Inspiration

In the realm of agriculture, synthetic biology offers exciting possibilities, especially when focusing on vital crops like rice. In our brainstorming phase, we had the privilege of attending a talk by Dr. Kutubuddin A Molla, an expert in genome engineering using CRISPR-Cas systems for rice plants. His talk sparked our interest in exploring CRISPR technology and its application to tackle various diseases affecting rice plants. Given our proximity to rice farming communities, we took the initiative to engage with local rice farmers and understand the challenges they face. We discovered alarming issues like pesticide misuse and the adverse effects of chemical pesticides on rice crops. Many of our team members have family members involved in farming, which deepened our understanding of the problems at hand. Through these conversations, we identified a concerning issue - bacterial blight, caused by the Xanthomonas oryzae pv oryzae bacteria, posing a significant threat to rice cultivation.

The Problem - Xanthomonas Oryzae

Infections caused by Xanthomonas species, particularly Xanthomonas oryzae pv oryzae, pose a significant problem in agriculture, specifically in rice farming. This bacterium is responsible for bacterial blight, a devastating disease affecting rice plants. Bacterial blight manifests as water-soaked lesions on leaves, eventually leading to wilting, browning, and death of the plant's tissue. The disease disrupts photosynthesis and nutrient transportation, severely impacting crop yield and quality. In Southeast Asian countries, yield losses are expected to range from 10%–20% in middling conditions to up to 50% in favorable conditions. The earlier the disease manifests itself, the greater the yield loss.

There are about 27 different plant species that are affected by Xanthomonas infections. These species display high levels of genetic diversity, damaging up to 400 different plant species (3). Hence, during our design, we had in mind to design our bio-control agent not just limited to Xanthomonas oryzae, but also to target it against other virulence factors across the conserved species. One of the well-known interesting facts is the bacteriophytochrome-mediated mechanism used by the Xanthomonas infection method. It is hypothesised that day-night variations play a crucial role in Xanthomonas infections. If these changes are altered due to climate change there can be a significant rise in the infections taking place.(13) We wanted to help our farmers in Agricultural field by doing something that would reduce bacterial blight infections and in turn reduce losses they suffer due to it. This motivated us to create our novel solution:

Xanthacinator

Xanthacinator is a two fold solution against Bacterial Blight. Xanthacinator has been designed around the feedback from stakeholders of the agriculture industry to make it user friendly. It involves a Detection kit and a novel Biocontrol Agent.

Detection

In order to curb the crucial lack of knowledge to properly distinguish between nutrient deficiency and bacterial blight, we developed a detection kit that uses an engineered E coli. The engineered E. coli is capable of detecting quorum sensing molecules, Diffusible Signal Factors (DSF) released by Xoo. In presence of DSF molecules, it activates the downstream genes which gives bright purple fluorescence visible to naked eyes.

Biocontrol agent

1. Outer Membrane Vesicles (OMVs):

1. Our project is inspired from nature. Escherichia coli being a gram negative bacteria has a natural property of production of Bacterial Outer Membrane Vesicles (OMVs).
2. OMVs are used to transfer various biomolecules, including DNA, toxins, and virulence factors for intra- and inter-species communication. [4].
3. An advantage of OMVs as a delivery vector is their protective role for DNA, RNA, and proteins, safeguarding them from harsh conditions. [5]
4. We decided to utilize this intrinsic property of E. coli and use it for our needs as a delivery vector of our target molecules which is the FnCas12a/crRNA complex.

Figure: Schematic representation of Bacterial Outer Membrane Vesicles [7]

2. FnCas12a

Another amazing natural machinery of nature is CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats). In contrast to the more popular CRISPR-Cas9, Cas12a offers many advantages:

1. Cas12a can indiscriminately degrade single-stranded DNA in addition to its primary function of cleaving double-stranded DNA
2. It has a smaller size of ~130 kDa than Cas9 which has a size of ~160 kDa [7, 8]
3. Unlike Cas9, Cas12a processes its own mature crRNA without requiring any tracrRNA [9]
4. And hence makes it advantageous to use it for multiplex genome editing
5. Our engineered E. coli releases OMVs loaded with CRISPR-FnCas12a with crRNA complex.The Crispr-RNAs are designed against multiple pathogenic genes of Xanthomonas.

Figure: The diagram illustrates Cas12a (Cpf1) alongside a crRNA that is bound and targeting a specific genomic location. [14]

3. Twin-Arginine Translocation Pathway- for loading into OMVs

1. Twin Arginine Translocation (Tat) pathway is an effective pathway to transport folded proteins across the inner membrane, preserving their native conformation.
2. The Tat machinery in Escherichia coli consists of three essential proteins: TatA, TatB, and TatC. The TatBC complex interacts with Tat substrate proteins by recognizing their signal peptides.
3. Once activated. TatA can create an aqueous channel for substrate transport. [10]
4. Our recombinant protein is fused with sptorA signal peptide for its efficient transport across the membrane into periplasmic space. Once loaded into the periplasmic space, the recombinant proteins can get loaded into the Outer Membrane Vesicles. [11]

Figure: Mechanism of how TAT complex works in translocating proteins fused with a Tat signal peptide from cytoplasm to periplasmic space [15]

How does our Biocontrol agent work?

Our chassis, Escherichia coli USML2, is a rice plant growth-promoting endophyte. It grows in the inner leaf surfaces and the interior of the plant tissues and thus shares the same niche as Xoo (pathogen) Upon sensing the presence of Xoo, our 'chassis' releases OMVs that carry CRISPR Fncas12a/crRNA ribonucleoprotein complex. These packages work like tiny missiles to target and eliminate the harmful Xanthomonas pathogen.

PROJECT POTENTIAL AND POSSIBILITIES

We have shown application of our project for one such model phytopathogen, i.e. Xoo. However our project can be expanded to be used against many more phytopathogens. Not only this, our project sets a primer to use Bacterial OMVs as delivery vehicles. However they can be used for many more applications like:

OMVs have diverse biomedical applications, including vaccines, adjuvants, cancer immunotherapy, drug delivery, anti-bacterial adhesion agents, and antibacterial therapy.
OMVs can serve as effective vaccines against pathogenic bacteria, inducing both cellular and humoral immune responses.
As adjuvants, OMVs enhance immune responses when mixed with antigens in vaccine preparations.
In cancer immunotherapy, OMVs can induce immune responses against tumor tissues or serve as nanocarriers for loading chemotherapeutic agents. [12]