The Problem

Huanglongbing disease is the most important disease in citrus worldwide and doesn’t currently have a cure. It is caused by the bacterium CLas (Candidatus liberibacter asiaticus). Because bacterial growth depends on nutrients from the plant phloem, and it is scattered in different concentrations inside the plant, its isolation and culture in laboratory environments has not been possible yet.
Candidatus liberibacter asiaticus is transmitted through Diaphorina citri ’s salivary glands. Bacterial transmission works as a cycle. Since the bacteria is hosted in the psyllid’s salivary glands, when it feeds on the plant the saliva is delivered into the plant’s phloem via its stylet, favoring the infection. Afterwards, the now infected citrus plant has the potential to transmit the bacteria to healthy psyllids when they feed, spreading the disease.

Its Impact

Infected plants are characterized by:

Yellow spotting on leaves
Reduced fruit yield per tree
Deformed, lopsided fruits with dark seeds and bitter taste, which are unsuitable for commercialization.
Reduced lifespan of trees

In Mexico there are about half a million hectares of citrus crops across 23 states. This creates jobs and provides a living for thousands of mexican families. In Mexico, a loss of almost half a million tons of sweet citrus production is expected in case HLB spreads across the country, causing losses of 126,439 jobs after a period of five years (Salcedo et al., n.d.). In Florida, orange production due to HLB has decreased in half over the past decade, accounting for losses of $9 billion in the past year (Wernau, 2017). Almost 100 million trees have been affected and destroyed in many countries of South and Southeast Asia, Indonesia, Philippines, India, Arabian Peninsula, and South Africa (Gottwald et al., 2007).

Our Solution

RNAi technology has been used extensively in research for gene silencing, and has been specifically proven to be effective to control plagues such as D. citri (El-Shesheny et al., 2013). However, current methods require direct application to specimens, making them unfeasible in real world environments.
Our team created a novel siRNA production mechanism. The device produces siRNA in E. coli . This device helped us produce the required siRNA sequences to target four different D. citri genes, effectively silencing their expression and preventing it from spreading HLB disease.

siRNA targets

Awd: When silenced, causes increased mortality in nymphs and prevents adults from developing normal wings, making them unable to fly and move.
WNT: When silenced, psyllids develop without wings, leading to increased mortality and less ability to live and oviposit on plants.
Rac I: Its upregulation in D. citri during CLas infection suggests the bacteria need the protein produced, so silencing it will make the bacteria less likely to survive inside the psyllid. Additionally, its downregulation might lead to increased mortality because it is involved in immunity processes.
SOD: It is involved in detoxification of superoxide radicals, so its silencing will increase mortality by preventing cell metabolism from functioning correctly.

E. coli production method

We developed a novel part that produces any small interfering RNA (siRNA), which is transcripted attached to a mRNA for expressing blue chromoprotein as a reporter. This part is a universal expression cassette for small siRNA in E. coli. This part can express any desired siRNA sequence, as it includes BamHI and HincII restriction sites limiting the region where the siRNA sequence is to be inserted. This method, designed by team TecCEM 2017, is named BSLA, which stands for Blue chromoprotein, Sense, Loop, Antisense; as it is its structure.

Proposed Encapsulation

Chitosan nanoparticles were prepared to encapsulate each of our siRNA sequences. Chitosan is a biodegradable polysaccharide which can be easily uptaken by cells. Because of its positive charge, it allows siRNA molecules to bind and prevents them from being degraded by nucleases inside the cell, while allowing eventual release of the RNA material.
Our formulation is designed to be applied directly to the underside of leaves and stems in citrus trees, where psyllids will ingest it at the time of feeding from the plant’s phloem.

In a nutshell

References

Salcedo, D., Mora, G., Covarrubias, I., Cíntora, C., Hinojosa, R., DePaolis, F. & Mora, S. (n.d.). Assessment of the economic impact of Huanglongbing (HLB) disease on Mexico’s citrus chain. (Comuniica, pp. 43-44.
Wernau, J. (2017). Why Your Orange Juice Might Be From Brazil: Florida’s Trees Are Dying. (The Wall Street Journal. Retrieved from https://www.wsj.com/articles/floridas-orange-industry-symbol-of-a-state-is-dying-1506437044
Gottwald, T. R., Graça, J. V. & Bassanezi, R. B. (2007). Citrus Huanglongbing: The Pathogen and Its Impact. (Plant Health Progress. doi:10.1094/PHP-2007-0906-01-RV.
El-Shesheny, I., Hajeri, S., El-Hawary, I., Gowda, S. & Killiny, N. (2013) Silencing Abnormal Wing Disc Gene of the Asian Citrus Psyllid, Diaphorina citri Disrupts Adult Wing Development and Increases Nymph Mortality. (PLOS ONE 8(5): e65392. https://doi.org/10.1371/journal.pone.0065392

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