Gene silencing of Diatraea saccharalis using an RNA interference (RNAi) technique

Author(s)

Bruna Garbatti Factor

• Document type: Doctoral Thesis
• Press: Piracicaba.
• Institution: Universidade de São Paulo (USP). Escola Superior de Agricultura Luiz de Queiroz (ESALA/BC)
• Defense date: 2022-12-05

Examining board members

Antonio Vargas de Oliveira Figueira; Eduardo Chumbinho de Andrade; Andrea Soares da Costa Fuentes; Maria Carolina Quecine Verdi

• Advisor: Antonio Vargas de Oliveira Figueira

Abstract

The RNAi technique is an innovative strategy that can be integrated into managing critical agricultural pests. Diatraea saccharalis is considered one of the main pests of sugarcane. The economic damage to the culture is quite considerable, generating R$ 5 billion of losses occur in Brazil due to the attack of this pest each season. Combining these factors implies, therefore, a demand for other control methods. Gene silencing by RNAi is a potential use for the control of D. saccharalis. Therefore, this work aimed to evaluate the transcriptome of D. saccharalisand identify the RNAi machinery related in this species, as well as potential target genes for gene silencing. In addition, to evaluate the silencing of D. saccharalis genes, the method of dsRNA delivery via the bacterium E. coli HT115 (DE3) was provided in an artificial diet. Furthermore, we looked to identify, characterize, and knockdown the gene related to a dsRNA degradation protein in insects, mainly lepidopterans, the REase of D. saccharalis, aiming at a more significant accumulation of dsRNA and better RNAi efficiency in the insect. Also, the creation of a dsRNA delivery method for D. saccharalis creation of a dsRNA delivery method for D. saccharalis larvae via endophytic bacteria Pantoea agglomerans 33.1 was studied. For this, it was necessary first to perform the knockout of the rnc gene of the microorganism and subsequent transformation for expression of dsRNA. Overall, homologous contigs of all components of the RNAi pathway were found. In addition, genes involved in hormone pathways, insecticide resistance, and insect digestive tract were identified as candidates for gene silencing via RNAi. From the evaluation of biological parameters (duration of the larval stage, larval mortality, and pupae weight), we were able to demonstrate, with the delivery of dsRNA against specific target genes of the insect, expressed in E. coli HT115 (DE3) bacteria, the absence of evidence of gene silencing effects. Assuming that, as in most insects of the order Lepidoptera, D. saccharalis shows recalcitrance to RNAi mechanisms. In addition, the identification and knockdown of the Ds_up56 gene, which encodes REase in D. saccharalis, reduced the accumulation of CHI transcripts by 3.5 times, demonstrating the contribution of this gene to the degradation of transcripts of the target gene and increasing RNAi efficiency and justifying the recalcitrance of D. saccharalis to RNAi. Finally, with the knockout of the rnc gene of P. agglomerans 33.1 and the transformation of this microorganism with a pdag silencing vector to express dsRNA against a specific gene of D. saccharalis, it was demonstrated that the occurrence of dsRNA accumulation in the bacterial host, proposing the use of P. agglomerans 33.1Δrnc as an RNAi delivery vector for insect pest control, proving to be a potential alternative for protection of relevant crops. (AU)

• FAPESP's process: 17/17882-7 - Gene silencing of Diatraea saccharalis using the interference RNA technique (RNAi)
• Grantee: Bruna Garbatti Factor
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)