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Knockout of the ipdC gene in Bacillus sp. (RZ2MS9) with a CRISPR-Cas9 and influence on the IAA biosynthesis L-tryptophan dependent

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Author(s):
Everthon Fernandes Figueredo
Total Authors: 1
Document type: Master's Dissertation
Press: Piracicaba.
Institution: Universidade de São Paulo (USP). Escola Superior de Agricultura Luiz de Queiroz
Defense date:
Examining board members:
Maria Carolina Quecine Verdi; Luis Eduardo Aranha Camargo; Aline Silva Romão-Dumaresq
Advisor: Maria Carolina Quecine Verdi
Abstract

Among the mechanisms related to the bacterium-plant interaction, the bacterial biosynthesis of indole acetic acid (AIA) plays a fundamental role in the promotion of plant growth, since it is capable of influencing innumerable physiological processes in plants. Different metabolic pathways are used by bacteria for the biosynthesis of IAA, with the indole-3-pyruvic acid (IPyA) pathway being the most commonly described. In this pathway, the indole-3-pyruvate decarboxylase (ipdC) gene has a vital role in the production of IAA using the amino acid L-tryptophan as a precursor. In this context, molecular studies about the metabolic pathways and the genes involved in this process are preponderant for the understanding of the interrelationship of the regulatory pathways with the phytormonium synthesis. The rhizobacterium Bacillus sp. (RZ2MS9) has been showing satisfactory activity in promoting plant growth. The sequencing of its genome pointed to the presence of a wide range of genes related to growth promotion, especially genes encoding auxins. Thus, the objective of the present study was to verify the function of the ipdC gene in the IAA biosynthesis L-tryptophan dependent through the knockout of the ipdC in the plant growth-promoting rhizobateria (PGPR) Bacillus sp. (RZ2MS9). Therefore, the knockout was realized using the CRISPR-Cas9. The knockout of the ipdC gene was efficient, generating disruptive mutants for the said gene. IAA biosynthesis by the ΔipdC strain showed reductions in phytormonium concentrations, according to the growth time, being 87.96% in 24 hours, 88.25% in 48 hours and 58.27% in 72 hours of growth compared to the Wild Type (WT). In addition, the biosynthesis of IAA in the absence of the amino acid L-tryptophan was also evaluated, with no phytormonium synthesis being observed at any growth time, both in the wild type and ΔipdC strain. The present study pioneered the knockout of the ipdC gene in a Bacillus strain using the CRISPR-Cas9. The results obtained contribute to a better understanding of the influence of the ipdC gene and the IPyA pathway in the IAA biosynthesis through the RZ2MS9 strain, and its role in plant growth promoting will be demonstrated in the future. (AU)

FAPESP's process: 17/11026-1 - Bacterial biosynthesis of indole acetic acid L-tryptophan dependent and influence on the modulation of vegetal growth
Grantee:Everthon Fernandes Figueredo
Support type: Scholarships in Brazil - Master