduce the incidence of leaves to a certain extent, but the effect is not obvious. The spraying effect of shRNA was better than that of siRNA, but it could not inhibit the infection of Rhizoctonia solani. After spraying shRNA bound with CNT, it can be found that the incidence of leaves has been greatly reduced, indicating that shRNA CNT can well inhibit and kill Rhizoctonia solani.

For the quantification of the lesion area, we also measured the image after analysis.View more information on measurement.

2.3 Detection of inhibition effect by qRT-PCR

2.3.1 Inhibition of siRNA

After the infection experiment was carried out, the leaves sprayed with siRNA region were cut every day. After the RNA was extracted from the leaves, it was reverse transcribed and qRT-PCR was performed to detect the inhibition effect of siRNA on the target gene of the mycelia in the infected leaves (Fig. 3-10).

Figure 3-10. Inhibition effect of siRNA on target gene.

It can be seen from the results that the spraying of siRNA can reduce the target gene of Rhizoctonia solani in leaves, but the effect is still not satisfactory. We speculate that it may be that siRNA is not stable at room temperature, and its exposed ends may be digested by contacting RNase in the air at any time. When we detect its inhibition effect, it is likely that siRNA has degraded most of it. Therefore, we decided to test the inhibition effect of more stable shRNA in spraying experiments.

2.3.2 Inhibition of shRNA

For shRNA, we also verified its inhibition by the same method as for siRNA (Figure 3-11.a, b). At the same time, after binding CNTs, we also tested their effects (Figure 3-11.c, d).

Figure 3-11. Inhibition effect of shRNA on target genes. Target genes: gfp(green fluorescent protein), PG, RPMK1-1, RPMK1-2, C6(RNAPol III subunit C6), Psm1(cohesin complex subunit Psm1), β1(Importin β1). (a) Silencing of target genes by spraying shRNA targeting key genes during infection without binding CNT. (b) Silencing of target genes by spraying shRNA targeting key survival genes of Rhizoctonia solani without CNT binding. (c) Silencing of target genes by spraying shRNA targeting key genes in the infection process after binding CNT. (d) Silencing effect of spraying shRNA targeting key survival genes of Rhizoctonia solani after binding CNT on target genes..

The results showed that when CNT was not bound, the average silencing efficiency of shRNAs targeting key genes in the infection process was about 0.33 compared with the control group, while the average silencing efficiency of shRNAs targeting key genes in the survival of Rhizoctonia solani was about 0.37; However, after the same amount of shRNA was bundled with CNT and sprayed, the expression of target genes was significantly inhibited compared with the control group and the group without CNT, and the average inhibition efficiency of the two types of shRNA was 0.59 and 0.66, respectively. To sum up, our shRNA inhibition effect is good, and binding CNT promotes the inhibition of shRNA against R.solani.

RNAi products testing

In order to ensure that our RNAi products can function for a long time and will not be degraded, it is necessary for us to conduct stability tests on the produced sRNAs. After spraying different sRNAs, let Rhizoctonia solani infect rice, and detect the expression level of rice target genes for 5 consecutive days to test the time when our sRNAs stably play the role of silencing (Figure 3-12).

Figure 3-12. Change of target gene expression level within 5 days after spraying sRNA.

In this test, both siRNA and shRNA target genes are PG. From the results, siRNA and shRNA can achieve the best silencing effect on the third day, but after that, the effect is rapidly weakened. After binding CNT, shRNA silences target genes more obviously, and silences almost all PG genes on the fourth day, and can play a more continuous role than siRNA and shRNA.

From the above results, we can be more sure that our shRNA has the potential to be sprayed as RNAi products in the field to treat rice sheath blight after binding CNT.

Matrix materials
TACE

2022 SZU-China designed TACE（Trichoderma's acest carrier ever) as a product in the prevention stage of RiceAide and conducted a successful iteration. We have proved that TACE 2.0 is superior to TACE 1.0 in most aspects through quantitative experiments. Next, we want to verify the functionality of TACE2.0 and examine its advantages as a commodity.

1. Observation of spore growth

To observe whether spores wrapped by TACE1.0 could be released and grown normally, we inoculated 40 mesh TACE1.0 wrapped spores, 80 mesh TACE1.0 wrapped spores, and Trichoderma clumps into PDA culture medium, observed the growth status of Trichoderma spores in above three groups. The results showed that the growth of spores wrapped by TACE1.0 was similar to that of Trichoderma clumps inoculated with normal methods, indicating that TACE1.0 was friendly to spores and had no effect on their normal growth and reproduction (Figure 4-1).

Figure 4-1. Morphological changes of Trichoderma atroviride in different groups (40 mesh TACE1.0 wrapped spores, 80 mesh TACE1.0 wrapped spores, Trichoderma clumps into PDA culture medium).

2. TACE helps Trichoderma with colonization

We decided to use 6mm TACE2.0 to verify the function of TACE2.0 in helping Trichoderma atroviride colonize rice stems on the aquatic surface. Thanks to our previous cultivation of a large amount of rice in the laboratory, we are able to use rice of about ten days after the transplanting period as the object, and took samples around the adhesion points of rice after 6h and 48h respectively for microscopic examination to observe the release and development of Trichoderma spores.

Figure 4-2. Adhesion of TACE 2.0 to Rice Stem.

Figure 4-3. (A) Inside the gel，most of the spores have not yet germinated.; (B) 2cm away from the adhesion point level，the spores are elongated and appear to be about to germinate 6h later; (C) 2 cm below the adhesion point level，a considerable portion of the spores germinated into hyphae 6h later.

The outer surface of TACE 2.0 is gelatinized and spores are gradually released, while the interior remains dry. Within a certain period of time, the interior of the gel is not suitable for spore germination. The released spores spread out of the gel layer and begin to germinate due to the appropriate external environment such as glucose concentration. With the passage of time, on the one hand, the spores in the gel spread to the horizontal direction; on the other hand, the spores settled naturally. Therefore, we observed that a high concentration of spores could still be detected around the adhesion point, and the spores 2 cm below the adhesion point might have a higher germination rate because of longer exposure to water.

Figure 4-4. A. The TACE gel was filled with Trichoderma mycelium two days later B&C. Trichoderma mycelium colonization was observed on the epidermis of rice stems

After 2 days, TACE2.0 has basically finished working. We collected the residual gel on the rice stem for observation, and found that there were a large number of Trichoderma hyphae in the TACE gel. We then tore the rice stem epidermis at the attachment point and observed it under the microscope, and found the traces of Trichoderma mycelium attachment. This proved that TACE 2.0 successfully helped Trichoderma to colonize the surface of rice. Our design for TACE works!