N-acetylcysteine absorption and its potential dual effect improve fitness and fruit yield in Xylella fastidiosa infected plants.

BACKGROUND: Xylella fastidiosa is a multi-host bacterium that can be detected in hundreds of plant species including several crops. Diseases caused by X. fastidiosa are considered a threat to global food production. The primary method for managing diseases caused by X. fastidiosa involves using insecticides to control the vector. Hence, it is necessary to adopt new and sustainable disease management technologies to control not only the insect but also the bacteria and plant health. We demonstrated that N-acetylcysteine (NAC), a low-cost cysteine analogue, is a sustainable molecule that can be used in agriculture to decrease the damage caused by X. fastidiosa and improve plant health. RESULTS: Using 15N-NAC we proved that this analogue was absorbed by the roots and transported to different parts of the plant. Inside the plant, NAC reduced the bacterial population by 60-fold and the number of xylem vessels blocked by bacterial biofilms. This reflected in a recovery of 0.28-fold of the daily sap flow compared to health plants. In addition, NAC-treated citrus variegated chlorosis (CVC) plants decreased the oxidative stress by improving the activity of detoxifying enzymes. Moreover, the use of NAC in field conditions positively contributed to the increase in fruit yield of CVC-diseased plants. CONCLUSION: Our research not only advances the understanding of NAC absorption in plants, but also indicates its dual effect as an antimicrobial and antioxidant molecule. This, in turn, negatively affects bacterial survival while improving plant health by decreasing oxidative stress. Overall, the positive field-based evidence supports the viability of NAC as a sustainable agricultural application. © 2024 Society of Chemical Industry.

Bio-Prospecting of Crude Leaf Extracts from Thirteen Plants of Brazilian Cerrado Biome on Human Glioma Cell Lines.

(1) Background: Malignant gliomas are aggressive tumors characterized by fast cellular growth and highly invasive properties. Despite all biological and clinical advances in therapy, the standard treatment remains essentially palliative. Therefore, searching for alternative therapies that minimize adverse symptoms and improve glioblastoma patients' outcomes is imperative. Natural products represent an essential source in the discovery of such new drugs. Plants from the cerrado biome have been receiving increased attention due to the presence of secondary metabolites with significant therapeutic potential. (2) Aim: This study provides data on the cytotoxic potential of 13 leaf extracts obtained from plants of 5 families (Anacardiaceae, Annonaceae, Fabaceae, Melastomataceae e Siparunaceae) found in the Brazilian cerrado biome on a panel of 5 glioma cell lines and one normal astrocyte. (3) Methods: The effect of crude extracts on cell viability was evaluated by MTS assay. Mass spectrometry (ESI FT-ICR MS) was performed to identify the secondary metabolites classes presented in the crude extracts and partitions. (4) Results: Our results revealed the cytotoxic potential of Melastomataceae species Miconia cuspidata, Miconia albicans, and Miconia chamissois. Additionally, comparing the four partitions obtained from M. chamissois crude extract indicates that the chloroform partition had the greatest cytotoxic activity against the glioma cell lines. The partitions also showed a mean IC50 close to chemotherapy, temozolomide; nevertheless, lower toxicity against normal astrocytes. Analysis of secondary metabolites classes presented in these crude extracts and partitions indicates the presence of phenolic compounds. (5) Conclusions: These findings highlight M. chamissois chloroform partition as a promising component and may guide the search for the development of additional new anticancer therapies.

Application of highly ozonated sunflower oil does not improve palatal wound healing: A randomized controlled clinical trial.

BACKGROUND: Ozone is a molecule that plays an important role in dentistry, specially for wound healing. The aim of the present study was to clinically and immunologically evaluate the effect of ozonated oil on the healing of palatal wounds. METHODS: This is a prospective, longitudinal, triple-blind, randomized, placebo-controlled clinical trial. The groups were divided as follows: Test group (n = 14): after removal of the free gingival graft (FGG), the palatal wound was treated with ozonized seed sunflower oil with a peroxide index between 510 and 625 meq O2 /kg; Control group (n = 14): after removal of the FGG, the palatal wound was treated with non-ozonated sunflower oil (placebo). The treatments were applied three times a day, for 7 days. RESULTS: There were no significant differences in the measurements of wound area (mm2 ) between the test and control groups in the different periods evaluated (0, 3, 7, and 14 days; p > 0.05). The intra-group analysis showed a significant decrease in wound size over the course of days (0, 3, 7, and 14 days; p < 0.05). Vascular endothelial growth factor (VEGF; pg/mL) presented a significant reduction at 7 days (p < 0.05) compared to day 3 in the test group (p < 0.05). There was a statistical difference for malondialdehyde (MDA; pg/mL) in the test group between 3 and 7 days post-treatment (p < 0.05) and between test and control groups on the 7th day (p < 0.05). CONCLUSIONS: The application of highly ozonated sunflower oil did not improve the remaining scar area of the palate, decreasing the VEGF and increasing the oxidative stress marker MDA.

Physiochemically Distinct Surface Properties of SU-8 Polymer Modulate Bacterial Cell-Surface Holdfast and Colonization.

SU-8 polymer is an excellent platform for diverse applications due to its high aspect ratio of micro/nanostructure fabrication and exceptional physicochemical and biocompatible properties. Although SU-8 polymer has often been investigated for various biological applications, how its surface properties influence the interaction of bacterial cells with the substrate and its colonization is poorly understood. In this work, we tailor SU-8 nanoscale surface properties to investigate single-cell motility, adhesion, and successive colonization of phytopathogenic bacteria, Xylella fastidiosa. Different surface properties of SU-8 thin films have been prepared using photolithography processing and oxygen plasma treatment. A more significant density of carboxyl groups in hydrophilic plasma-treated SU-8 surfaces promotes faster cell motility in the earlier growth stage. The hydrophobic nature of pristine SU-8 surfaces shows no trackable bacterial motility and 5-10 times more single cells adhered to the surface than its plasma-treated counterpart. In addition, plasma-treated SU-8 samples suppressed bacterial adhesion, with surfaces showing less than 5% coverage. These results not only showcase that SU-8 surface properties can impact the spatiotemporal bacterial behavior but also provide insights into pathogens' prominent ability to evolve and adapt to different surface properties.

Molecular basis for host responses to Xanthomonas infection.

MAIN CONCLUSION: This review highlights the most relevant and recent updated information available on the defense responses of selected hosts against Xanthomonas spp. Xanthomonas is one of the most important genera of Gram-negative phytopathogenic bacteria, severely affecting the productivity of economically important crops worldwide, colonizing either the vascular system or the mesophyll tissue of the host. Due to its rapid propagation, Xanthomonas poses an enormous challenge to farmers, because it is usually controlled using huge quantities of copper-based chemicals, adversely impacting the environment. Thus, developing new ways of preventing colonization by these bacteria has become essential. Advances in genomic and transcriptomic technologies have significantly elucidated at molecular level interactions between various crops and Xanthomonas species. Understanding how these hosts respond to the infection is crucial if we are to exploit potential approaches for improving crop breeding and cutting productivity losses. This review focuses on our current knowledge of the defense response mechanisms in agricultural crops after Xanthomonas infection. We describe the molecular basis of host-bacterium interactions over a broad spectrum with the aim of improving our fundamental understanding of which genes are involved and how they work in this interaction, providing information that can help to speed up plant breeding programs, namely using gene editing approaches.

Overexpression of CsSAMT in Citrus sinensis Induces Defense Response and Increases Resistance to Xanthomonas citri subsp. citri.

Citrus canker is a destructive disease caused by Xanthomonas citri subsp. citri, which affects all commercial sweet orange (Citrus sinensis [L.] Osbeck) cultivars. Salicylic acid (SA) and systemic-acquired resistance (SAR) have been demonstrated to have a crucial role in mediating plant defense responses against this phytopathogen. To induce SAR, SA is converted to methyl salicylate (MeSA) by an SA-dependent methyltransferase (SAMT) and translocated systemically to prime noninfected distal tissues. Here, we generated sweet orange transgenic plants (based on cvs. Hamlin and Valencia) overexpressing the SAMT gene from Citrus (CsSAMT) and evaluated their resistance to citrus canker. We obtained four independent transgenic lines and confirmed their significantly higher MeSA volatilization compared to wild-type controls. Plants overexpressing CsSAMT showed reduced symptoms of citrus canker and bacterial populations in all transgenic lines without compromising plant development. One representative transgenic line (V44SAMT) was used to evaluate resistance response in primary and secondary sites. Without inoculation, V44SAMT modulated CsSAMT, CsNPR1, CsNPR3, and CsWRKY22 expression, indicating that this plant is in a primed defense status. The results demonstrate that MeSA signaling prompts the plant to respond more efficiently to pathogen attacks and induces immune responses in transgenic plants at both primary and secondary infection sites.

Controlled spatial organization of bacterial growth reveals key role of cell filamentation preceding Xylella fastidiosa biofilm formation.

The morphological plasticity of bacteria to form filamentous cells commonly represents an adaptive strategy induced by stresses. In contrast, for diverse human and plant pathogens, filamentous cells have been recently observed during biofilm formation, but their functions and triggering mechanisms remain unclear. To experimentally identify the underlying function and hypothesized cell communication triggers of such cell morphogenesis, spatially controlled cell patterning is pivotal. Here, we demonstrate highly selective cell adhesion of the biofilm-forming phytopathogen Xylella fastidiosa to gold-patterned SiO2 substrates with well-defined geometries and dimensions. The consequent control of both cell density and distances between cell clusters demonstrated that filamentous cell formation depends on cell cluster density, and their ability to interconnect neighboring cell clusters is distance-dependent. This process allows the creation of large interconnected cell clusters that form the structural framework for macroscale biofilms. The addition of diffusible signaling molecules from supernatant extracts provides evidence that cell filamentation is induced by quorum sensing. These findings and our innovative platform could facilitate therapeutic developments targeting biofilm formation mechanisms of X. fastidiosa and other pathogens.

Bioprospecting of Natural Compounds from Brazilian Cerrado Biome Plants in Human Cervical Cancer Cell Lines.

Cervical cancer is the third most common in Brazilian women. The chemotherapy used for the treatment of this disease can cause many side effects; then, to overcome this problem, new treatment options are necessary. Natural compounds represent one of the most promising sources for the development of new drugs. In this study, 13 different species of 6 families from the Brazilian Cerrado vegetation biome were screened against human cervical cancer cell lines (CCC). Some of these species were also evaluated in one normal keratinocyte cell line (HaCaT). The effect of crude extracts on cell viability was evaluated by a colorimetric method (MTS assay). Extracts from Annona crassiflora, Miconia albicans, Miconia chamissois, Stryphnodendron adstringens, Tapirira guianensis, Xylopia aromatica, and Achyrocline alata showed half-maximal inhibitory concentration (IC50) values < 30 µg/mL for at least one CCC. A. crassiflora and S. adstringens extracts were selective for CCC. Mass spectrometry (Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (ESI FT-ICR MS)) of A. crassiflora identified fatty acids and flavonols as secondary compounds. One of the A. crassiflora fractions, 7C24 (from chloroform partition), increased H2AX phosphorylation (suggesting DNA damage), PARP cleavage, and cell cycle arrest in CCC. Kaempferol-3-O-rhamnoside and oleic acid were bioactive molecules identified in 7C24 fraction. These findings emphasize the importance of investigating bioactive molecules from natural sources for developing new anti-cancer drugs.

Overexpression of Citrus reticulata SAMT in Nicotiana tabacum increases MeSA volatilization and decreases Xylella fastidiosa symptoms.

MAIN CONCLUSION: Nicotiana tabacum overexpressing CrSAMT from Citrus reticulata increased production of MeSA, which works as an airborne signal in neighboring wild-type plants, inducing PR1 and increasing resistance to the pathogen Xylella fastidiosa. Xylella fastidiosa is one of the major threats to plant health worldwide, affecting yield in many crops. Despite many efforts, the development of highly productive resistant varieties has been challenging. In studying host plant resistance, the S-adenosyl-L-methionine: salicylic acid carboxyl methyltransferase gene (SAMT) from Citrus reticulata, a X. fastidiosa resistant species, was upregulated in response to pathogen infection. SAMT is involved with the catalysis and production of methyl salicylate (MeSA), an airborne signal responsible for triggering systemic acquired resistance. Here we used tobacco as a model system and generated transgenic plants overexpressing C. reticulata SAMT (CrSAMT). We performed an in silico structural characterization of CrSAMT and investigated its biotechnological potential in modulating the immune system in transgenic plants. The increase of MeSA production in transgenic lines was confirmed by gas chromatography (GC-MS). The transgenic lines showed upregulation of PR1, and their incubation with neighboring wild-type plants activated PR1 expression, indicating that MeSA worked as an airborne signal. In addition, transgenic plants showed significantly fewer symptoms when challenged with X. fastidiosa. Altogether, these data suggest that CrSAMT plays a role in host defense response and can be used in biotechnology approaches to confer resistance against X. fastidiosa.

Persistence in Phytopathogenic Bacteria: Do We Know Enough?

Phytopathogenic bacteria affect a wide range of crops worldwide and have a negative impact in agriculture due to their associated economic losses and environmental impacts. Together with other biotic and abiotic stress factors, they pose a threat to global food production. Therefore, understanding bacterial survival strategies is an essential step toward the development of new strategies to control plant diseases. One mechanism used by bacteria to survive under stress conditions is the formation of persister cells. Persisters are a small fraction of phenotypic variants within an isogenic population that exhibits multidrug tolerance without undergoing genetic changes. They are dormant cells that survive treatment with antimicrobials by inactivating the metabolic functions that are disrupted by these compounds. They are thus responsible for the recalcitrance of many human diseases, and in the same way, they are thought to contribute to the survival of bacterial phytopathogens under a range of stresses they face in the environment. It is believed that persister cells of bacterial phytopathogens may lead to the reoccurrence of disease by recovering growth and recolonizing the host plant after the end of stress. However, compared to human pathogens, little is known about persister cells in phytopathogens, especially about their genetic regulation. In this review, we describe the overall knowledge on persister cells and their regulation in bacterial phytopathogens, focusing on their ability to survive stress conditions, to recover from dormancy and to maintain virulence.

Determination of the Influence of Chronic Periodontitis on Pulp Sensibility by Means of Electric and Thermal Cold Testing.

INTRODUCTION: Communication between pulp and periodontal tissue has been well established. However, it is unknown when periodontal disease begins to affect the clinical response of pulp tissue. The aim of this study was to assess the influence of periodontal severity on pulp sensibility by means of electric and thermal cold testing. METHODS: The teeth assessed in this study were allocated into 3 groups considering radiographic alveolar bone loss (ABL) as follows: slight periodontitis (SP, ABL ≤7 mm without reaching the apex, n = 25), moderate periodontitis (ABL >7 mm without reaching the apex, n = 23), and severe periodontitis (SvP, ABL >7 reaching the apex, n = 8). Gingival recession (GR), probing depth (PD), and clinical attachment level (CAL) were also measured. RESULTS: The results showed higher levels of PD and CAL in the SvP group compared with the SP group (P < .05), with no significant difference in GR (P > .05). The SvP group showed significant ABL compared with the other groups (P > .05). The SP group showed a significant number of teeth with a positive pulp response, whereas the SvP group showed a significant number of teeth with a negative pulp response (P < .05); no significant differences were observed between the thermal cold and electric tests (P > .05). CONCLUSIONS: Within the limits of this study, it can be concluded that pulp clinical involvement with a negative response to thermal cold and electric testing occurs only in the most advanced stage of chronic periodontitis with apical involvement.

Draft Genome Sequence of 11399, a Transformable Citrus-Pathogenic Strain of Xylella fastidiosa.

The draft genome of Xylella fastidiosa subsp. pauca strain 11399, a transformable citrus-pathogenic strain, is reported here. The 11399 genome size is 2,690,704 bp and has a G+C content of 52.7%. The draft genome of 11399 reveals the absence of four type I restriction-modification system genes.

LRR-RLK family from two Citrus species: genome-wide identification and evolutionary aspects.

BACKGROUND: Leucine-rich repeat receptor-like kinases (LRR-RLKs) represent the largest subfamily of plant RLKs. The functions of most LRR-RLKs have remained undiscovered, and a few that have been experimentally characterized have been shown to have important roles in growth and development as well as in defense responses. Although RLK subfamilies have been previously studied in many plants, no comprehensive study has been performed on this gene family in Citrus species, which have high economic importance and are frequent targets for emerging pathogens. In this study, we performed in silico analysis to identify and classify LRR-RLK homologues in the predicted proteomes of Citrus clementina (clementine) and Citrus sinensis (sweet orange). In addition, we used large-scale phylogenetic approaches to elucidate the evolutionary relationships of the LRR-RLKs and further narrowed the analysis to the LRR-XII group, which contains several previously described cell surface immune receptors. RESULTS: We built integrative protein signature databases for Citrus clementina and Citrus sinensis using all predicted protein sequences obtained from whole genomes. A total of 300 and 297 proteins were identified as LRR-RLKs in C. clementina and C. sinensis, respectively. Maximum-likelihood phylogenetic trees were estimated using Arabidopsis LRR-RLK as a template and they allowed us to classify Citrus LRR-RLKs into 16 groups. The LRR-XII group showed a remarkable expansion, containing approximately 150 paralogs encoded in each Citrus genome. Phylogenetic analysis also demonstrated the existence of two distinct LRR-XII clades, each one constituted mainly by RD and non-RD kinases. We identified 68 orthologous pairs from the C. clementina and C. sinensis LRR-XII genes. In addition, among the paralogs, we identified a subset of 78 and 62 clustered genes probably derived from tandem duplication events in the genomes of C. clementina and C. sinensis, respectively. CONCLUSIONS: This work provided the first comprehensive evolutionary analysis of the LRR-RLKs in Citrus. A large expansion of LRR-XII in Citrus genomes suggests that it might play a key role in adaptive responses in host-pathogen co-evolution, related to the perennial life cycle and domestication of the citrus crop species.

The MqsRA Toxin-Antitoxin System from Xylella fastidiosa Plays a Key Role in Bacterial Fitness, Pathogenicity, and Persister Cell Formation.

Through the formation of persister cells, bacteria exhibit tolerance to multidrug and other environmental stresses without undergoing genetic changes. The toxin-antitoxin (TA) systems are involved in the formation of persister cells because they are able to induce cell dormancy. Among the TA systems, the MqsRA system has been observed to be highly induced in persister cells of Xylella fastidiosa (causal agent of citrus variegated chlorosis-CVC) activated by copper stress, and has been described in Escherichia coli as related to the formation of persister cells and biofilms. Thus, we evaluated the role of this TA system in X. fastidiosa by overexpressing the MqsR toxin, and verified that the toxin positively regulated biofilm formation and negatively cell movement, resulting in reduced pathogenicity in citrus plants. The overexpression of MqsR also increased the formation of persister cells under copper stress. Analysis of the gene and protein expression showed that this system likely has an autoregulation mechanism to express the toxin and antitoxin in the most beneficial ratio for the cell to oppose stress. Our results suggest that this TA system plays a key role in the adaptation and survival of X. fastidiosa and reveal new insights into the physiology of phytopathogen-host interactions.

Type II Toxin-Antitoxin Distribution and Adaptive Aspects on Xanthomonas Genomes: Focus on Xanthomonas citri.

Prokaryotic toxin-antitoxin (TA) systems were first described as being designed to prevent plasmid loss in bacteria. However, with the increase in prokaryotic genome sequencing, recently many TAs have been found in bacterial chromosomes, having other biological functions, such as environmental stress response. To date, only few studies have focused on TA systems in phytopathogens, and their possible impact on the bacterial fitness. This may be especially important for pathogens like Xanthomonas spp., which live epiphytically before entering the host. In this study, we looked for TA systems in the genomes of 10 Xanthomonas strains. We verified that citrus-infecting pathovars have, on average, 50% more TAs than other Xanthomonas spp. and no genome harbors classical toxins such as MqsR, RelB, and HicA. Only one TA system (PIN_VapC-FitB-like/SpoVT_AbrB) was conserved among the Xanthomonas genomes, suggesting adaptive aspects concerning its broad occurrence. We also detected a trend of toxin gene loss in this genus, while the antitoxin gene was preferably maintained. This study discovers the quantitative and qualitative differences among the type II TA systems present in Xanthomonas spp., especially concerning the citrus-infecting strains. In addition, the antitoxin retention in the genomes is possibly related with the resistance mechanism of further TA infections as an anti-addiction system or might also be involved in regulation of certain specific genes.

Nanowire Arrays as Cell Force Sensors To Investigate Adhesin-Enhanced Holdfast of Single Cell Bacteria and Biofilm Stability.

Surface attachment of a planktonic bacteria, mediated by adhesins and extracellular polymeric substances (EPS), is a crucial step for biofilm formation. Some pathogens can modulate cell adhesiveness, impacting host colonization and virulence. A framework able to quantify cell-surface interaction forces and their dependence on chemical surface composition may unveil adhesiveness control mechanisms as new targets for intervention and disease control. Here we employed InP nanowire arrays to dissect factors involved in the early stage biofilm formation of the phytopathogen Xylella fastidiosa. Ex vivo experiments demonstrate single-cell adhesion forces up to 45 nN, depending on the cell orientation with respect to the surface. Larger adhesion forces occur at the cell poles; secreted EPS layers and filaments provide additional mechanical support. Significant adhesion force enhancements were observed for single cells anchoring a biofilm and particularly on XadA1 adhesin-coated surfaces, evidencing molecular mechanisms developed by bacterial pathogens to create a stronger holdfast to specific host tissues.

The Antitoxin Protein of a Toxin-Antitoxin System from Xylella fastidiosa Is Secreted via Outer Membrane Vesicles.

The Xylella fastidiosa subsp pauca strain 9a5c is a Gram-negative, xylem-limited bacterium that is able to form a biofilm and affects citrus crops in Brazil. Some genes are considered to be involved in biofilm formation, but the specific mechanisms involved in this process remain unknown. This limited understanding of how some bacteria form biofilms is a major barrier to our comprehension of the progression of diseases caused by biofilm-producing bacteria. Several investigations have shown that the toxin-antitoxin (TA) operon is related to biofilm formation. This operon is composed of a toxin with RNAse activity and its cognate antitoxin. Previous reports have indicated that the antitoxin is able to inhibit toxin activity and modulate the expression of the operon as well as other target genes involved in oxidative stress and mobility. In this study, we characterize a toxin-antitoxin system consisting of XfMqsR and XfYgiT, respectively, from X. fastidiosa subsp. pauca strain 9a5c. These proteins display a high similarity to their homologs in X. fastidiosa strain Temecula and a predicted tridimensional structure that is similar to MqsR-YgiT from Escherichia coli. The characterization was performed using in vitro assays such as analytical ultracentrifugation (AUC), size exclusion chromatography, isothermal titration calorimetry, and Western blotting. Using a fluorometric assay to detect RNAses, we demonstrated that XfMqsR is thermostable and can degrade RNA. XfMqsR is inhibited by XfYgiT, which interacts with its own promoter. XfYgiT is known to be localized in the intracellular compartment; however, we provide strong evidence that X. fastidiosa secretes wild-type XfYgiT into the extracellular environment via outer membrane vesicles, as confirmed by Western blotting and specific immunofluorescence labeling visualized by fluorescence microscopy. Taken together, our results characterize the TA system from X. fastidiosa strain 9a5c, and we also discuss the possible influence of wild-type XfYgiT in the cell.

In vitro Determination of Extracellular Proteins from Xylella fastidiosa.

The phytopathogen Xylella fastidiosa causes economic losses in important agricultural crops. Xylem vessel occlusion caused by biofilm formation is the major mechanism underlying the pathogenicity of distinct strains of X. fastidiosa. Here, we provide a detailed in vitro characterization of the extracellular proteins of X. fastidiosa. Based on the results, we performed a comparison with a strain J1a12, which cannot induce citrus variegated chlorosis symptoms when inoculated into citrus plants. We then extend this approach to analyze the extracellular proteins of X. fastidiosa in media supplemented with calcium. We verified increases in extracellular proteins concomitant with the days of growth and, consequently, biofilm development (3-30 days). Outer membrane vesicles carrying toxins were identified beginning at 10 days of growth in the 9a5c strain. In addition, a decrease in extracellular proteins in media supplemented with calcium was observed in both strains. Using mass spectrometry, 71 different proteins were identified during 30 days of X. fastidiosa biofilm development, including proteases, quorum-sensing proteins, biofilm formation proteins, hypothetical proteins, phage-related proteins, chaperones, toxins, antitoxins, and extracellular vesicle membrane components.

Characterization of the LysR-type transcriptional regulator YcjZ-like from Xylella fastidiosa overexpressed in Escherichia coli.

The Xylella fastidiosa 9a5c strain is a xylem-limited phytopathogen that is the causal agent of citrus variegated chlorosis (CVC). This bacterium is able to form a biofilm and occlude the xylem vessels of susceptible plants, which leads to significant agricultural and economic losses. Biofilms are associated with bacterial pathogenicity because they are very resistant to antibiotics and other metal-based chemicals that are used in agriculture. The X. fastidiosa YcjZ-like (XfYcjZ-like) protein belongs to the LysR-type transcriptional regulator (LTTR) family and is involved in various cellular functions that range from quorum sensing to bacterial survival. In the present study, we report the cloning, expression and purification of XfYcjZ-like, which was overexpressed in Escherichia coli. The secondary folding of the recombinant and purified protein was assessed by circular dichroism, which revealed that XfYcjZ-like contains a typical α/ß fold. An initial hydrodynamic characterization showed that XfYcjZ-like is a globular tetramer in solution. In addition, using a polyclonal antibody against XfYcjZ-like, we assessed the expression profile of this protein during the different developmental phases of X. fastidiosa in in vitro cultivated biofilm cells and demonstrated that XfYcjZ-like is upregulated in planktonic cells in response to a copper shock treatment. Finally, the ability of XfYcjZ-like to interact with its own predicted promoter was confirmed in vitro, which is a typical feature of LysR. Taken together, our findings indicated that the XfYcjZ-like protein is involved in both the organization of the architecture and the maturation of the bacterial biofilm and that it is responsive to oxidative stress.

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.