Project Description
The Problem
Citrus greening, or Huanglongbing (HLB), is an agricultural disease in citrus of global impact. The far-ranging distributions, tricky spread pathway, and currently untreatable pathogen together make this disease a huge problem to farmers. Chronic symptoms gradually show up in infected citrus plants, usually leaves dotted with yellowish mottles appeared in the early stage, followed by branches dieback and, finally, reduce fruit quality from aspects of fresh weight, nutrient contents, juice production, and even acidity [1,2]. First described in 1929 and reported in 1943, HLB is considered to originated from Aisa and Africa [3]. Decades later, HLB spread to America, especially Florida where citrus is a great industry, due to the facilitation of transportation internationally [1]. Currently about 90% of citrus trees in Florida was infected, while most manufacturing regions in China, including Fujian, Guangdong, and Zhejiang, are influenced by this deadly plant disease [4]. The gram-negative bacteria “Candidatus Liberibacter asiaticus” (CLas), along with the affiliated family members like africanus (CLaf) and americanus (CLam), was detected and identified as the main pathogen of HLB [5]. However, the CLas was currently unculturable due to unknown reasons, which tremendously hinders relevant research [6]. CLas stays in pest D. citri for the pre-infection stage of life cycle and enters citrus tree vascular system for the major infection stage through stinging [6]. Being the major vector responsible for HLB spread, D. citri has obtained tremendous habitats globally, ranging from the US to Brazil, and almost all HLB-influenced regions in China [1].
Citrus farmers are those influenced most profoundly in face of citrus greening. Mr. Huang, a farmer in Jiangxi who owns a citrus farm, claimed a cost of over 300 RMB for chemical drugs, pesticides, and labor to prevent his citrus trees away from this terrible disease per mu (a Chinese unit for area, 1mu = 667m2) and per week. In summary, about 100000 RMB would be wasted on citrus greening prevention per mu per growth period, which is equal to the sum of all other costs. Besides, should any trees be infected would the whole farm renew all citrus trees in prevention of further loss. A more feasible and economic approach to prevent and treat citrus greening thus holds a great potential to make a real difference on local and even global citrus greening prevention patterns.
Efforts to End Citrus Greening
To date, a few attempts have been made in the field of synthetic biology to seek approaches to HLB treatment. Agricultural company Southern Gardens Citrus in the US once proposed an engineered citrus tristeza virus (CTV) to eliminate the pathogen [7]. The plant virus that originated from the citrus is now engineered on its genome so that a special spinach defense protein can be produced in infected citrus, which is expected to be lethal to the pathogens. Although the researchers claimed no influence in the quality of orange fruits produced from treated citrus trees, doubts regarding food safety are still in the air.
Other attempts were made to block the transmission host, psyllid D. citri, of the pathogen bacteria. RNA interference technology utilizes the mechanism of RNA directed gene silencing to knock down the expression of target genes. Small RNAs designed to turn off pathogen transmission related genes were synthesized to help psyllid get rid of pathogens [8,9]. However, the storage and delivery of small RNAs are still left unsolved.
Some previous iGEM teams also worked on this topic. For instance, the team “FSU” in 2019 focused their attention on the pathogen bacteria CLas. Antimicrobial peptides (AMP) are oligopeptides that have novel capacities in removing bacteria without leading to serious antibiotic resistance issues. In their project, a cell-free synthesis system was developed to produce several types of AMPs or mixtures, which were collected and injected into citrus trees with a nicely designed dose control system.
Engineered Phage for SAFE Bio Control
In consideration of the safety issue such as those in CTV virus method. We proposed to create a device that can exclusively act and execute functions in the pathogen or vector. One of the top options is an obligatory bacterial phage on CLas since the phage only infects bacteria and pose less threat to human the the citrus plants. After iterations of literature review, a phage that was named as CLasMV1 was shown to possess infection specificity to CLas bacteria [10,11]. More importantly, the phage obtains a symbiosis relationship with the CLas bacteria out of unknown reasons [11]. This feature confers the opportunity and time for the foreign genetic circuit introduced by the engineered phage to execute its functions. Therefore, CLasMV1 was selected as our chassis phage for engineering.
The art of CLas bacteria life cycle adds versatility to engineered phage. On one hand, the phage can be engineered to directly eliminate the pathogenic bacteria; On the other hand, the phage can be engineered to synthesize substances for the vector psyllids elimination since it can be brought inside of the pest with CLas. Inspired by previous ideas of using AMP and RNA to target CLas and D. citri, we proposed to engineer our phage for the synthesis of these two substances in different scenarios.
Despite the fact that CLasMV1 infects CLas bacteria, it still has the concern for foreign gene contamination and spreading in the environment. The potential risks of microbes other than CLas take in engineered phage genome poses the threat of local microbiome homeostasis disorder. In consideration of this bio-safety issue, an active biosafety device to our genetic circuit so that the expression of foreign genes can only be allowed under the circumstance that it is in CLas bacteria. Transcriptional factors (TF) are excellent biomarkers to be sensed in a intracellular circumstance like this. The MarR family TF LdtR was identified as a unique and important transcriptional regulator in CLas bacteria [5]. Therefore, LdtR was taken as the marker of CLas, which specifically activates the biosensor, B488_05770 promoter (P_B488_05770), to start the transcription of downstream substances. The signal sensed by P_B488_05770 was amplified by RinA amplification system, which induces intense expression of downstream AMP or RNA.
Optimized AMP and dsRNA for EFFICIENT Bio Control
Although previous teams and researchers showed the usefulness of AMP and dsRNA in CLas and D. citri eliminations, it is still noticed that the lethality of their AMPs are unsatisfactory, so as dsRNA, which has off-target effects in some cases. In an attempt to remove the targets in a fast and clear way, it is necessary to introduce fundamental improvements to the substances.
Due to the lack of peptide optimization tools for small peptides like AMP and task as ours, we developed a peptide sequence and structural optimization tool, ProteinOpti. Taking aspects including lyticity, hydrophobicity, peptide charge, and 3D hydrophobic moment into account, ProteinOpti excellently accomplished the task of small peptide evolution towards instructed directions. The optimized SAMP_6 showed greater lethality to the CLas alternative, which provides improved performance in pathogen removal.
One of the most popular reasons for dsRNA off-target is the imperfect base pairing between dsRNA and its targets [12]. The common genome references currently in NCBI databank only represents the D. citri populations of the authors locations, which could be less or more divergent from our local D. citri populations. This deviation could potentially influence the efficiency of our dsRNA and increase the off-target effects. As counter-measurement, we sequenced the whole genome and transcriptome of our local D. citri for specific dsRNA gene design. From which we could hopefully make our engineered phage a highly efficient one.
References
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9: Yu X, Gowda S, Killiny N. Double-stranded RNA delivery through soaking mediates silencing of the muscle protein 20 and increases mortality to the Asian citrus psyllid, Diaphorina citri. Pest Manag Sci. 2017 Sep;73(9):1846-1853. doi: 10.1002/ps.4549. Epub 2017 Mar 14. PMID: 28195429.
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12: Ray P, Sahu D, Aminedi R, Chandran D. Concepts and considerations for enhancing RNAi efficiency in phytopathogenic fungi for RNAi-based crop protection using nanocarrier-mediated dsRNA delivery systems. Front Fungal Biol. 2022 Sep 8;3:977502. doi: 10.3389/ffunb.2022.977502. PMID: 37746174; PMCID: PMC10512274.