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RNA interference Design of RNAi molecules Improvement
Plant immune pathway Inducible factors Engineered B. subtilis Suicide switch

Overview


Plant fungal disease is one of the main culprits of crop damage, it usually first infects a small part of the crop, and then quietly infects the surrounding crop without being detected, and eventually leads to a large reduction of crop production. In order to prevent the onset and transmission of plant fungal diseases, people have tried variety of means. Such as crop rotation, spacing planting, chemical fungicides, seedling cultivation, the adoption of biological control bacteria and so on. However, these measures are not effective enough to stop diseases, and its worldwide impact can't be ignored. Tomato gray mold is a kind of plant fungal disease caused by Botrytis cinerea, by taking tomato gray mold as a starting point, we hope to find a solution for the control of all plant fungal diseases.


In recent years, RNAi biopesticides have attracted much attention. By silencing the target genes of pathogenic bacteria by RNAi technology, chemical fungicides can be replaced by an effective, non-toxic and environmentally friendly way. At the same time, during the process of integrated human practices , we have paid attention to the concept of "prevention is more than cure" and the development of plant immune preparations. Therefore, we decided to integrate the two aspects of RNAi pesticides and plant immunity into our experimental design.


During B. cinerea infects tomatoes, it will secrete virulence factors to help the attack on tomatoes, and tomatoes will secrete immune substances to defend under stress. At this point, RNAi pesticides and plant immune agents can play a role in the interaction between B. cinerea and the tomato.


Figure 1. The interaction between B. cinerea and Tomato

  • - Blocking B. cinerea infection: if the tomato has been infected by B. cinerea, the RNAi molecules can silence the virulence factor genes helping to infect and the key genes relating to fungus's own growth

  • - Enhancing tomato immunity: if the tomato has not been infected, applying a signal to the tomato to stimulate its immunity can improve its anti-fungal ability.

Figure 2. Tomato beats gray mold with the help of SZU-China

Finally, by combining the two parts and wrapping them into a bead, the final product "PolycoBead" is formed.


Figure 3. Composition of PolycoBead

RNAi therapy


RNA interference and SIGS

RNA interference (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 4. Schematic diagram of RNAi mechanism process

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.


Figure 5. SIGS process diagram of naked shRNA and CPP-shRNA

Choosing RNAi technology has the following advantages:


  • - Specific targeting
    Only the essential genes of B. cinerea are targeted and silenced without causing harm to other organisms.

  • - Environmental friendliness
    RNAi molecules degrade easily, so they are environmentally friendly.

  • - Controllability
    RNAi biopesticides do not cause permanent changes in plant gene expression and are controllable.

Design of RNAi molecules

Short hairpin RNA (shRNA) consists of two short reverse complementary sequences and a loop sequence to form the hairpin structure. shRNA can be actively absorbed by B. cinerea, then enter into its cells to be processed into siRNA, and further specifically target mRNA to achieve degradation. Or, entering plant cells, the related proteins in the cell will process and deliver shRNAs to B. cinerea, which can also achieve the effect of silencing mRNA, reducing the level of its specific protein, and finally forming the inhibition of the pathogen. According to the key genes and self-survival genes of B. cinerea, we selected 6 targets and designed corresponding shRNAs according to these targets.


Inhibiting infectivity:

Figure 6(a). The PME expressed by the Bcpme1 hydrolyzes tomato cell walls

- The cell wall is the main interface for plant and microbial interaction, limiting the invasion of pathogens and the spread of infections. When B. cinerea invades the plant host, it synthesizes exogenous enzymes that degrade pectin, a major component of the plant cell wall. Pectin methylesterase (PME) is a hydrolase that catalyzes the hydrolysis of α ester bonds on pectin molecules in plant cell walls. By silencing Bcpme1, an important gene for the expression of PME by B. cinerea, it is capable to prevent B. cinerea from harming the plant cell wall and blocking its invasion from the early stage of infection.


Figure 6(b). The BcOAH1 helps produce oxalic acid and infects the tomato

- During infection, B. cinerea will express oxaloacetate hydrolase, which catalyzes the hydrolysis of oxaloacetate in B. cinerea to oxalic acid. After oxalic acid enters the host plant, the free calcium ions in the host body can form calcium oxalate crystals, which is easy to cause the symptoms of plant blockage and. The release of oxalic acid can also inhibit the outbreak of reactive oxygen species (ROS) in plants, which is not conducive to the plant to defense the infection of B. cinerea. Silencing the gene BcOAH of B. cinerea can reduce the production of oxalic acid and prevent further infection and spread of pathogens after invasion.


Figure 6(c). The DCL1/2 interferes with the tomato's immune system

- Dicer-like proteins DCL1 and DCL2 of B. cinerea participate in the synthesis of siRNA. Then siRNAs will be delivered to host plant cells to participate in the formation of RISC complexes in the host RNAi mechanism, and subsequently silence and inhibit host immune-related genes, making plant cells more susceptible to infection. The shRNA was designed to silence B. cinerea genes BcDCL1 and BcDCL2, preventing them from synthesizing siRNA that interferes with the plant cell's immune system, thus allowing the plant to effectively defend itself against B. cinerea.


Killing pathogen:

  • - Ergosterol (ERG) is a C28 sterol that is particularly present in fungal cell membranes and plays a crucial role in the structure and function of fungal cell membranes. Ergosterol is responsible for maintaining membrane fluidity, regulating membrane permeability, affecting membrane-related enzyme activity, and influencing fungal cell growth. Silencing Bccyp51, a key gene in the ergosterol biosynthesis pathway, can destroy the cell membrane of B. cinerea and produce killing effect on it.

  • - Chitin is the main structural component of the fungal cell wall, and chitin synthase (CHS) catalyzes the synthesis of chitin. By silencing the important chitin synthase gene BcCHSIIIa, the formation of the cell wall of B. cinerea could be affected, thus killing pathogenic fungi.

Figure 7. Ergosterol (ERG) is the helmet, and chitin synthase (CHS) helps make the armor

After confirming the selection of the above six targets Bcpme1, BcOAH1, BcDCL1, BcDCL2, Bccyp51, and BcCHSIIIa, we searched the cDNA library of B. cinerea according to the sequences or primes provided in the literature, and found the homologous cDNA sequence of B. cinerea. Then, the sequence was input into the National Center for Biotechnology Information (NCBI) website for analysis and prediction, and the CDS sequence of the target gene was input into the total nucleic acid database BLAST to query the homologous similarity of neighboring species. siRNA sequences were designed in non-conserved regions to ensure species-specific and biosafety of our shRNAs.


Next, we used a professional siRNA design website to predict the siRNA sequences that would effectively target the mRNA, and then screened out siRNA fragments with high potential activity in a series of predictions based on shRNA design principles. For biosafety reasons, we BLAST the candidate siRNA fragments into the total mRNA database to ensure that it does not target any genes of common species (such as human, tomato, dog, rice, wheat, etc.), ensuring sequence specificity.


Figure 8. shRNA Molecular Design Process

Finally, we assembled the selected siRNA sequence into our shRNA expression fragment in the sequence of siRNA sense strand - loop - reversed siRNA antisense strand and attached the sequence to the pET-28a(+) vector. The recombinant vector was transferred into RNase-deficient E. coli HT115 (DE3), and the large-scale fermentation production of shRNA in E. coli could be achieved by induction of IPTG. In our experiment, the results of treatment of different shRNAs at both phenotypic and molecular levels were analyzed to screen out the effective shRNAs.


Click Proof of concept for more details.


Improvement

However, even though many targets have been proven feasible, RNAi biopesticides have not been effectively promoted in the agricultural field so far. The main problems are as follows:


  • - RNAi molecules are unable to efficiently enter the target cell and play their roles.

  • - RNAi may be less effective because of the target.

  • - The production of RNAi molecules relies more on cell fermentation, making it difficult to reduce costs.

Delivery of shRNA molecules


Cell-penetrating peptides (CPPs) is a short chain amino acid composed of about 30 amino acids, including basic amino acids and R groups, which can be used to deliver biological molecules such as siRNA, pDNA, plasmid and protein, and has been relatively mature in the medical field. Engineered CPP and shRNA molecules interact via positively charged amino acid residues such as arginine and lysine with the phosphate skeleton of negatively charged nucleic acids to form submicron peptide-nucleic acid complexes. This non-covalent binding method has stability and reversibility, thus realizing the transfer and function of biomolecules.


BP100-(KH)9 is a CPP that fuses the highly potent cell-penetrating peptide BP100 with the biomolecular binding domain KH9 to improve the delivery efficiency of biomolecules into plant cells. BP100-(KH)9 shows a spherical shape under the Atomic force microscope (AFM) and can wrap shRNA molecules to form spherical complexes. The combination of BP100-(KH)9 and shRNA is simple, just mix the two substances in a certain proportion, and let them stand at room temperature for a period of time to obtain the complex of CPP-shRNA. By spraying CPP-shRNAs on tomatoes infected by B. cinerea, shRNAs can enter plant cells more efficiently under the effect of CPP. Then, siRNAs are formed after processing of Dicer / Dicer-like proteins in cells, which will be next delivered to B. cinerea, to achieve the effect of silencing the key genes of B. cinerea infection and its own survival genes.


Figure 9. CPP-shRNAs spray and action diagram

Concatenated shRNA molecules


In the case of only spraying shRNA molecules against one target gene, the shRNA may not play the best effect due to the problem of target sequence, siRNA sequence and so on. However, after concatenating shRNAs targeting different mRNAs using specific sequences, the resulting shRNAs molecules can be simultaneously processed into different siRNAs to function. The bifunctive shRNA (bi-shRNA) was thus used, resulting in a lower effective dose than the siRNA/shRNA with the same effect to minimize the possibility of non-target side effects and to provide a more sustained effect.


Figure 10. bi-shRNA schematic

Production of RNAi molecules


The current production of RNAi molecules is dominated by fermentation. The production of RNA molecules under this method is prone to the following problems:


  • - The fermentation system is easily polluted, and the environment needs to be strictly controlled.

  • - The living bacteria in the fermentation system face the problem of easy aging.

  • - shRNAs extracted by fermentation are difficult to purify and are usually accompanied by small amounts of other RNA.

The above problems are the challenges that need to be addressed in the process of fermentation production of RNAi molecules, and people usually need to take other measures to eliminate these problems, which leads to an increase in the cost of production, making RNAi biopesticides difficult to apply. Therefore, we designed a cell-free RNAi molecules production system, hoping that the successful operation of this system can provide ideas for low-cost and high-efficiency application of RNAi molecules.


More details can be viewed in Hardware .


Plant immunity


Plant immune pathway

Plants grow in a complex environment full of harmful pathogenic microorganisms, including Botrytis cinerea. In the process of co-evolution with pathogens, plants have evolved a variety of mechanisms to resist the infection of pathogens, and gradually formed the plant immune system. In response to pathogen attacks, plants have developed two typical defense systems: Pathogen-associated molecular patterns Triggered Immunity (PTI), and Effector Triggered Immunity (ETI).


In the process of PTI, the pattern recognition receptors (PRRs) on the plant cell membrane recognize the pathogen-associated molecular patterns (PAMPs) to activate the plant's initial defense system, resulting in cell rapid defence responses, such as cell necrosis, reactive oxygen species (ROS) outbreak, cell wall enhancement, regulation of plant hormone pathways, and expression of defense-related genes and proteins. PAMPs include protein-inducing factors derived from pathogenic bacteria, such as harpin protein isolated from Erwinia amylovory and Pseudomonas syringae, glucan-1,4-α-glycosidase BcGS1 of Botris griseus, and flagellin of Pseudomonas aeruginosa etc.


After successful infection, the pathogen interferes with the plant's initial PTI response by secreting effector factors, and the plant evolves a resistance protein (R protein) that directly or indirectly recognizes the specific effectors AVR possessed by the pathogen and triggers a more intense and sustained immune response, also known as the ETI. Plant immune mechanism has signal transduction, including salictlic acid (SA) and ethylene (ET) mediated signal transmission pathway, and on this basis the whole plant defense network is formed, and finally the whole defense response against pathogens is generated.


Figure 11. Plants immunity pathways

Inducible factors of plant immunity

According to the characteristics of plant immune process, more and more plant immune-inducing factors have been developed and used, which have become a new way of plant disease control. These include biological and abiotic inducible factors, such as the fungus Metarhizium, Trichoderma, Paecilomyces, etc, the bacteria Bacillus subtilis, Pseudomonas, etc. And some microbial metabolites such as β-1,3-glucan, oligo-chitine and other oligosaccharide inducers from fungus, and protein inducer such as protein phosphomutase BvEP from Bacillus velezensis LJ02. Among them, protein phosphatase BvEP from Bacillus velezensis LJ02 has been shown being able to induce the PTI and ETI immune pathways in tomato fruits, stimulate ROS outbreak and related enzyme expression, and have no significant effect on the weight and nutrient content of tomato fruits.


Bacillus subtilis is one of the most widely accepted plant probiotics and has a high homology with Bacillus velezensis. It has been reported that B. subtilis strains have a control effect on different fungal diseases of different plants, such as Rice sheath blight in cereal crops, wheat holoses, and tomato gray mold in vegetable and fruit crops, and have a good control effect on a variety of crops and fungal diseases. B. subtilis has the advantages of strong protein expression system, strong stress resistance, fast growth rate, easy colonization in plant rhizosphere, etc. It can exert antibacterial and antifungal effect through spatial and nutritional competition or produce anti-pathogenic substances, and can produce more than 70 kinds of anti-pathogenic lipids and proteins, as well as various volatile anti-pathogenic substances such as dimethylamine, formic acid, propionic acid, dimethyldisulfide and propylamine. B. subtilis is an ideal plant probiotic. Due to the unstable effect of biocontrol microorganisms, the main application methods used are combining with chemical pesticides or coordinating with other antagonistic microorganisms for controling plant fungal diseases.


Engineered B. subtilis

After BLAST, we found that the nucleic acid sequence of BvEP has a high homology with B. subtilis, and B. subtilis has a simple genetic background and a strong protein expression system. Therefore, we combined B. subtilis and protein immune inducing factor BvEP and Flg22 to modify B. subtilis to express the Flg22 and BvEP to achieve the function of 1+1>2. The engineered bacteria can be used in farmland during the peak period of B. cinerea outbreak, so that Tomato gray mold can be controlled.


Figure 12. Engineered bacteria release immune inducing factors to stimulate tomato immunity

In our design, we use a strong promoter Pveg to express our immune-inducing factor BvEP. Pveg is the promoter of the constiutive expression target gene in B. subtilis, which contains two binding sites of the main σ factor a1 of B. subtilis, which can be simultaneously bound to make the gene efficient expression. At the same time, we select the 2x Fd Terminator to have a strong termination effect on the expression pathway. Finally, the pathway is assembled into pMA5, a commonly used protein expression vector of B. subtilis by seamless cloning, and transfer into B. subtilis WB800. During the experimental validation, we first verify the normal operation of this gene pathway in E. coli and ensure the effectiveness of the BvEP expressed by it in plants. We select pET-28a(+) as the vector of BvEP expression in E. coli, and transfer it to the proteinase-deficient E. coli BL21(DE3) for experimental verification.


Figure 13. The expression pathways of different immune inducing factors

In order to verify the effectiveness of protein-inducing factor BvEP, we extracted BvEP expressed by E. coli BL21 (DE3) after induction by IPTG and treated tomato leaves with it. The outbreak of ROS is one of the characteristics of successfully induced plant immune response. Therefore, the brown-red compound generated by the reaction of 3,3'-Diaminobenzidine tetrahydrochloride (DAB) with hydrogen peroxide (a type of ROS in plant cells) is used as an indicator, so as to locate the explosion intensity of hydrogen peroxide in tissues as a representation of the strength of plant immune pathways.


Leran more information in results.


Suicide switch

To prevent our engineered bacteria from flooding the field, we designed a sucrose-induced toxin-based/antitoxin system as a suicide switch, which includes the mazE and mazF genes. The mazF gene encodes the stable toxin protein MazF, which is capable of recognizing and cutting all ACA sequences of single-stranded mRNA, independent of ribosome mRNA endonuclease. Due to the high probability of occurrence of these three base sequences, the expression of mazF can break down almost all mRNA, thus inhibiting the release of ribosomes and protein synthesis on the cleaved mRNA, and finally leading to cells death.


The MazE antitoxin protein encoded by mazE is unstable. In the presence of sucrose, MazE is induced to be expressed and then degraded by the ClpPA complex (an ATP-dependent serine catalytic enzyme) to form a specific conformation. This particular conformation can recognize and bind to MazF toxin proteins, forming hexamers (FF-EE-FF) that inactivate them. In other words, after adding sucrose, MazE and MazF reached a stable state stopping cell death. But when the sucrose runs out, MazF regains its dominance and the cells die.


Figure 14. The suicide switch in B. substilis

PolycoBead


According to the feedback of integrated human practices, pesticide abuse is a widespread problem in agriculture today when farmers apply pesticides. The pesticide preparations that farmers can buy on the market are usually in large capacity, and even if the optimal application amount of pesticides has been marked on the package or in the instruction manual, farmers will subjectively add excessive amounts, resulting in increased pesticide residues in crops, and even spread to the environment. In addition, due to the instability of RNAi biopesticides, RNA molecules are easily degraded by nucleases in the environment, so it is very necessary to separate RNAi biopesticides from the environment, so as to reduce and avoid RNA degradation.


In view of the above problems, we hope to design a specific form to package our products and regulate the application standards of our biopesticide to avoid the phenomenon of pesticide abuse. In addition, we also try to simplify the form of products as much as possible, so that farmers can use them more simply and conveniently. Inspired by the laundry detergent capsules, we envision incorporating our product in a bead based on this fixed volume packaging and guiding farmers to use a certain amount of beads in tomato fields at a specific planting density to treat and prevent tomato gray mold. Thus, we design our product PolycoBead.


Figure 15. Polycobead

Figure 16. Polycobead

Polycobead uses a high alcoholysis degree water-soluble polyvinyl alcohol (PVA) as the outer film to wrap our CPP-shRNAs preparation and the engineered chassis-bacteria B. subtilis together to form a bead. Since PVA film is a water-soluble material, when farmers use our Polycobead, they only need to put it into a certain volume of water, and can be sprayed directly into the field after rapid dissolution.


Using PVA film has the following advantages:


  • - The high alcoholysis degree water-soluble PVA film has a large number of hydroxyl groups, so it has a strong hydrophilicity. Every 100 cm2, 75 μm thick PVA film can be completely dissolved by stirring in sufficient water at room temperature for about 1 minute.

  • - PVA film has been proved to have excellent biocompatibility and biodegradability, and it is the only vinyl polymer that can be used by bacteria as a carbon source. It can be decomposed by microorganisms and enzymes in nature, and it can be degraded by 75% in 46 days, and finally it can be degraded into CO2 and H2O, and it is proved to be non-toxic to life by biological tests.

  • - Based on its water-soluble and biodegradable properties, the liquid formed after the PVA film is dissolved in water can penetrate into the soil to increase the soil agglomeration, air permeability and water retention, which is conducive to the survival of plants.

  • - PVA film has high mechanical strength, good toughness and high tensile strength. Its tensile strength can reach 44.1 ~ 63.7MPa and its elongation can reach 400%.

  • - The barrier and anti-static properties of PVA film can help the contents to isolate the environment and prevent electrostatic dust from contaminating the product, so it can well avoid the degradation of RNA in contact with the nuclease from the environment.

  • - PVA film is very easy to process, low production cost and easy to obtain.

The CPP-shRNA complex we produce exists in an aqueous solution form. Although CPP can effectively slow down the degradation of RNA, it still cannot guarantee the stability of RNA molecules in long-term storage, so we use glycerin to provide a liquid environment in the bead. Glycerin, a polar solvent commonly used to preserve biological samples, can form hydrogen bonds with H2O molecules to reduce their activity, and can also form hydrogen bonds and van der Waals forces with proteins and nucleic acids to protect biological molecules from degradation and deformation. Glycerin is miscible with water in any proportion, which ensures the fastest release of the CPP-shRNAs complex once the Polycobead enters the water system. In addition, although a small amount of the glycerin-water mixture enters the natural enironment, it can be degraded by the microbial community in the soil.


Engineered B. subtilis exists in the form of concentrated bacterial liquid inside the PolycoBead. In order to reduce the interference of engineered bacteria on the CPP-shRNAs complex and ensure that B. subtilis can maintain its activity, alginate embedding method is used to mix engineered bacteria, calcium alginate embedding is one of the most widely used cell fixation methods, which has the advantages of high cell density, low toxicity to microorganisms, simple production process and high cell product release rate. The engineered bacteria is mixed with 1% sodium alginate solution and then dropped into Ca2+ solution. The sodium alginate then reacts with Ca2+ to form a water-insoluble calcium alginate shell on the outer layer, while the engineered bacterial solution is encased in calcium alginate to form smaller beads. When the calcium alginate - engineered bacteria complex is released into the environment, with the loss of calcium ions, the engineered bacteria in it will also be released into the soil environment at a slow rate, so as to colonization to the tomato roots to compete for the ecological niche and express the immune-inducing factor BvEP, eventually stimulating the tomato immune mechanism and resisting the infection of gray mold.


More details can be viewed in Proof of concept.



    1. [1] Fletcher SJ, Reeves PT, Hoang BT, Mitter N. A Perspective on RNAi-Based Biopesticides. Front Plant Sci. 2020 Feb 12;11:51. doi: 10.3389/fpls.2020.00051.
      [2] Niu D, Hamby R, Sanchez JN, Cai Q, Yan Q, Jin H. RNAs - a new frontier in crop protection. Curr Opin Biotechnol. 2021 Aug;70:204-212. doi: 10.1016/j.copbio.2021.06.005. Epub 2021 Jul 1.
      [3] Abdellatef E, Kamal NM, Tsujimoto H. Tuning Beforehand: A Foresight on RNA Interference (RNAi) and In Vitro-Derived dsRNAs to Enhance Crop Resilience to Biotic and Abiotic Stresses. Int J Mol Sci. 2021 Jul 19;22(14):7687. doi: 10.3390/ijms22147687.
      [4] Saurabh S, Vidyarthi AS, Prasad D. RNA interference: concept to reality in crop improvement. Planta. 2014 Mar;239(3):543-64. doi: 10.1007/s00425-013-2019-5. Epub 2014 Jan 9.
      [5] Bharathi JK, Anandan R, Benjamin LK, Muneer S, Prakash MAS. Recent trends and advances of RNA interference (RNAi) to improve agricultural crops and enhance their resilience to biotic and abiotic stresses. Plant Physiol Biochem. 2023 Jan;194:600-618. doi: 10.1016/j.plaphy.2022. 11.035. Epub 2022 Dec 10.
      [6] Islam MT, Sherif SM. RNAi-Based Biofungicides as a Promising Next-Generation Strategy for Controlling Devastating Gray Mold Diseases. Int J Mol Sci. 2020 Mar 18;21(6):2072. doi: 10.3390/ijms21062072.
      [7] Hoang BTL, Fletcher SJ, Brosnan CA, Ghodke AB, Manzie N, Mitter N. RNAi as a Foliar Spray: Efficiency and Challenges to Field Applications. Int J Mol Sci. 2022 Jun 14;23(12):6639. doi: 10.3390/ijms23126639.
      [8] Sarkar A, Roy-Barman S. Spray-Induced Silencing of Pathogenicity Gene MoDES1 via Exogenous Double-Stranded RNA Can Confer Partial Resistance Against Fungal Blast in Rice. Front Plant Sci. 2021 Nov 26;12:733129. doi: 10.3389/ fpls.2021.733129.
      [9] Qiao L, Lan C, Capriotti L, Ah-Fong A, Nino Sanchez J, Hamby R, Heller J, Zhao H, Glass NL, Judelson HS, Mezzetti B, Niu D, Jin H. Spray-induced gene silencing for disease control is dependent on the efficiency of pathogen RNA uptake. Plant Biotechnol J. 2021 Sep;19(9):1756-1768. doi: 10.1111/pbi.13589. Epub 2021 May 4.
      [10] Bofill-De Ros X, Gu S. Guidelines for the optimal design of miRNA-based shRNAs. Methods. 2016 Jul 1;103:157-66. doi: 10.1016/j.ymeth.2016.04.003. Epub 2016 Apr 12.
      [11] Han Y, Joosten HJ, Niu W, Zhao Z, Mariano PS, McCalman M, van Kan J, Schaap PJ, Dunaway-Mariano D. Oxaloacetate hydrolase, the C-C bond lyase of oxalate secreting fungi. J Biol Chem. 2007 Mar 30;282(13):9581-9590. doi: 10.1074/jbc.M608961200. Epub 2007 Jan 23.
      [12] Wang M, Weiberg A, Lin FM, Thomma BP, Huang HD, Jin H. Bidirectional cross-kingdom RNAi and fungal uptake of external RNAs confer plant protection. Nat Plants. 2016 Sep 19;2:16151. doi: 10.1038/nplants.2016.151.
      [13] Weiberg A, Wang M, Lin FM, Zhao H, Zhang Z, Kaloshian I, Huang HD, Jin H. Fungal small RNAs suppress plant immunity by hijacking host RNA interference pathways. Science. 2013 Oct 4;342(6154):118-23. doi: 10.1126/science.1239705.
      [14] Soulié MC, Perino C, Piffeteau A, Choquer M, Malfatti P, Cimerman A, Kunz C, Boccara M, Vidal-Cros A. Botrytis cinerea virulence is drastically reduced after disruption of chitin synthase class III gene (Bcchs3a). Cell Microbiol. 2006 Aug;8(8):1310-21. doi: 10.1111/j.1462-5822.2006.00711.x.
      [15] Soulié MC, Piffeteau A, Choquer M, Boccara M, Vidal-Cros A. Disruption of Botrytis cinerea class I chitin synthase gene Bcchs1 results in cell wall weakening and reduced virulence. Fungal Genet Biol. 2003 Oct;40(1): 38-46. doi: 10.1016/s1087-1845(03)00065-3.
      [16] Morcx S, Kunz C, Choquer M, Assie S, Blondet E, Simond-Côte E, Gajek K, Chapeland-Leclerc F, Expert D, Soulie MC. Disruption of Bcchs4, Bcchs6 or Bcchs7 chitin synthase genes in Botrytis cinerea and the essential role of class VI chitin synthase (Bcchs6). Fungal Genet Biol. 2013 Mar;52:1-8. doi: 10.1016/j.fgb.2012.11.011. Epub 2012 Dec 22.
      [17] Duanis-Assaf D, Galsurker O, Davydov O, Maurer D, Feygenberg O, Sagi M, Poverenov E, Fluhr R, Alkan N. Double-stranded RNA targeting fungal ergosterol biosynthesis pathway controls Botrytis cinerea and postharvest grey mould. Plant Biotechnol J. 2022 Jan;20(1):226-237. doi: 10.1111/pbi.13708. Epub 2021 Nov 18.
      [18] Thagun C, Horii Y, Mori M, Fujita S, Ohtani M, Tsuchiya K, Kodama Y, Odahara M, Numata K. Non-transgenic Gene Modulation via Spray Delivery of Nucleic Acid/Peptide Complexes into Plant Nuclei and Chloroplasts. ACS Nano. 2022 Mar 22;16(3):3506-3521. doi: 10.1021/acsnano.1c07723. Epub 2022 Feb 23.
      [19] Rao DD, Senzer N, Wang Z, Kumar P, Jay CM, Nemunaitis J. Bifunctional short hairpin RNA (bi-shRNA): design and pathway to clinical application. Methods Mol Biol. 2013;942:259-78. doi: 10.1007/978-1-62703-119-6_14.
      [20] Pruitt RN, Gust AA, Nürnberger T. Plant immunity unified. Nat Plants. 2021 Apr;7(4):382-383. doi: 10.1038/s41477-021-00903-3.
      [21] Hu J, Chang R, Yuan Y, Li Z, Wang Y. Identification of Key Residues Essential for the Activation of Plant Immunity by Subtilisin From Bacillus velezensis LJ02. Front Microbiol. 2022 Aug 15;13:869596. doi: 10.3389/fmicb. 2022.869596.
      [22] Yuan M, Ngou BPM, Ding P, Xin XF. PTI-ETI crosstalk: an integrative view of plant immunity. Curr Opin Plant Biol. 2021 Aug;62:102030. doi: 10.1016/j.pbi. 2021.102030. Epub 2021 Mar 5.
      [23] Abdul Malik NA, Kumar IS, Nadarajah K. Elicitor and Receptor Molecules: Orchestrators of Plant Defense and Immunity. Int J Mol Sci. 2020 Jan 31;21(3):963. doi: 10.3390/ijms21030963.
      [24] Zipfel C, Rathjen JP. Plant immunity: AvrPto targets the frontline. Curr Biol. 2008 Mar 11;18(5):R218-20. doi: 10.1016/j.cub.2008.01.016.
      [25] Yuan M, Jiang Z, Bi G, Nomura K, Liu M, Wang Y, Cai B, Zhou JM, He SY, Xin XF. Pattern-recognition receptors are required for NLR-mediated plant immunity. Nature. 2021 Apr;592(7852):105-109. doi: 10.1038/s41586-021-03316-6. Epub 2021 Mar 10.
      [26] Oh CS, Martin GB. Effector-triggered immunity mediated by the Pto kinase. Trends Plant Sci. 2011 Mar;16(3):132-40. doi: 10.1016/j.tplants.2010.11.001.
      [27] Chinchilla D, Zipfel C, Robatzek S, Kemmerling B, Nürnberger T, Jones JD, Felix G, Boller T. A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence. Nature. 2007 Jul 26;448(7152):497-500. doi: 10.1038/nature05999. Epub 2007 Jul 11.
      [28] Chen S, Cui L, Wang X. A plant cell wall-associated kinase encoding gene is dramatically downregulated during nematode infection of potato. Plant Signal Behav. 2022 Dec 31;17(1):2004026. doi: 10.1080/15592324.2021.2004026. Epub 2021 Dec 29.
      [29] Shah P, Powell AL, Orlando R, Bergmann C, Gutierrez-Sanchez G. Proteomic analysis of ripening tomato fruit infected by Botrytis cinerea. J Proteome Res. 2012 Apr 6;11(4): 2178-92. doi: 10.1021/pr200965c. Epub 2012 Mar 20.
      [30] Aziz A, Heyraud A, Lambert B. Oligogalacturonide signal transduction, induction of defense-related responses and protection of grapevine against Botrytis cinerea. Planta. 2004 Mar;218(5):767-74. doi: 10.1007/s00425-003-1153-x. Epub 2003 Nov 14.
      [31] Palomäki T, Saarilahti HT. The extreme C-terminus is required for secretion of both the native polygalacturonase (PehA) and PehA-Bla hybrid proteins in Erwinia carotovora subsp. carotovora. Mol Microbiol. 1995 Aug;17(3):449-59. doi: 10.1111/j. 1365-2958.1995.mmi_17030449.x.
      [32] Xiang T, Zong N, Zou Y, Wu Y, Zhang J, Xing W, Li Y, Tang X, Zhu L, Chai J, Zhou JM. Pseudomonas syringae effector AvrPto blocks innate immunity by targeting receptor kinases. Curr Biol. 2008 Jan 8;18(1):74-80. doi: 10.1016/j.cub.2007.12. 020. Epub 2007 Dec 27.
      [33] Ferrari S, Vairo D, Ausubel FM, Cervone F, De Lorenzo G. Tandemly duplicated Arabidopsis genes that encode polygalacturonase-inhibiting proteins are regulated coordinately by different signal transduction pathways in response to fungal infection. Plant Cell. 2003 Jan;15(1):93-106. doi: 10.1105/tpc.005165.
      [34] Sarangi S, Swain H, Adak T, Bhattacharyya P, Mukherjee AK, Kumar G, Mehetre ST. Trichoderma-mediated rice straw compost promotes plant growth and imparts stress tolerance. Environ Sci Pollut Res Int. 2021 Aug;28(32):44014-44027. doi: 10. 1007/s11356-021-13701-3. Epub 2021 Apr 12.
      [35] Peng D, Li S, Wang J, Chen C, Zhou M. Integrated biological and chemical control of rice sheath blight by Bacillus subtilis NJ-18 and jinggangmycin. Pest Manag Sci. 2014 Feb;70(2):258-63. doi: 10.1002/ps.3551. Epub 2013 Jun 18.
      [36] Ben Khedher S, Kilani-Feki O, Dammak M, Jabnoun-Khiareddine H, Daami-Remadi M, Tounsi S. Efficacy of Bacillus subtilis V26 as a biological control agent against Rhizoctonia solani on potato. C R Biol. 2015 Dec;338(12):784-92. doi: 10.1016/j. crvi.2015.09.005. Epub 2015 Nov 10.
      [37] Kourelis J, van der Hoorn RAL. Defended to the Nines: 25 Years of Resistance Gene Cloning Identifies Nine Mechanisms for R Protein Function. Plant Cell. 2018 Feb;30(2):285-299. doi: 10.1105/tpc.17.00579. Epub 2018 Jan 30.
      [38] Li Z, Hu J, Sun Q, Zhang X, Chang R, Wang Y. A novel elicitor protein phosphopentomutase from Bacillus velezensis LJ02 enhances tomato resistance to Botrytis cinerea. Front Plant Sci. 2022 Nov 29;13:1064589. doi: 10.3389/fpls.2022.1064589.
      [39] Frías M, Brito N, González C. The Botrytis cinerea cerato-platanin BcSpl1 is a potent inducer of systemic acquired resistance (SAR) in tobacco and generates a wave of salicylic acid expanding from the site of application. Mol Plant Pathol. 2013 Feb;14(2):191-6. doi: 10.1111/j.1364-3703.2012.00842.x. Epub 2012 Oct 16.
      [40] Engelberg-Kulka H, Hazan R, Amitai S. mazEF: a chromosomal toxin-antitoxin module that triggers programmed cell death in bacteria. J Cell Sci. 2005 Oct 1;118 (Pt 19):4327-32. doi: 10.1242/jcs.02619.
      [41] Jinbin Wang, Tong Yue, Chuan He, Yifan Zhou, Yinshuang Bai, Qingwei Li, Wei Jiang, Yanna Huang, Xiaofeng Liu, Biocontrol of tomato bacterial wilt by a combination of Bacillus subtilis GSJB-1210 and ningnanmycin, Scientia Horticulturae, Volume 321, 2023, 112296, ISSN 0304-4238, doi:/10.1016/j.scienta.2023.112296.
      [42] Coelho RV, de Avila E Silva S, Echeverrigaray S, Delamare APL. Bacillus subtilis promoter sequences data set for promoter prediction in Gram-positive bacteria. Data Brief. 2018 May 13;19:264-270. doi: 10.1016/j.dib.2018.05.025.
      [43] Zhang XZ, Yan X, Cui ZL, Hong Q, Li SP. mazF, a novel counter-selectable marker for unmarked chromosomal manipulation in Bacillus subtilis. Nucleic Acids Res. 2006 May 19;34(9):e71. doi: 10.1093/nar/gkl358.
      [44] Samaras A, Roumeliotis E, Ntasiou P, Karaoglanidis G. Bacillus subtilis MBI600 Promotes Growth of Tomato Plants and Induces Systemic Resistance Contributing to the Control of Soilborne Pathogens. Plants (Basel). 2021 May 31;10(6):1113. doi: 10. 3390/plants10061113.
      [45] Daudi A, O'Brien JA. Detection of Hydrogen Peroxide by DAB Staining in Arabidopsis Leaves. Bio Protoc. 2012;2(18):e263. Epub 2012 Sep 20.