Pathogen derived resistance mediated by the toxin of a toxin-antitoxin system of Xylella fastidiosa

Abstract

The sweet orange is the main citrus crop produced in Brazil, with great economic importance to the country with 53% of the world production of orange juice. The state of São Paulo is the main producer and exporter of the product, accounting for 98% of the national production of the product. Despite its importance to the Brazilian economy, the citrus industry has been suffering with phytosanitary problems due to the large number of pests and diseases in the culture. Among these diseases, the Citrus Variegated Chlorosis (CVC), caused by the bacterium Xylella fastidiosa, as well as citrus canker caused by Xanthomonas citri subsp. Citri, and the disease Huanglongbing (HLB, also known as greening) caused in Brazil by the bacteria Candidatus Liberibacter asiaticus and Candidatus Liberibacter americanus, are among the diseases that cause the most damage to the citrus industry. Biofilm formation is considered the main pathogenicity factor of the bacterium X. fastidiosa, but it is also used as a defense strategy by the bacterium, since it presents specific mechanisms that confer resistance to antimicrobial compounds. Among these mechanisms, there are the formation of persister cells, which are cells in a dormant state, where they do not multiply and do not die, and so, after stopping the treatment, the cells return to multiply and colonize the host. It was verified in several works that the formation of persister cells is associated with the expression of some genes belonging to the bacterial toxin-antitoxin system (TA). The TA system consists of two closely linked genes, in which one gene encodes a toxin that may impair the host cell and the other encode a cognate unstable antitoxin which protects the host from the lethal effect of the toxin. We recently demonstrated that X. fastidiosa is capable of forming persister cells under stress conditions by antimicrobial compounds, and that the expression of genes from the TA system may be associated with this phenotype. Among these genes, the XF2490, which is a toxin from a TA system of X. fastidiosa, showed the highest expression in the treatments with antimicrobial compounds. To determine whether the constitutive expression of this gene in the plant could provide "pathogen-derived resistance", the model plant Nicotiana tabacum has been transformed with this gene. The hypothesis of this work is that the plant overexpressing the XF2490 gene could induce the formation of persister cells and consequently keep the cells dormant, preventing systemic infection. The results obtained by previous work of the undergraduate student Reinaldo R. de Souza Neto demonstrated that some events of the transformed plants with the XF2490 gene showed tolerance to X. fastidiosa, with little or no incidence of symptoms compared to wild-type, suggesting that the gene is a good candidate for transformation into citrus. However, the plants were tested in the T1 generation, so there were no biological replicates. Thus, one of the objectives of this project is to evaluate at least five transformation events with the XF2490 gene, with ten biological replicates and study the factors involved in the interaction of the transgenic plant with the pathogen. The other objective of the project is to evaluate Citrus sinensis transformed with XF2490, which were already obtained by our group, infecting them with X. fastidiosa and X. citri, seeking possible tolerance to pathogens. Finally, the project aims to correlate the data obtained with the plant model and C. sinensis for functional and applied studies of genes that may confer resistance to pathogens.