Background: The Asian citrus psyllid, Diaphorina citri, is a vector of the plant pathogen Candidatus Liberibacter asiaticus (CLas), the causal agent of Huanglongbing disease. HLB represents the main threat to the citrus industry around the world due to its fast spreading, high infectivity, and incurability. An alternative for the control of this plague in Rutaceae is the implementation of RNA interference (RNAi). Here, we propose a novel method to produce small interfering RNA (siRNA) in Escherichia coli. The method includes a reporter system to visually demonstrate dsRNA expression. We produced and tested siRNAs against three D. citri genes: Abnormal Wing Disk (AWD), Superoxide dismutase 1 (SOD), and Wingless (WNT). These genes play key roles in psyllid development and maturity, and thus, represent promising targets for potential vector control. Results: The blue coloration in bacterial cultures was easily observable, and it corroborated the generation of the dsRNA that is in the same transcript. We evaluated the dosage efficiency of siRNA using the AWD siRNA. The RNAi treatment was evaluated, and all the siRNAs tested were able to induce silencing (-3.05 for AWD, -2.60 for SOD and -2.57 for WNT). Conclusions: The novel bacterial plasmid effectively produces siRNAs, and the blue color reporter is visually facilitated to check the expression of each dsRNA. Treating D. citri with the produced siRNAs resulted in a decrease in gene expression. In addition, psyllid mortality was observed, being the highest when treated with WNT-siRNA. Our results suggest the potential of siRNA treatment as a method for controlling Huanglongbing. How to cite: Rueda-Silva JC, González-Campos LI, Durán-Armenta LF, et al. Novel bacterial plasmid produces small interfering RNAs (siRNAs) that induce effective gene silencing in the Asian citrus psyllid Diaphorina citri. Electron J Biotechnol 2023;64. https://doi.org/10.1016/j.ejbt.2023.03.006.
Bibliographical noteFunding Information:
We express our gratitude to the Southeastern Regional Laboratory of Tamarixia radiata Massive Reproduction, especially to Dr. Francisco Javier Trujillo and Engr. Maria Dolores García Cancino for providing the Diaphorina citri specimens and training us on the safe and ethical maintenance and handling of the psyllids. We also thank Dr. Rosa Estela Navarro González for donating us the E. coli strain HT115 (DE3). We thank our former teammates: Itzel Guadalupe Becerril Rojas, Frida Sofía García Castro, Fernanda Valdés Legorreta, Philippe Monnier Aguilar, and Jacqueline Liszeth Oliva-Ramírez for their kind help and involvement in the project. Thanks to Roberto Delgado Durán and Ramón Eduardo Rivero Aranda for their technical assistance and advice in the laboratory for biosafety care and equipment management. We also thank Leonardo Cañete-Sifuentes for helping us generate the LATEXversion of this paper. We thank Dr. Olga Zamudio Prieto for her support in the grammar, spelling, and style revision of the manuscript.
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