Our project system has achieved a paradigm shift from passive response to proactive intervention through the in-depth collaboration of three core modules: using engineered Bacillus subtilis as a "biosensor" to convert sucrose in aphid honeydew into a remotely detectable methyl salicylate signal; According to the signal instruction, the RNAi delivery system performs defensive functions, while the engineered Metarhizium exerts dual killing effects through external infection and internal gene silencing. These three modules are seamlessly connected via a digital platform, jointly constructing a complete biological defense network that spans from early warning to precise intervention.

For the monitoring system, we designed a sucrose-responsive methyl salicylate pathway, which consists of three processes:

1. Sucrose in honeydew enters Bacillus subtilis, enters its natural shikimate pathway, and ultimately synthesizes the key intermediate chorismic acid.
2. The pchB and pchA gene clusters introduced from Pseudomonas aeruginosa express isochorismate synthase (BBa_25QMMMDA) and isochorismate pyruvate lyase (BBa_25ESDI6C) to catalyze the conversion of chorismic acid to salicylic acid.
3. The BSMT gene introduced from Petunia nyctaginiflora encodes a salicylic acid methyltransferase (BBa_25CQF6CS) that catalyzes the methylation of salicylic acid, converting it into volatile methyl salicylate.

Finally, the components are integrated into composite part BBa_25PYPDA5 to perform the sucrose-responsive function. Subsequently, methyl salicylate volatilizes into the air and is recognized by sensors, and the aphid population density is inferred based on its concentration.

We have compiled a plug-and-play RNAi prevention and control toolkit—the "Plug-and-Play RNAi Toolkit" collection (UUID: 2d306d88-673e-4aa8-b895-ed7842a9059b)—for teams planning to use RNAi for pest and disease control in the future. We hope this toolkit will help them quickly develop safe, efficient, and targeted green agricultural solutions. The toolkit includes:

1. RNAi therapeutics
2. targetable delivery VLPs (Virus-Like Particles)
3. templates for high-efficiency in vitro dsRNA (double-stranded RNA) production

For three key target genes of the brown citrus aphid (Toxoptera citricida), we designed a total of 7 small RNAs during the engineering implementation process for the specific control of aphids.

1. For dsRNAs: We screened dsRNA targeting single genes, including dsCHS (BBa_25DD7Y31), dsCYP450 (BBa_25AI7XC8), and dsCP (BBa_25ZZCL83). These were compared with dsRNA (BBa_25EZA8EJ) to analyze the feasibility of designing dsF3 molecules with multi-target gene fusion.

2. For tandem RNAs: We developed tri-shRNAs (BBa_25SVHEZZ) and bi-amiRNAs (BBa_2524PT52), which contribute to RNA stability and correct recognition by RNAi-related proteins.

Thus, we completed the iteration from single-gene to multi-gene, and from dsRNA to shRNA to amiRNA (see engineering for details). Meanwhile, we designed and screened the bidirectional triple terminator template with the highest transcription efficiency (BBa_2576URGK), achieving an in vitro transcription system with high yield, high purity, and low time consumption.

Through the design of various small RNAs, we have not only addressed the issues that may arise from fixed sequences in traditional miRNA design, but also fully leveraged the advantages of the miRNA processing mechanism to enhance the effect of multi-gene targeted silencing. We expect that the above small RNA designs will significantly improve the efficiency of aphid control and provide more effective lethal effects.

To ensure the stability of the designed RNAi molecules in the complex plant-insect interaction environment, we selected MS2 virus-like particles (VLPs) as protective delivery carriers. MS2 VLPs can achieve self-assembly into structurally stable complexes through specific recognition between capsid proteins and target RNAs, thereby maintaining the integrity and functionality of RNAi molecules after application.

1. Based on the natural assembly mechanism of MS2 virus, we developed a recombinant MS2 VLP delivery system. This system retains the self-assembly and pac site recognition functions of capsid proteins, but completely removes the viral genome to ensure it has no replication or infection capabilities. We introduced a pac site into the C-terminus of target RNAi molecules, enabling them to be specifically recognized by capsid proteins. Using an in vivo assembly strategy, we co-expressed MS2 capsid protein dimers and RNAi molecules containing pac sites (BBa_25DO50CT) in Escherichia coli BL21(DE3), allowing them to directly self-assemble into complete VLP particles intracellularly. Finally, VLP-RNAi complexes with uniform structure and complete RNA encapsulation were obtained through purification.

2. By fusing TAT cell-penetrating peptide and GBP3.1 aphid intestinal targeting peptide with VLP particles (BBa_25GBKM7R, BBa_25KD6QSQ), the targeted delivery capability of VLP-RNAi complexes in aphids was further enhanced.

In response to potential local outbreaks of aphid populations, we have designed a targeted therapeutic agent based on engineered Metarhizium anisopliae. This agent can not only infect pests externally like natural Metarhizium anisopliae, but also serve as a living biological factory, continuously producing and delivering our carefully designed triple-fusion RNAi molecule (pBar_PtrpC-dsF3：BBa_25WG3A50) inside aphids, achieving a "double blow" to pests and rapidly eliminating the aphid populations that have erupted in the environment.