Alessandra A. de Souza

The genome sequence of the gram-positive sugarcane pathogen Leifsonia xyli subsp. xyli.

The genome sequence of Leifsonia xyli subsp. xyli, which causes ratoon stunting disease and affects sugarcane worldwide, was determined. The single circular chromosome of Leifsonia xyli subsp. xyli CTCB07 was 2.6 Mb in length with a GC content of 68% and 2,044 predicted open reading frames. The analysis also revealed 307 predicted pseudogenes, which is more than any bacterial plant pathogen sequenced to date. Many of these pseudogenes, if functional, would likely be involved in the degradation of plant heteropolysaccharides, uptake of free sugars, and synthesis of amino acids. Although L. xyli subsp. xyli has only been identified colonizing the xylem vessels of sugarcane, the numbers of predicted regulatory genes and sugar transporters are similar to those in free-living organisms. Some of the predicted pathogenicity genes appear to have been acquired by lateral transfer and include genes for cellulase, pectinase, wilt-inducing protein, lysozyme, and desaturase. The presence of the latter may contribute to stunting, since it is likely involved in the synthesis of abscisic acid, a hormone that arrests growth. Our findings are consistent with the nutritionally fastidious behavior exhibited by L. xyli subsp. xyli and suggest an ongoing adaptation to the restricted ecological niche it inhabits.

Differentiation of strains of Xylella fastidiosa by a variable number of tandem repeat analysis.

ABSTRACT Short sequence repeats (SSRs) with a potential variable number of tandem repeat (VNTR) loci were identified in the genome of the citrus pathogen Xylella fastidiosa and used for typing studies. Although mono- and dinucleotide repeats were absent, we found several intermediate-length 7-, 8-, and 9-nucleotide repeats, which we examined for allelic polymorphisms using PCR. Five genuine VNTR loci were highly polymorphic within a set of 27 X. fastidiosa strains from different hosts. The highest average Neis measure of genetic diversity (H) estimated for VNTR loci was 0.51, compared to 0.17 derived from randomly amplified polymorphic DNA (RAPD) analysis. For citrus X. fastidiosa strains, some specific VNTR loci had a Hvalue of 0.83, while the maximum value given by specific RAPD loci was 0.12. Our approach using VNTR markers provides a high-resolution tool for epidemiological, genetic, and ecological analysis of citrus-specific X. fastidiosa strains.

Development of a Molecular Tool for the Diagnosis of Leprosis, a Major Threat to Citrus Production in the Americas

Citrus leprosis virus (CiLV), a tentative member of the Rhabdoviridae family, affects citrus trees in Brazil, where it is transmitted by mites Brevipalpus spp. It also occurs in other South American countries and was recently identified in Central America. This northbound spread of CiLV is being considered a serious threat to the citrus industry of the United States. However, despite its importance, difficulties related to the biology of CiLV have hindered much of the progress regarding its accurate detection, leaving both the analyses of symptoms and electron microscopy as the only tools available. An attempt to overcome this problem was made by constructing a cDNA library from double-stranded RNA extracted from leprosis lesions of infected Citrus sinensis (sweet orange) leaves. After cloning and sequencing, specific primers were designed to amplify putative CiLV genome regions with similarity to genes encoding the movement protein and replicase of other plant viruses. RNA from infected citrus plants corresponding to different varieties and locations were amplified by reverse transcription-polymerase chain reaction (RT-PCR) using the two pairs of primers. Amplified products were purified, cloned in pGEM-T, and sequenced. The sequences confirmed the genomic regions previously associated with CiLV. The results demonstrate that RT-PCR was specific, accurate, rapid, and reliable for the detection of CiLV.

The role of conditioning film formation and surface chemical changes on Xylella fastidiosa adhesion and biofilm evolution

Biofilms are complex microbial communities with important biological functions including enhanced resistance against external factors like antimicrobial agents. The formation of a biofilm is known to be strongly dependent on substrate properties including hydrophobicity/hydrophilicity, structure, and roughness. The adsorption of (macro)molecules on the substrate, also known as conditioning film, changes the physicochemical properties of the surface and affects the bacterial adhesion. In this study, we investigate the physicochemical changes caused by Periwinkle wilt (PW) culture medium conditioning film formation on different surfaces (glass and silicon) and their effect on X. fastidiosa biofilm formation. Contact angle measurements have shown that the film formation decreases the surface hydrophilicity degree of both glass and silicon after few hours. Atomic force microscopy (AFM) images show the glass surface roughness is drastically reduced with conditioning film formation. First-layer X. fastidiosa biofilm on glass was observed in the AFM liquid cell after a period of time similar to that determined for the hydrophilicity changes. In addition, attenuation total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy supports the AFM observation, since the PW absorption spectra increases with time showing a stronger contribution from the phosphate groups. Although hydrophobic and rough surfaces are commonly considered to increase bacteria cell attachment, our results suggest that these properties are not as important as the surface functional groups resulting from PW conditioning film formation for X. fastidiosa adhesion and biofilm development.

A kinetic model for Xylella fastidiosa adhesion, biofilm formation, and virulence

Xylella fastidiosa is the causal agent of citrus variegated chlorosis and Pierces disease which are the major threat to the citrus and wine industries. The most accepted hypothesis for Xf diseases affirms that it is a vascular occlusion caused by bacterial biofilm, embedded in an extracellular translucent matrix that was deduced to be the exopolysaccharide fastidian. Fourier transform infrared spectroscopy analysis demonstrated that virulent cells which form biofilm on glass have low fastidian content similar to the weak virulent ones. This indicates that high amounts of fastidian are not necessary for adhesion. In this paper we propose a kinetic model for X. fastidiosa adhesion, biofilm formation, and virulence based on electrostatic attraction between bacterial surface proteins and xylem walls. Fastidian is involved in final biofilm formation and cation sequestration in dilute sap.

Copper resistance of biofilm cells of the plant pathogen Xylella fastidiosa

Xylella fastidiosa is a phytopathogen that causes diseases in different plant species. The development of disease symptoms is associated to the blockage of the xylem vessels caused by biofilm formation. In this study, we evaluated the sensitivity of biofilm and planktonic cells to copper, one of the most important antimicrobial agents used in agriculture. We measured the exopolysaccharides (EPS) content in biofilm and planktonic cells and used real-time reverse transcription polymerase chain reaction to evaluate the expression of the genes encoding proteins involved in cation/multidrug extrusion (acrA/B, mexE/czcA, and metI) and others associated with different copper resistance mechanisms (copB, cutA1, cutA2, and cutC) in the X. fastidiosa biofilm formed in two different media. We confirmed that biofilms are less susceptible to copper than planktonic cells. The amount of EPS seems to be directly related to the resistance and it varies according to the media where the cells are grown. The same was observed for gene expression. Nevertheless, some genes seem to have a greater importance in biofilm cells resistance to copper. Our results suggest a synergistic effect between diffusion barriers and other mechanisms associated with bacterial resistance in this phytopathogen. These mechanisms are important for a bacterium that is constantly under stress conditions in the host.

Expression of Pathogenicity-Related Genes of Xylella fastidiosa In Vitro and In Planta

Xylella fastidiosa is responsible for several economically important plant diseases. It is currently assumed that the symptoms are caused by vascular occlusion due to biofilm formation. Microarray technology was previously used to examine the global gene expression profile of X. fastidiosa freshly isolated from symptomatic plants or after several passages by axenic culture medium, and different pathogenicity profiles have been obtained. In the present study the expression of some pathogenicity-related genes was evaluated in vitro and in planta by RT-PCR. The results suggest that adhesion is important at the beginning of biofilm formation, while the genes related to adaptation are essential for the organism’s maintenance in planta. Similar results were observed in vitro mainly for the adhesion genes. The pattern of expression observed suggests that adhesion modulates biofilm formation whereas the expression of some adaptation genes may be related to the environment in which the organism is living.

Spatiotemporal distribution of different extracellular polymeric substances and filamentation mediate Xylella fastidiosa adhesion and biofilm formation

Microorganism pathogenicity strongly relies on the generation of multicellular assemblies, called biofilms. Understanding their organization can unveil vulnerabilities leading to potential treatments; spatially and temporally-resolved comprehensive experimental characterization can provide new details of biofilm formation, and possibly new targets for disease control. Here, biofilm formation of economically important phytopathogen Xylella fastidiosa was analyzed at single-cell resolution using nanometer-resolution spectro-microscopy techniques, addressing the role of different types of extracellular polymeric substances (EPS) at each stage of the entire bacterial life cycle. Single cell adhesion is caused by unspecific electrostatic interactions through proteins at the cell polar region, where EPS accumulation is required for more firmly-attached, irreversibly adhered cells. Subsequently, bacteria form clusters, which are embedded in secreted loosely-bound EPS, and bridged by up to ten-fold elongated cells that form the biofilm framework. During biofilm maturation, soluble EPS forms a filamentous matrix that facilitates cell adhesion and provides mechanical support, while the biofilm keeps anchored by few cells. This floating architecture maximizes nutrient distribution while allowing detachment upon larger shear stresses; it thus complies with biological requirements of the bacteria life cycle. Using new approaches, our findings provide insights regarding different aspects of the adhesion process of X. fastidiosa and biofilm formation.

RNA-Seq analysis of Citrus reticulata in the early stages of Xylella fastidiosa infection reveals auxin-related genes as a defense response

BackgroundCitrus variegated chlorosis (CVC), caused by Xylella fastidiosa, is one the most important citrus diseases, and affects all varieties of sweet orange (Citrus sinensis L. Osb). On the other hand, among the Citrus genus there are different sources of resistance against X. fastidiosa. For these species identifying these defense genes could be an important step towards obtaining sweet orange resistant varieties through breeding or genetic engineering. To assess these genes we made use of mandarin (C. reticulata Blanco) that is known to be resistant to CVC and shares agronomical characteristics with sweet orange. Thus, we investigated the gene expression in Ponkan mandarin at one day after infection with X. fastidiosa, using RNA-seq. A set of genes considered key elements in the resistance was used to confirm its regulation in mandarin compared with the susceptible sweet orange.ResultsGene expression analysis of mock inoculated and infected tissues of Ponkan mandarin identified 667 transcripts repressed and 724 significantly induced in the later. Among the induced transcripts, we identified genes encoding proteins similar to Pattern Recognition Receptors. Furthermore, many genes involved in secondary metabolism, biosynthesis and cell wall modification were upregulated as well as in synthesis of abscisic acid, jasmonic acid and auxin.ConclusionsThis work demonstrated that the defense response to the perception of bacteria involves cell wall modification and activation of hormone pathways, which probably lead to the induction of other defense-related genes. We also hypothesized the induction of auxin-related genes indicates that resistant plants initially recognize X. fastidiosa as a necrotrophic pathogen.

Global Expression Profile of Biofilm Resistance to Antimicrobial Compounds in the Plant-Pathogenic Bacterium Xylella fastidiosa Reveals Evidence of Persister Cells

Investigations of biofilm resistance response rarely focus on plant-pathogenic bacteria. Since Xylella fastidiosa is a multihost plant-pathogenic bacterium that forms biofilm in the xylem, the behavior of its biofilm in response to antimicrobial compounds needs to be better investigated. We analyzed here the transcriptional profile of X. fastidiosa subsp. pauca in response to inhibitory and subinhibitory concentrations of copper and tetracycline. Copper-based products are routinely used to control citrus diseases in the field, while antibiotics are more widely used for bacterial control in mammals. The use of antimicrobial compounds triggers specific responses to each compound, such as biofilm formation and phage activity for copper. Common changes in expression responses comprise the repression of genes associated with metabolic functions and movement and the induction of toxin-antitoxin systems, which have been associated with the formation of persister cells. Our results also show that these cells were found in the population at a ca. 0.05% density under inhibitory conditions for both antimicrobial compounds and that pretreatment with subinhibitory concentration of copper increases this number. No previous report has detected the presence of these cells in X. fastidiosa population, suggesting that this could lead to a multidrug tolerance response in the biofilm under a stressed environment. This is a mechanism that has recently become the focus of studies on resistance of human-pathogenic bacteria to antibiotics and, based on our data, it seems to be more broadly applicable.