We noticed that current agrochemicals solutions face many problems. Lack of specificity and environmental damage are some of the main, but far from all. A way to ensure a change to these problems is the use of antimicrobial peptides. These molecules emerged as an alternative to antibiotics to boost the plant's innate immune system. Unlike chemical antibiotics, susceptible strains of microorganisms rarely become resistant when using AMPs (Shams et al., 2019). Another big problem we noticed is the strength of the pathogen since it is able to kill a plant in three days (Pérez et al., 2017). Due to these reasons, we decided to use strong AMPs to enhance our solution's effectiveness. Our choses were:

These peptides were chosen due to their mechanism of action. When combined with the power of siRNAs, we can attack the phytopathogen during the important stages of its development.

(CBD)2-DrsB1

Dermaseptin B1 (DrsB1) is a 3.2 kDa antimicrobial peptide isolated from Phyllomedusa bicolor and it is one of the strongest AMPs known among all AMPs. It supplies both antibacterial and antifungal protection to a broad range of plant pathogens but shows no toxic effects on plant and mammalian cells (Khademi et al., 2020). The fusion with a tandem repeat of a chitin binding domain (CBD) helps the peptide attach to the fungal cell wall chitin, thereby increasing the lytic activity of the catalytic domain. The resultant molecule turns out to be 22.1 kDa.

Action mechanism: Although chitin is a major component of the fungal cell wall, oomycetes contain none or very little amounts. Yet, zoospores and released sporangia are the only stages in which P. capsici presents chitin (Cheng et al., 2019). During this phase, the CBD helps the peptide to attach to the surface of chitin polymer. Thus, it promotes the destruction of chitin by hydrolyzing -1,4 glycosidic bonds (Khademi et al., 2020). Furthermore, once DrsB1 binds to the zoospore, it destabilizes lipid membranes of pathogens. Thus, it causes disorders in the formation of the membrane. Consequently, accumulation of cationic AMPs punch transient holes and, hence, may result in cell lysis (Shams et al., 2019).

PcOSM

Piper colubrinum is one of the few organisms with natural resistance to many fungal diseases. This also includes wilt caused by P. capsici. It has been reported that this osmotin gene is differentially overexpressed in response to the infection of P. capsici (Mani & Manjula, 2010). The osmotin-like AMP is a 24 kDa multifunctional stress responsive cationic protein. It belongs to the pathogenesis related-5 (PR-5) family, which accumulates in response to both biotic and abiotic stresses.

Action mechanism: This peptide has the ability to intervene since the beginning of the pathogen's life cycle. It has an influence on spore germination, spore lysis, and reduction of spore viability. However, if the oospore manages to germinate by emitting a short hyphae (the germ tube) PcOSM has the opportunity to act. This is due to its antifungal activity through inhibition of hyphal growth. PcOSM has disruptive effects that increase membrane permeability. Thus, it causes morphological changes, increases ROS production, and leads to the oomycete's cell death (Geetha et al., 2021).

siRNA DESIGN

The suppression of the plant's natural defense system is one characteristic that makes P. capsici such an aggressive pathogen (Li et al., 2020; Fan et al., 2018). This is possible thanks to the RXLR effectors (Liang et al., 2021). Thus, we decided to use siRNA technology to improve biotreatment efficacy. This way, we can reduce the pathogenicity of microorganisms by silencing key genes (such as the RXLR effectors of P. capsici). In addition, this technology allows us to generate different molecules to attack other microorganisms that are part of the complex that causes wilt. Hence, it is a great add-on to combat wilting as mentioned by our stakeholders in our integrated human practices.

Several authors have demonstrated the ability of fungi and oomycetes to take up siRNAs from the environment (Cheng et al., 2022; Werner et al., 2020; Qiao et al., 2021). Thus, we decided to integrate siRNAs as one of the ingredients of our biofungicide.

The plan is that Agrocapsi will contain synthetic gene-specific siRNAs produced through synthetic biology strategies. These siRNAs will enter through the pathogen membrane. Once inside the cell, the molecules will be processed and will silence the RXLR1 gene of P. capsici. Thus, production of effector proteins that attack the plant will stop (Figure 3). Click on the picture to know more!

Once we chose to silence the RXLR1 gene, we began to design the sequence of the siRNA we planned to produce. For this we used the online tool E-RNAi (Horn & Boutros, 2010).

For siRNA selection we followed the following considerations (Fakhr et al., 2016):

• The first nucleotide of the siRNA sequence can either be an A or a G.

• Choose sequences with low GC content

• Avoid 5' and 3'UTRs

• Select a ~21 nt sequences in the target mRNA

Also, in collaboration with the Tec-Monterrey team, we performed an analysis of the thermodynamic properties of our sequence. For this we used a tool they developed to evaluate the relative efficiency of siRNAs, where the worst siRNAs have an efficiency of 0 and the best ones have an efficiency close to 100. Our siRNA showed an efficiency of 93%. You can read more on the siRNA registration page.

A summary of the genes we seek to silence is shown in Table 1.

Organism	Gene	Justification	Reference
P. capsici	RXLR	The RXLR effectors of P. capsici are critical for its pathogenicity. This is mainly due to the fact that this effector proteins are able to suppress the plant immune system.	(He et al., 2018; Cheng et al., 2022)
F. oxysporum	Ornithine Decarboxylase	This protein is essential for the growth and development of the fungus. In addition, iRNA silencing of this gene is reported to reduce disease incidence in plants.	(Singh et al., 2020)

Once the peptides and siRNAs were chosen it was time to design the bacterial constructs for their recombinant expression. Escherichia coli is the strain of excellence for recombinant expression of bioproducts. This is because of its simplicity and the transformation is not very complicated. It also has a faster growth rate compared to other microorganisms such as yeasts and mammalian cells. Additionally, it is worth mentioning that it is less expensive than other microorganisms. siRNA production must be in another chassis since certain strains have reported better results due to the absence of RNAases.

On the Parts page you can take a look at the constructs we have designed to make Agrocapsi a reality. Below you can read a justification of the choice of each of the parts used for the development of our project.

CHASSIS

To produce both peptides and siRNAs we have chosen two chassis:

Escherichia coli BL21(DE3)

E. coli BL21 (DE3) chassis was selected for PcOSM and DrsB1 peptides because it contains the λDE3 prophage that includes the T7 polymerase gene under the control of the lacUV5 promoter and is inducible by IPTG. This chassis is recommended for the recombinant expression of genes that include the lac promoter such as in our case. We wanted the lac promoter for our protein production, so we can induce its expression and have control of it. Equally important, this strain offers the lack of proteases such as Lon and OmpT, which ensures a good production of our peptides (González & Fillat, 2018).

Escherichia coli HT115

This chassis was chosen due to its several features that help the iRNA production. It avoids enzymatic hydrolysis in the accumulated genetic material, a mutation is generated in the gene encoding RNase III. Also, it presents the RNA polymerase T7 (inducible either by IPTG or lactose), allowing the production of RNAs starting from a T7 promoter. Something extremely important was to avoid the siRNAs to degrade, so this train of E. coli is deficient of RNase III (Papic et al., 2015).

PROMOTER

LacI regulated promoter

The LacI regulated promoter is very similar to T7, but the main difference is that this one is weaker. Sometimes T7 may be a little too strong in its expression and that can cause protein misfolding, toxicity or other mistakes. This promoter is also positively induced by IPTG.

T7 promoter

As mentioned earlier, we were searching for an inducible promoter because that way we control the expression of the proteins and siRNAs. This promoter is induced with IPTG.

DsbA

We included a coding sequence for DsbA from P. aeruginosa for the two peptides, which would help in the formation of disulfide bonds and the proper folding of the peptides.

To achieve this, we have sought to design a solution around the needs of the people affected by the problem. We wanted to make a solution focused on the development of society, useful and accepted in our country and in the world. You can read more about our way to achieve this on the Integrated Human Practices page.

DELIVERY METHOD: CHITOSAN NANOCAPSULES

After a long research with the iGEM UAM team, we have decided that the delivery method of our biotreatment will be chitosan nanocapsules.

Nanoencapsulation is a technique that enables the regulated delivery of bioactive substances such as antimicrobial peptides, iRNA, active compounds, and others that are beneficial for the plant (Patiño, 2017).

Characteristics

Due to their biodegradability, biocompatibility, and low toxicity, our chitosan-based nanoparticles (NP) have distinguished themselves (Sharifi-Rad et al., 2021). They enable the stability and focus of these compounds. The resulting amphiphilic nanocapsule has components that are both hydrophobic and hydrophilic. This characteristic encourages the development of micellar structures. They also create a stabilizing contact between the nanoparticle's core and the surrounding aqueous medium.

Toxicity

Prokaryotes, animals, plants, yeasts, and prokaryotic cells contain a range of enzymes that may break down chitosan. Amino-sugars or non-toxic substances are released when this breakdown takes place. Chitosan has no negative effects when it comes into contact with living things. In comparison to the toxicity of salt and sugar, an LD50 toxicity of 16 g/kg has been recorded; this is regarded as mild toxicity and is safe for both plants and animals (National Toxicology Program, 2017).

Biodegradation

Due to their general toxicity and low environmental degradability, synthetic nanoparticles produced by nanotechnology have been heralding an uncertain future for agriculture. In order to combat the impacts of synthetic nanoparticles, several nanoparticles have been developed, such as those based on chitosan, which are biocompatible and low in toxicity, in contrast to their non-biodegradable counterparts.

Chitosan nanoparticles' ability to avoid the issue of bioaccumulation makes this one of their most researched properties, making them suitable for use in the field (Hofmann et al., 2020).

Chitosan-based nanoencapsulation manufacturing

There are numerous ways to create chitosan NPs, and the process is quite simple. The protocol to apply depends on the financial requirements for industrial manufacturing. Ionic gelation is a process that lowers expenses and doesn't need sophisticated lab equipment or reactive substances.

Chitosan must first be dissolved in aqueous acetic acid at a concentration of 1% wt. or hydrochloric acid (HCI) at a concentration of 0.1% wt. NaOH 1 M is added to the solution to bring the pH level down to pH 5. followed by 40 minutes of stirring or ultrasonication of the solution.

The cross-linking agent preparation can be done concurrently. In deionized water, TPP is made as a 0.1% wt. solution adding 0.1 M of HCl to the solution to change the pH.

When both solutions are complete, the chitosan solution is dissolved in a polyanionic solution (TPP) to produce the chitosan cation. It is also possible, but not required, to add a stabilizing agent such as Poloxamer. The precipitation forms the NPs after the chitosan/TTP solution has been gently stirred for 10 minutes at room temperature. The unreacted chitosan and TPP are then removed from the suspension by centrifuging. One option for resuspending the nanoparticle pellet is in water (ChitoLytic, 2022).

There are no guidelines as to how much or at what concentration each substance should be. Those parameters could alter according to the properties of any potential NPs. Their size or porosity may fluctuate with an alteration in concentration or time. Chitosan NPs are able to encapsulate many molecules thanks to the capacity to make these changes.

Chitosan-based nanoencapsulation manufacturing

Chitosan nanoparticles must have previously been present in a solution at a concentration of 3.0 g/L1 in order to encapsulate. Then, 1000 mL of a solution containing 700 mL of TPP (1.2 g/L) and 300 mL of siRNA (550 g/L) will be added dropwise to the chitosan solution while being stirred magnetically. This remedy will be carried out, although centrifugation might be used to separate the nanoparticles (de Britto et al., 2014).

LIFE CYCLES OF THE DIFFERENT ORGANISMS THAT CAUSES WILT

Throughout the development of our Integrated Human Practices, farmers mentioned to us the impact of wilt. However, although P. capsici is one of the most devastating pathogens causing this disease, it is not usually the only one found when there are signs of disease. To help meet the needs of growers, we have decided to implement the use of siRNAs that inhibit the growth and/or reduce the pathogenicity of each microorganism. To achieve this, the first step is to understand the life cycle of each of these microorganisms.

Fusarium oxysporum

Fusarium oxysporum is considered a threat to the chilli crop and has caused losses of up to 40% of total chilli production (Shaheen et al., 2021).

The life cycle of the pathogen is of two phases: a soprophytic and a parasitic phase (Singh & Vyas, 2021). In the parasitic phase, the fungus occurs in the presence of a host when temperature and moisture conditions are optimal (Worku & Sahe, 2018). The fungus enters the plant through the root, mechanical damage, or hail injury (Shaheen et al., 2021).

The fungus initiates its growth in the vascular tissues of roots and stems. It enters the roots of plants using its mycelium or sporangial germ. When it reaches the xylem, it begins to grow in the vessels and reduces the amount of water and nutrients in the plant. Wilt symptoms appear as a result of the collapse of the affected vessels and clogging by the mycelium. Also, the reduction of water causes wilting and death of the plant (Gordon, 2017) ( Figure 5).

Rhizoctonia solani

Rhizoctonia solani is a fungus that does not produce asexual spores. The fungus survives mainly by its mycelial and sclerotial forms in crop residues and soil. Sclerotia can survive in soil without a host for 8-10 years (Shu et al., 2019).

Under favorable conditions, R. solani sclerotia germinate to form hyphae in the soil, which can grow both inside the plant and on the surface. The conducive condition for infection requires humidity greater than 25% and a temperature range of 20°C-35°C (Haque & Parvin, 2021).

The fungus can penetrate the host by forming an infection cushion. Also it can produce an appressoria that penetrates through the cell wall and takes nutrients from the plant cell. Wilting and complete collapse of cotyledons and immature seedling death occur. R. solani causes damping-off to young seedlings and root and crown rot older plants.(Haque & Parvin, 2021; Espinoza-Ahumada et al., 2019).