Probing the Application of OmpA-Derived Peptides to Disrupt the Acquisition of 'Candidatus Liberibacter asiaticus' by Diaphorina citri.

Huanglongbing (HLB) is currently the most devastating disease of citrus worldwide. Both bacteria 'Candidatus Liberibacter asiaticus' (CLas) and 'Candidatus Liberibacter americanus' (CLam) are associated with HLB in Brazil but with a strong prevalence of CLas over CLam. Conventionally, HLB management focuses on controlling the insect vector population (Diaphorina citri; also known as Asian citrus psyllid [ACP]) by spraying insecticides, an approach demonstrated to be mostly ineffective. Thus, development of novel, more efficient HLB control strategies is required. The multifunctional bacterial outer membrane protein OmpA is involved in several molecular processes between bacteria and their hosts and has been suggested as a target for bacterial control. Curiously, OmpA is absent in CLam in comparison with CLas, suggesting a possible role in host interaction. Therefore, in the current study, we have treated ACPs with different OmpA-derived peptides, aiming to evaluate acquisition of CLas by the insect vector. Treatment of psyllids with 5 µM of Pep1, Pep3, Pep5, and Pep6 in artificial diet significantly reduced the acquisition of CLas, whereas increasing the concentration of Pep5 and Pep6 to 50 µM abolished this process. In addition, in planta treatment with 50 µM of Pep6 also significantly decreased the acquisition of CLas, and sweet orange plants stably absorbed and maintained this peptide for as long as 3 months post the final application. Together, our results demonstrate the promising use of OmpA-derived peptides as a novel biotechnological tool to control CLas.

Overexpression of mqsR in Xylella fastidiosa Leads to a Priming Effect of Cells to Copper Stress Tolerance.

Copper-based compounds are widely used in agriculture as a chemical strategy to limit the spread of multiple plant diseases; however, the continuous use of this heavy metal has caused environmental damage as well as the development of copper-resistant strains. Thus, it is important to understand how the bacterial phytopathogens evolve to manage with this metal in the field. The MqsRA Toxin-Antitoxin system has been recently described for its function in biofilm formation and copper tolerance in Xylella fastidiosa, a plant-pathogen bacterium responsible for economic damage in several crops worldwide. Here we identified differentially regulated genes by X. fastidiosa MqsRA by assessing changes in global gene expression with and without copper. Results show that mqsR overexpression led to changes in the pattern of cell aggregation, culminating in a global phenotypic heterogeneity, indicative of persister cell formation. This phenotype was also observed in wild-type cells but only in the presence of copper. This suggests that MqsR regulates genes that alter cell behavior in order to prime them to respond to copper stress, which is supported by RNA-Seq analysis. To increase cellular tolerance, proteolysis and efflux pumps and regulator related to multidrug resistance are induced in the presence of copper, in an MqsR-independent response. In this study we show a network of genes modulated by MqsR that is associated with induction of persistence in X. fastidiosa. Persistence in plant-pathogenic bacteria is an important genetic tolerance mechanism still neglected for management of phytopathogens in agriculture, for which this work expands the current knowledge and opens new perspectives for studies aiming for a more efficient control in the field.

A Simple Defined Medium for the Production of True Diketopiperazines in Xylella fastidiosa and Their Identification by Ultra-Fast Liquid Chromatography-Electrospray Ionization Ion Trap Mass Spectrometry.

Diketopiperazines can be generated by non-enzymatic cyclization of linear dipeptides at extreme temperature or pH, and the complex medium used to culture bacteria and fungi including phytone peptone and trypticase peptone, can also produce cyclic peptides by heat sterilization. As a result, it is not always clear if many diketopiperazines reported in the literature are artifacts formed by the different complex media used in microorganism growth. An ideal method for analysis of these compounds should identify whether they are either synthesized de novo from the products of primary metabolism and deliver true diketopiperazines. A simple defined medium (X. fastidiosa medium or XFM) containing a single carbon source and no preformed amino acids has emerged as a method with a particularly high potential for the grown of X. fastidiosa and to produce genuine natural products. In this work, we identified a range of diketopiperazines from X. fastidiosa 9a5c growth in XFM, using Ultra-Fast Liquid Chromatography coupled with mass spectrometry. Diketopiperazines are reported for the first time from X. fastidiosa, which is responsible for citrus variegated chlorosis. We also report here fatty acids from X. fastidiosa, which were not biologically active as diffusible signals, and the role of diketopiperazines in signal transduction still remains unknown.