Silenciamento do gene da β-1,3-Glucanase de Carica papaya por CRISPR-Cas9

Abstract

Callose deposition in plasmodesma is directly associated with viral movement. The prediction of micro RNAs from papaya infected by the papaya meleira virus viral complex (PMeV complex) revealed a high presence of miRNAs repressing the post transcriptional expression of genes involved in the synthesis of β-1,3-glucanases. In this sense, our study aimed to evaluate the silencing of the β-1,3-glucanase enzyme gene as a form of resistance to the PMeV complex that causes the papaya sticky disease. For this purpose, the new CRISPR/Cas9 gene-editing technology was used. To attest to the functioning of the CRISPR/Cas9 technique in papaya tissues, we sought to develop a visual marker interrupting chlorophyll biosynthesis by silencing the phytoene desaturase gene in papaya leaves, cells and protoplasts. For the transformation of papaya tissues by agroinfiltration, the binary vector pKSE401 was used, which contains the Cas9 enzyme gene and a customizable sequence referring to the structure of the guide RNA. The new plasmids generated were inserted by electroporation into Agrobacterium tumefaciens GV3101, which was renamed AtpKSPD and AtpKSBG for silencing of phytoene desaturase and β-1,3-glucanase, respectively. As a result, all papaya leaves that were submitted to agroinfiltration by AtpKSPD showed depigmentation due to the inactivation of the phytoene desaturase gene and the same was observed in embryos regenerated from transformed cells. The agroinfiltrated tissues expressed the customized gRNA together with Cas9. The same method was used for AtpKSBG. The results obtained attest that the use of CRISPR/Cas9 technology can be successfully applied in Carica papaya. We emphasize that so far there is no established protocol for editing C. papaya by CRISPR technology, thus, our work is a pioneer in the application of the technique in papaya, also highlighting the pioneering in β-1,3-glucanase and phytoene desaturase gene silencing in this crop, the second being a promising visual marker of the technique's functioning for C. papaya