| Congresso Brasileiro de Microbiologia 2023 | Resumo: 1049-1 | ||||
Resumo:Trichoderma harzianum is the main fungal biological control agent (BCA) used as biopesticide for the management of a diversity of plant diseases. Despite of the relevant knowledge avail-able regarding its mode of action, there is still a lack of detailed genetic information. The CRISPR systems revolutionized the genetic manipulation of living organisms, including plants, animals and bacteria, but it is an underexplored tool regarding filamentous fungi. Furthermore, CRISPR systems have the potential to help researchers to uncover the gene functions of these fungi. However, the absence of an appropriate selection marker impairs the successful application of the technology in BCAs studies. The aim of this work was to evaluate for the first time the use of a self-replicative and non-integrative CRISPR vector containing sulfonylu-rea resistance gene (sur - acetolactate synthase resistant) enabling the application of chlo-rimuron ethyl as a selection marker in T. harzianum. In the first assay, the sensibility of T. harzianum BRM 67436 strain to chlorimuron ethyl (sulfonylurea) was determined. The chlorimuron ethyl concentrations from 0 to 100 µg/mL were tested against seven different concentrations of the fungus conidia (101 to 107 conidia/mL). The Petri dishes were incubated at 25°C during five days and fungus growth ability was evaluated. In the second assay, T. harzianum proto-plasts were genetically transformed with the empty pM11 plasmid (did not containing func-tional sgRNA). Protoplasts were obtained after incubation of fresh T. harzianum mycelia (48h growth in YPD broth) with T. harzianum lysing enzyme at 30°C for four hours. Protoplast con-centration was adjusted to 1x107 protoplasts/mL in Trichoderma Transformation Buffer and subsequently transformed with 3µg of pM11. The protoplasts and pM11 mix were spread in Transformation Media, containing or not chlorimuron ethyl in different previously selected concentrations and incubated at 25°C during seven days. The treatments were: T1 = control (protoplasts); T2 = protoplasts + 100 µg/mL de chlorimuron; T3 = protoplasts + 200 µg/mL de chlorimuron; T4 = protoplasts + 300 µg/mL de chlorimuron; T5 = protoplasts + 100 µg/ml de chlorimuron + pM11; T6 = protoplasts + 200 µg/ml de chlorimuron + pM11; T7 = protoplasts + 300 µg/ml de chlorimuron + pM11. Colony growth in each treatment was compared. In conclu-sion, the chlorimuron resistance due to transient expression of sur gene guarantee that pM11 was self-replicated successfully in T. harzianum. Although, new assays are necessary to con-firm pM11 as a new tool for fungal-BCAs gene edition.
Palavras-chave: biocontrol, CRISPR, gene edition, plasmid Agęncia de fomento:Ministério da Agricultura e Pecuária - MAPA – BRAZIL. | |||||