Background

The typical inoculum for rice sheath blight (ShB) is sclerotia from infected plants. These sclerotia lie dormant in the soil for many years and re-infect healthy rice plants during subsequent crop seasons. Because of their buoyancy, sclerotia can float over long distances in the field, thus contributing to the spread of disease.

The wide and rapid spread of the disease means that paddy fields face the possibility of large outbreaks of ShB every year. Therefore, prevention is a necessary part of rice management. We learned from field investigation that farmers will spray a wide range of compound pesticides in the prevention stage, which causes fertilizer loss and environmental pollution. At the same time, excessive nitrogen application was more likely to cause rice sheath blight.

Because ShB is harmful and has strong transmission ability, early detection of diseased rice is very important. The primary infection site of ShB is the base of rice crop. It is extremely difficult to detect the disease by visual observation. By the time farmers notice obvious symptoms, R.solani is likely to have spread to other nearby plants. Existing disease detection methods are laborious and costly, require professional and sophisticated instruments, which are not suitable for our target users.

The existing treatments of rice sheath blight have their own defects: Agricultural control methods are time-consuming and laborious, and can not cure the disease completely; Existing pesticides are either easy to cause drug resistance of R.solani or pollute the environment; rice with reliable levels of ShB resistance has not been found.

In conclusion, for rice sheath blight, RiceAide proposed a combination management scheme from three aspects: prevention, detection and treatment.

Prevention - Trichoderma atroviride

Why take precautions?

The remaining dominant sclerotia in the soil is an important source of R.solani spread and infection in rice fields. Once the spread and resuscitation process of sclerotia is unimpeded, the rice field will face a huge disaster. Therefore, early prevention is particularly important for the control of rice sheath blight.

Selection of chassis organism

Trichoderma is a group of fungi widely distributed in soil. Over the past few decades, many Trichoderma species have been used as biocontrol agents to control damage caused by soil-borne diseases.It has the following mechanisms in crop protection:

- For pathogenic fungi

Entangling and parasitism pathogens, Trichoderma secretes chitinase and protease to degrade and penetrate the cell wall, absorbing nutrients to inhibit R.solani. Occupying the niche of R.solani through rapid growth and proliferation, Trichoderma forms protective biofilms in the susceptible parts of rice, such as stems and roots, to prevent the infection of R.solani.

- For crops

Having a significant contribution to the sustainability of ecosystems and compelling improvements in the quality and quantity of agricultural produce, Trichoderma-based biological control agents(BCAs) remarkably improve photosynthesis, plant growth, and nutrient use efficiency for impressive crop yields.

Trichoderma atroviride is a commonly used biological control agent, which has been proven to effectively inhibit the growth of soil-transmitted pathogens and induce enhanced defense activity of crops. Meanwhile, Trichoderma atroviride has also been reported to be able to achieve genetic engineering through a variety of transformation methods. Therefore, Trichoderma atroviride was finally selected as our chassis organism after many considerations.

However, the effect of wild type Trichoderma in rice field was relatively mild, and it could only exert a limited inhibitory effect on R.solani. Therefore, we hope to arm Trichoderma species by means of genetic engineering to greatly improve their competitive advantage against R.solani.

Design overview

If the R.solani lurking in the soil waiting to spread is compared to the enemy submarines, Trichoderma atroviride are the soldiers who guard rice territory, and to effectively defeat the enemy, they need more advanced, powerful equipment. In order to better win this war, we have prepared three kinds of equipment for Trichoderma atroviride army:

The deck of the Trichoderma ship: Epl-1

As an amphiphilic fungal protein with hydrophobic characteristics, Epl-1 can easily self-assemble at the aquatic interface and form a protein layer, which can be re-dissolved in water. When Trichoderma atroviride emerges from water, the aerial hyphae and conidia are covered with a layer of hydrophobic proteins, making them hydrophobic. We hoped that over-expression of Epl-1 could help T.atroviride float near the aquatic interface of rice, enhance T.atroviride's hyperparasitism against R.solani and induce rice's resistance.

Figure 1.Gene pathway design and mechanism of Epl 1

Type I torpedo: Prb 1

The main mechanism of inhibitory ability of Trichoderma to fungal pathogens is the release of lytic enzymes including Prb1. The serine protease Prb1 plays an important role in the destruction of the plant pathogens. During the interaction, Trichoderma penetrates into the host mycelium, by partial degradation of its cell wall. Prb1 is widely found in various Trichoderma species which attack various of phytopathogenic fungi responsible for major crop diseases. The production of Prb1 in Trichoderma is induced by the presence of a phytopathogenic fungus, or its cell walls, and repressed by glucose. Evidence suggests that over-expression of Prb1 improved antagonism of Trichoderma.

Figure 2.Gene pathway design and mechanism of Prb 1

Type II torpedo: Snakin-1

Snakin-1, which was isolated from potato (Solanum tuberosum), is a cysteine-rich antimicrobial peptide. As a vital component of the innate and the adaptive immune responses in plants, Snakin-1 helps deliver broad spectrum resistance against a wide variety of phytopathogens. Snakin-1 was indeed effective against the rice sheath blight fungus without affecting the normal growth and development of rice. There is a hypothesis that Snakin-1 affects the fungus by triggering apoptosis via multiple pathways. The Snakin-1 alters the cell membrane permeability and its cell surface hydrophobicity, therefore affecting its adhesion capabilities. The combination of all these factors accounts for its antifungal action against Rhizoctonia solani.

Figure 3.Gene pathway design and mechanism of Snakin 1

Having been proven to be effective control agents against R.solani, the above proteins will be expressed in Trichoderma atroviride to enhance its inhibitory effect on Rhizoctonia solani. Through homologous recombination, the coding sequence of the above proteins will be presented on plasmid pCAMBIA1302, which is composed of strong constitutive promoter CaMV 35S and NOS terminator. In addition, we ligated 6× His tag at the end of CDS to facilitate protein purification in subsequent experiments.

Condition triggered suicide switch

We designed a suicide switch, which is condition-triggered, for Trichoderma atroviride.

cbh1 promoter, which was first discovered in Trichoderma reesei, is one of the strongest inducible promoters in the fungal kingdom. It is often used to construct expression vectors. The cbh1 promoter sequence contains cellulose and sophorose inducible binding sites and glucose feedback inhibitory binding sites. These sites are in different positions, which provide multiple possibilities for us to regulate the expression of genes downstream of the promoter. We selected a known 1360bp cbh1 promoter sequence from the cbh1 gene of Trichoderma koningii, retaining the inducible regulatory site of polysaccharide and glucose. We also retained the 17bp leader sequence between the cbh1 promoter and cbh1 gene. Correspondingly, we used a 790bp cbh1 terminator from Trichoderma reesei in the circuit of our Trichoderma suicide switch.

The Pcbh1-cbh1ls-mazEF-Tcbh1 circuit was integrated into pCAMBIA1302 vector by homologous recombination. We transformed the recombinant plasmid into Trichoderma atroviride by Agrobacterium-mediated transformation in order to achieve condition-triggered suicide of T.atroviride ultimately.

• 1. In the lab and inside the wrapping materials, we cultured T.atroviride under glucose-rich conditions. Glucose within high concentration inhibited the cbh1 operon, rendering this suicide pathway unexpressed.
• 2. When T. atroviride are released to the field and glucose concentration gradually reduces in the the wrapping materials and surrounding environment, the inhibitory effect of glucose on the suicide switch reduces correspondingly. At the same time, the utilization of carbon sources by T. atroviride converts to many kinds of saccharides such as cellulose and sophorose. These saccharides induced the expression of cbh1 operon, leading to the suicide of T.atroviride.

Figure 4.Condition triggered suicide switch for Trichoderma atroviride

TACE the carrier

In order to precisely block the transmission of R.solani sclerotia from aquatic interface and soil, we designed TACE(Trichoderma's acest carrier ever) and completed its iteration, which is used as encapsulation carrier for Trichoderma atroviride spore.

Figure 5.Upgraded TACE of different particle sizes

The upgraded TACE is hydrophilic sustained-release matrix preparation with Hypromellose (HPMC) as the main component and T.atroviride spores as the effective component. In addition, glucose was added to TACE2.0 to inhibit the condition triggered suicide switch of engineered T.atroviride.

Figure 6.The functioning process of TACE

Once disseminated in paddy fields, the floating TACE can quickly adhere to the rice stems at aquatic interface contrapuntally. Due to the exposure to water, the outer layer of TACE is hydrated into gel, and the spores are released slowly, forming a continuous local high concentration environment of T. atroviride spores. With the help of TACE, T.atroviride colonizes the susceptible parts of rice, so as to block and contain the transmission of the R.solani sclerotia from aquatic interface and soil. The HPMC matrix of TACE will mostly dissolve in at least 2 days, leaving no residue in paddy fields eventually.

Vedio 1. Field application of TACE

As the downstream product of engineered T.atroviride, TACE has been proven a great success. See more about TACE in product.

Detection - LAMP system

Overview

Our LAMP detection system consists of two main steps:

• The first step is to amplify DNA samples using primers labeled with biotin.
• The second step is to hybridize the amplified product with a target-specific ssDNA probe labeled with fluorescein amidite and load it to a lateral flow device for detection.

To verify and test the LAMP detection system, we went through the whole process of experimental exploration, as shown in the following mind map.

Figure 7. Overview of LAMP system design

LAMP Amplification

LAMP, loop-mediated isothermal amplification, is a novel nucleic acid amplification method, characterized by four specific primers designed for six regions of the target gene. In the catalyzation of strand replacement DNA polymerase (Bst DNA polymerase) under the action of 60-65°C constant temperature, 15-60 minutes amplification, LAMP reaction is able to achieve 10^9~10^10 times of nucleic acid amplification.

LAMP has the advantages of simple operation, strong specificity, high sensitivity and convenient product detection.

Before the experiments, we found that if LAMP detection is conducted in the laboratory, the method of DNA extraction is essential to the quality of DNA sample and the success of amplification reaction. In order to explore the best method for DNA extraction of Rhizoctonia solani and guide our subsequent experiments, we listed the common laboratory extraction methods, including crude extraction, protocols of three easy methods, CTAB protocol, TPS protocol (Table 1).

Table 1. Common laboratory extraction methods

After all the experiments, including the PCR and LAMP reactions with DNA samples extracted by different extraction methods, we compared all the methods according to the experimental process and results (Table 2).

Table 2. Comparison of laboratory extraction methods for the extraction of R.solani DNA

Based on the above comparison, the detection limit of DNA extracted by TPS method is the lowest, and the concentration and purity are high. In addition, the operation steps and reagents used in TPS protocol are not excessive. Thus, TPS protocol is recommended for R.solani DNA extraction in laboratory tests.

In addition to DNA samples, appropriate primers and systems are also very important for amplification reaction. Therefore, LAMP primers and systems for different Rhizoctonia solani were determined by sequence alignment and literature search. PCR primers and systems were also explored for comparison with LAMP reaction to show the advantages of LAMP reaction.

We selected two strains of R.solani, AG-1 and AG-3, for our experiments. ITS sequences of them are conserved (GenBank KY884015.1, MT177251.1, MT177252.1). The ITS sequences of both are highly similar, with only a few base differences. We use the following universal primers for fungi and related PCR systems to amplify ITS sequences of R.solani though PCR reaction.

Table 3. Primers for R.solani ITS sequences in PCR reaction

We use the following primers to amplify ITS sequences of R.solani though LAMP reaction.

Table 4. Primers for R.solani ITS sequences in LAMP reaction

Figure 8. ITS (ITS1 and 5.8 s rRNA) sequence of Rhizoctonia solani AG-1 and AG-3, showing location of LAMP primers. RSAG-1FIP, RSAG-3FIP, RSAGBIP binds two sites (F1C and F2, B1C and B2, respectively) in R.solani ITS sequence.

In order to make the detection limit of our LAMP protocol more visual, we compared the detection limits between PCR and LAMP reactions and between different DNA extraction methods. Our PCR system had the highest sensitivity to DNA extracted by TPS protocol. After LAMP amplification, ladder-like bands can be observed even in samples diluted to the lowest concentration. In addition, the LAMP reaction was more sensitive than the PCR reaction.

Table 5. Dilution gradient of DNA samples extracted by different methods and the quality of DNA in the amplification reaction.

More results can be viewed on the Results page.

LAMP-LFD Detection

Lateral flow devices (LFD) are widely used for detection of diseases since they have strength including portability, easy operation and high accuracy. In our experiments, we utilized a universal LFD to detect rice sheath blight. For researchers, LAMP-LFD increases the convenience of laboratory testing and can be carried to rice fields for field testing. Therefore, we simulated the implementation of LAMP-LFD in field detection.

In implementation of LAMP-LFD detection, biotin-labeled primers were used for amplification reaction, and the final LAMP amplicon was labeled with biotin. It was hybridized to a target-specific ssDNA probe labeled with a fluorescein-containing label (fluorescein amidite, purple in figure). After dilution, the amplification product was added to the LFD in drops, and the amplicon captured latex beads coated with anti-biotin antibodies. The latex bead /LAMP complex is selectively captured and enriched by anti-fluorescein antibodies at the test line "T" as it passes through the LFD device, showing red color. The unbound excess latex bead/antibiotin antibody complex is captured and enriched by biotin at the control line "C".

Figure 9. LAMP-LFD Detection.

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. We conduct these procedures to imitate actual field detection and successfully verified the feasibility of our LAMP-LFD detection for rice sheath blight, which created the foundation for its future application.

More results can be viewed on the Results page.

Application

Through multiple surveys in Human Practice, we have learned that in China, scientific research institutions guide most of the pesticide use and sowing techniques in rice fields, and farmers also trust scientific research institutions. Therefore, LAMP-LFD detection targets farmers and researchers.

Figure 10. Application of LAMP-LFD detection.

Laboratory testing

Field testing

In order to make field testing more convenient for researchers, we conceived a portable field testing kit based on lyophilized microsphere technology. This kit integrates all the devices and reagents required for field testing. Researchers can just carry this kit to conduct field tests on rice crops. If properly operated, the test results will appear within about 1.5 hours, which quickly diagnose whether there is rice sheath blight in the paddy field.

Figure 11. Field testing kit.

Treatment - RNAi

Overview

1.RNAi Technology

RNAi is a natural reaction process, which is relatively conserved in evolution. It can silence the expression of target genes by specifically targeting the mRNA of cells, belonging to post transcriptional regulation of genes (PTGS) . RNAi usually realizes this regulation process through small RNA (sRNA). RNAi is started by microRNA or siRNA, and then formed by Dicer or Dicer like (DCL) protein and combined to Ago protein to form RNAi silencing complex (RISC), which specifically silences the expression of target genes . RNAi has broad application prospects in crop disease control due to its specificity, efficiency and stability. In addition to autogenesis in organisms, there is also cross-border RNAi, that is, sRNAs transfer between different species and trigger RNAi process.

Figure 12. RNAi silence process.

2.SIGS

At present, RNAi is mainly used in crop protection through two ways: host induced gene silencing (HIGS) and spray induced gene silencing (SIGS). To adopt HIGS means to transform crops through transgenic means, however, people's acceptance of Genetically Modified products is still not high in China, so this method can not be well applied in real life. We soon noticed SIGS, which is an emerging, non transgenic RNAi strategy. Under the condition that pathogenic fungi can absorb RNAi molecules from outside, RNAi molecules can also play a good role in cross-kingdom silencing.

3.Binding with nanomaterials

Nanomaterials such as lipid nanoparticles have been shown to deliver drugs and biomolecules to animal cells, but their application potential in plant cells remains to be developed. Some studies have shown that carbon nanotubes (CNTs) can effectively deliver biological molecules in plant cells, and have realized internalization into mature plant cells after binding DNA. Because CNT is non-toxic, efficient and stable in delivering biomolecules , we decided to bind our shRNA molecules to CNT in the hope that it can make shRNA more stable in the environment and better absorbed by R.solani cells to start the RNAi process.

In addition, in the study of nano clay flakes (LDH), it was proved that they can carry dsRNA to target and silence the target gene of tobacco virus. LDH is a kind of designable, non-toxic and degradable nanomaterial. Its existence ensures the stability and continuous release of dsRNA, and can provide long-term RNAi protection for leaves. Since we hope our RNAi products can be sprayed into the field environment to play a stable role without easy degradation, the characteristics of LDH deeply attracted us.

Design of shRNA molecules

The shRNA (short hairpin RNA) consists of two short inverted repeats and a loop sequence, forming a hairpin structure. The designed shRNA can precisely bind to the specific mRNA of Rhizoctonia solani AG1-IA and realize the degradation of the mRNA through RNA interference mechanism. This blocks the translation of a particular protein, resulting in a decrease in its level, which ultimately leads to inhibition of pathogens. In the treatment stage of RiceAide, we will use carbon nanotube/nanoclay bundled with shRNA as pesticide to spray on rice infected with rice sheath blight. 2022 SZU-China designed 10 shRNAs from the perspectives of inhibiting infectivitity and killing pathogens.

Inhibiting infectivitity

Rhizoctonia solani has an efficient infectious ability, which makes it one of the most harmful diseases to rice. We first found three gene suppression targets that can specifically inhibit the infection ability of Rhizoctonia solani.

Killing pathogens

Further, we want to kill Rhizoctonia solani through RNA interference, so we found four gene suppression targets for housekeeping gene of Rhizoctonia solani AG1-IA. Housekeeping gene is a kind of gene that is stably expressed in most cells of organism, and its encoded protein is essential for maintaining the basic life activities of cells. Housekeeping genes usually maintain a low level of methylation and remain in an active transcription state. We found four coding sequences in the literature, which are housekeeping gene that have been applied to inhibit Rhizoctonia solani.

Construction of shRNA expression vector

We have identified PG1, RPMK1-1, and RPMK1-2, which are critical to the infectivity of Rhizoctonia solani, and RNAPolIII subunit C6, cohesin complex subunit Psm1, Ubiquitin ligase E3, and Importin `\beta`1，which are critical to the survival of Rhizoctonia solani, as our RNAi targets. We searched the cDNA database of Rhizoctonia solani AG1-IA according to the sequences provided by the literature, and found the homologous cDNA sequences of AG1-IA strain.

Next, the sequences found by method above were analyzed, queried or predicted on the National Center for Biotechnology Information (NCBI) website. Also, the total nucleic acid database blast was carried out on the target CDS to query the similarity of homologous genes in adjacent species, and shRNA was designed in non-conserved regions to improve the species specificity of our shRNA and ensure biological safety.

Figure.13 shRNA Molecular Design Process.

And then by professional siRNA Design websites to analyze these gene fragments. Based on the principle of shRNA design, we selected the fragments with high potential siRNA activity from a series of sequences. At the same time, we try to select shRNA fragments that inhibit the target in the first third of the length of the target mRNA. Because according to the mechanism of RNAi, the dsRNA synthesised is longer, producing more small siRNAs subsequently. It can better amplify the secondary inhibition effect.

For biosafety, the candidate RNAi fragments were submitted to the total mRNA database for blast, and the sequence similarity was compared. Focus on species with more than 90% similarity and their nucleic acid fragments to ensure that there is no matching of common species (human, rice, dog, wheat, etc.) to ensure the specificity of the sequence.

Finally, the chosen RNAi fragment is assembled in the order of RNAi fragment — loop — the reverse complement of the sense RNAi fragment.

Figure.14 shRNA production device and RNA interference.

We have confirmed that this siRNA sequence has a good binding ability to Rhizoctonia solani AG1-IA. The intrinsic order of this sequence is RNAi fragment—loop—Reverse complement of the sense RNAi fragment. This sequence was assembled in the pET28a (+) plasmid containing the IPTG-inducible phage T7 promoter and subsequently transferred into RNase-deficient E.coli HT115. In our project, shRNA will be industrially produced by E.coli on a large scale, and shRNA will be purified and sprayed on rice fields to inhibit Rhizoctonia solani.

R-body

What is R-body？

R-body(Refractile inclusion bodies) is an amazing protein complex with refractive properties. It originated from bacteria of the genus Caedibacter or Caedimonas, obligate endosymbionts of Paramecium. R-body is composed of approximately 12 kDa monomers that polymerize to form supercoiled bands approximately 0.5 microns in diameter. When exposed to a pH of 6.5 or less, it unfolds in a telescoping manner into a 10-20-micron long needle-like structure (about 10 times the length of the E.coli). The natural R-body gene cluster contains four genes in the order of RebA-RebB-RebD-RebC.

Table 6. Functions of four genes in the R-body gene cluster

Figure.15 Original R-body gene cluster expression device.

The assembly of the R-body is roughly divided into "major" and "secondary" assembly phases. The R-body is acidic during the major assembly phase and becomes more basic as the assembly proceeds. In the secondary assembly stage, the R-body is basic, and the assembly speed increased. While all three (Reb A, Reb B, Reb C) proteins are required for the major assembly event, only RebB and RebC are required for the basic assembly process. RebD is not necessary for the synthesis of R-body.

Figure. 16 Scanning electron microscope image and cartoon image of a single R-body in a fully curled and fully extended state.

Expression of R-body

We carefully examine the properties of R-body (Refractile inclusion bodies) and find some interesting points. Based on it，we delete RebD to ensure the simplification of the gene cluster of R-body and move RebB to the first place, so as to up-regulate the expression of RebB, and make the synthesis and assembly of R-body in E.coli more efficient. In summary, we obtained a modified version of the R-body gene cluster, in which the gene are arranged in the order of RebB-RebA-RebC. This modified version of the R-body will be used as a conditioning self-cracking device for engineered E.coli.

See more about modified R-body gene cluster in Improvement.

Figure.17 Improved R-body gene cluster expression device

The original and modified versions of R-body gene clusters were placed on the plasmid pRSFDuet1，controlled by the arabinose promoter. In the design of treatment stage in our project, we engineered E.coli HT115(DE3) to produce RNAi molecules and synthesize R-bodies.In the first step of production, we induced E.coli HT115(DE3) to produce RNAi molecules by IPTG; in the second step, we used arabinose to induce E.coli to produce R-body, and then we acidify the bacterial medium below pH6.5 to crack E.coli, leading to the release of RNAi molecular.

RNAi products

The following is the production process of our RNAi products.

• (1)Transforming the constructed shRNA expression plasmid into E.coli HT115(DE3).
• (2)Adding IPTG to induce Escherichia coli to produce shRNA molecules.
• (3)After obtaining enough shRNA, arabinose is added to induce the production of R-body.
• (4)Add sufficient CO2 to acidify the bacterial culture medium to a pH below 6.5, so that E.coli can be cracked.
• (5)The shRNA isolated from E.coli was obtained by centrifugation and incubated with CNT for 30 min to obtain shRNA CNT products.
• (6)The shRNA-CNT produced will be packaged in spray bottles.
• (7)The spray bottle is assembled on the UAV to complete the large area spraying of RNAi products in the field.

Figure.18 RNAi product production process

Actual spraying

For the actual spraying of our RNAi products, we hope to verify its results in four aspects. We mainly check whether RNAi products can play a good role through the growth of mycelia on leaves, the degree of leaf disease, and the expression level of target genes. For RNAi products that can target the key survival genes of R.solani, we also carried out the growth inhibition experiment of R.solani in the culture medium.

Figure.19 RNAi experiments

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