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First things first, what is Agrocapsi? Take a look at the importance of chilli in Mexico and worldwide, the devastating wilt caused by Phytophthora capsici and our solution: a mix of antimicrobial peptides and interference RNA.
Agriculture is the basis of today's society. Over ten thousand years ago, humankind adapted agriculture and livestock into its lifestyle (Bula, 2020). With this, larger social groups formed as they became independent from hunting and gathering plants. This paved the way for the beginning of civilizations.
In 2018, agriculture accounted for 4% of the global gross domestic product (GDP). On the other hand, in some least developed countries, this percentage was up to 25% of GDP (World Bank, 2016). As we can see, this activity has become crucial to economic growth worldwide. Meanwhile, in 2021 the IBRD destined $6.7 billion USD for agriculture-related commitments. Also, there were 157 projects helping over 11 million farmers with agricultural assets and adopting improved agricultural technology (World Bank, 2016).
For our country, agriculture is a pillar of the economy. Currently, 13% of the population in Mexico works in agriculture (>3.3 million farmers). This means that 3.7% of the country's GDP comes from a wide variety of crops in agriculture (Organización de las Naciones Unidas para la Alimentación y la Agricultura, n.d.). In addition, agriculture is an activity passed from generation to generation. This brings food to the table, jobs, incomes, and impulses our culture and traditions. We harvest many of those ingredients that make our dishes unique, and proud to say they represent us. Some of the main crops Mexico focuses on are (González-Trinidad et al., 2020):
Mexicans work, cry, smile, live, and sing together. We appreciate our traditions, flavors, parties and culture, all represented in the way we cook. We maintain a wide variety and tastefulness in our meals. This traces its origins to Aztecs, Zapotecs, and many other pre-hispanic civilizations (Mendoza & Sánchez, 2020). A great number of our plates revolve around chilli. It gives that fiery, yet satisfying response to our bodies. Although it may not be for everyone, 90% of our traditional dishes include chilli (proving we love intensity).
Our culinary traditions are so meaningful that UNESCO declared them World Heritage (Presidencia de la República, 2015). It has such importance to us that it has become a symbol of our identity around the globe. Hence, a great income relies on its production (Figure 3). Around 150,000 hectares produce chilli and we export up to 30% of it, creating a national income of 1.6 million dollars (Sánchez-Toledano et al., 2022; SAGARPA, 2017).
Our team is proud to say that Chihuahua is the biggest producer of chilli in Mexico (López et al., 2021). Therefore, we are glad to contribute by developing a solution to one of the biggest problems on chilli crops.
Agriculture is a fundamental activity, but it's no easy task. It faces several challenges such as droughts, soil erosion and climate change. Nonetheless, one of the most concerning problems for farmers are plagues and diseases. In chilli production, a notorious disease is wilt caused by the oomycete Phytophthora capsici. This oomycete is one of the most destructive pathogens of chilli plants. Its impact is so severe that it is responsible for up to 100% of losses in production (Nabor-Romero et al., 2020). This is due to its ability to quickly overcome the host defense system (Ali et al., 2019).
P. capsici also causes foliar blighting, damping-off, root, stem, and fruit rot of the host. (Hausbeck & Lamour, 2004):
P. capsici infects the plants via haustoriums that enter the cell. Then, the oomycete releases effector proteins which inhibit the plant´s natural defense. Aside from this mechanism, it also consumes intracellular nutrients (Zhang et al., 2022).
The oomycete has a life cycle with both sexual and asexual phases. Sexual reproduction occurs when both A1 and A2 mycelium hybridize in the same field. This union gives place to the formation of antheridium and oogonium. When these two parts fuse together, they form oospores. These oospores germinate in two different ways: producing germ tubes that branch, giving rise to the typical mycelium; or producing sporangium. On the other hand, asexual reproduction happens under favorable environmental conditions. By this type of reproduction the oomycete can produce a large number of sporangia on the surface of the affected tissue. Zoospores develop massively in the sporangium and are released into the environment under rain or irrigation conditions (Ristaino et al., 1999).
Treatments against this pathogen consist mainly in the use of agrochemicals. One of the most used is mefenoxam, which is toxic to humans, causes environmental damage, and promotes the arisal of resistant pathogen populations (Sánchez-Gurrola et al., 2019 ; Tomah et al., 2020).
Wilt has become a great concern for producers since (Granke et al., 2012):
Many of our team members have spent their lives alongside the field. That’s why we wanted to focus on agriculture, specifically on chilli, a fruit that represents us. Our goal was to deal with this problem in a safe and responsible manner. Through synthetic biology, we realized that we could generate a solution. This is how Agrocapsi was born: A biofungicide which combines the power of two antimicrobial peptides and interfering RNA to safeguard the food safety of our country and the world.
After a lengthy literature review, we concluded that antimicrobial peptides (AMPs) could be useful to fight P. capsici in a sustainable manner. AMPs not only succeed in inhibiting the growth of certain microorganisms but also, microorganisms rarely generate resistance (Varasteh et al., 2019). To achieve the attack of the oomycete at different stages, we selected two antimicrobial peptides: DrsB1, a dermaseptin, and PcOSM, an osmotin-like protein (Khademi et al., 2019; Geetha et al., 2021). We also sought to reduce the pathogenicity of P. capsici to make the biofungicide more effective. Hence, we aimed to silence the genes coding for the RXLR effector proteins through small interfering RNA (iRNAs).
Agrocapsi's active ingredients are:
An osmotin from Piper colubrinum.
(CBD)2-DrsB1. A dermaseptin from Phyllomedusa bicolor fused with a tandem repeat of a chitin binding domain (CBD).
A specific siRNA against the gene coding for the effector protein RXLR1.
BUT, WHY THESE MOLECULES?
In addition to inhibiting the growth of P. capsici and helping prevent infection without endangering the health of users or the environment, these molecules have complementary action mechanisms:
It is an osmotin produced by Piper colubrinum. This is one of the few plants with a natural resistance against P. capsici (Malik & George, 2018). PcOSM is a non-toxic 24 kDa protein that destabilizes the oomycete’s cell membrane. Once inside the cell, it induces reactive oxygen species which help to eradicate the oomycte (Geetha et al., 2021). This peptide is able to act at different stages of the oomycete life cycle: inhibits hyphal (germ tube) growth and reduces spore germination by reducing spore viability (Geetha, et al., 2021).
DrsB1 comes from Phyllomedusa bicolor. DrsB1 is a defensin that also inhibits P. capsici by destabilizing the cell membrane (Khademi et al., 2020). Also, the CBD protects the AMP from degradation and improves the union with zoospores (Varasteh et al., 2019). Although P. capsici lacks chitin in the membrane, zoospores do have it (Cheng et al., 2019).
The application of these molecules will reduce the pathogenicity of P. capsici through the silencing of key genes, such as the RXLR effectors of P. capsici (Cheng et al., 2022; Edwards et al., 2020; Demirer et al., 2020). The RXLR effector proteins are responsible for suppressing the plant's defense system, so silencing this gene reduces the pathogenicity of the oomycete and gives the plant the opportunity to defend itself against infection (Fan et al., 2018).
The combination of these three molecules increases the effectiveness of Agrocapsi, since they attack P. capsici at different key stages as shown in Figure 7. You can read more in depth about the action mechanism of each of these molecules in the Design page.
Our goal is to ease farmers' jobs and offer a solution against one of the toughest problems in chilli crops. With Agrocapsi, we want to protect our culture, GDP, families, workers, farmlands, and identity. To achieve this, we plan to express both peptides and siRNA in E. coli BL21 (DE3) and HT115, respectively. Then, encapsulate our molecules, generating a product that can be easily applied through irrigation. To make our project go from an idea to a reality, we plan to follow the methodology shown in Figure 8.
We are Tec Chihuahua, and we believe that synthetic biology can enhance sustainability in crop production while rescuing an icon of our identity.
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